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Sample records for ago2 immunoprecipitation identifies

  1. Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

    PubMed Central

    Swerdel, Mavis R.; Moore, Jennifer C.; Cohen, Rick I.; Wu, Hao; Sun, Yi E.; Hart, Ronald P.

    2009-01-01

    Background MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. Methodology/Principal Findings SOLiD ultra-deep sequencing identified >107 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. Conclusions/Significance Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation. PMID:19784364

  2. Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses.

    PubMed

    Fujisawa, Masaki; Shima, Yoko; Higuchi, Naoki; Nakano, Toshitsugu; Koyama, Yoshiyuki; Kasumi, Takafumi; Ito, Yasuhiro

    2012-06-01

    The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.

  3. Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum

    PubMed Central

    Cai, Pengfei; Piao, Xianyu; Hou, Nan; Liu, Shuai; Wang, Heng; Chen, Qijun

    2012-01-01

    Background The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored. Methodology/Principal Findings Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons. Conclusions/Significance In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular

  4. Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors.

    PubMed

    Wagner, Meike; Jung, Johannes; Koslowski, Michael; Türeci, Özlem; Tiwari, Vijay K; Sahin, Ugur

    2016-01-01

    DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly by interaction with other DNA-bound factors. Chromatin immunoprecipitation (ChIP) assay is a powerful method to study interactions of proteins and a specific genomic DNA region. Recruitment of ERα to promoters of estrogen-dependent genes is a common mechanism to activate or enhance gene transcription in breast cancer thus promoting tumor progression. In this chapter, we demonstrate a stepwise protocol for ChIP assay using binding of ERα to its genomic targets after stimulation with 17β-estradiol (E2) in breast cancer cells as an example.

  5. RNA-Seq and RNA Immunoprecipitation Analyses of the Transcriptome of Streptomyces coelicolor Identify Substrates for RNase III

    PubMed Central

    Gatewood, Marcha L.; Bralley, Patricia; Weil, M. Ryan

    2012-01-01

    RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative protein-coding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by ≥2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by ≥2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage. PMID:22389483

  6. Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors.

    PubMed

    Heisig, Julia; Weber, David; Englberger, Eva; Winkler, Anja; Kneitz, Susanne; Sung, Wing-Kin; Wolf, Elmar; Eilers, Martin; Wei, Chia-Lin; Gessler, Manfred

    2012-01-01

    HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.

  7. From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data

    PubMed Central

    Bottini, Silvia; Hamouda-Tekaya, Nedra; Tanasa, Bogdan; Zaragosi, Laure-Emmanuelle; Grandjean, Valerie; Repetto, Emanuela

    2017-01-01

    Abstract Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification. PMID:28108660

  8. Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: Identifying ChIP-quality p300 monoclonal antibodies

    PubMed Central

    Gasper, William C.; Marinov, Georgi K.; Pauli-Behn, Florencia; Scott, Max T.; Newberry, Kimberly; DeSalvo, Gilberto; Ou, Susan; Myers, Richard M.; Vielmetter, Jost; Wold, Barbara J.

    2014-01-01

    Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1. PMID:24919486

  9. Chromatin immunoprecipitation scanning identifies glucocorticoid receptor binding regions in the proximal promoter of a ubiquitously expressed glucocorticoid target gene in brain.

    PubMed

    van der Laan, Siem; Sarabdjitsingh, R Angela; Van Batenburg, Marcel F; Lachize, Servane B; Li, Hualing; Dijkmans, Thomas F; Vreugdenhil, Erno; de Kloet, E Ron; Meijer, Onno C

    2008-09-01

    While the actions of glucocorticoids on brain functions have been comprehensively studied, the underlying genomic mechanisms are poorly understood. In this study, we show that glucocorticoid-induced leucine zipper (GILZ) mRNA is strongly and ubiquitously induced in rat brain. To decipher the molecular mechanisms underlying these genomic effects, it is of interest to identify the regulatory sites in the promoter region. Alignment of the rat GILZ promoter with the well-characterized human promoter resulted in poor sequence homology. Consequently, we analyzed the rat 5' flanking sequence by Matrix REDUCE and identified two high-affinity glucocorticoid response elements (GRE) located 2 kb upstream of the transcription start site. These findings were corroborated using the glucocorticoid receptor (GR) expressing Ns-1 PC12 rat cell-line. In these cells, dexamethasone treatment leads to a progressive increase of GILZ mRNA expression levels via a GR-dependent mechanism. Subsequently, using chromatin immunoprecipitation assays we show that the two high-affinity GREs are located within the GR-binding regions. Lastly, we demonstrate using multiple tissue in situ hybridization a marked increase in mRNA expression levels in spleen, thymus, heart, lung, liver, muscle, testis, kidney, colon, ileum, as well as in brain and conclude that the GILZ gene can be used to study glucocorticoid effects in many additional rodent tissues.

  10. Strong reduction of AGO2 expression in melanoma and cellular consequences

    PubMed Central

    Völler, D; Reinders, J; Meister, G; Bosserhoff, A-K

    2013-01-01

    Background: Processing of microRNAs (miRNAs) is a highly controlled process. Deregulation of miRNA expression was observed in several types of cancer but changes in the miRNA-processing enzymes have not been analysed until today. In this study, we analysed Argonaute2 (AGO2, EIF2C2), as one main factor of the miRNA processing ensemble, in the context of cancer development, especially in melanoma. Methods: We determined the AGO2 expression level in melanoma, as well as in other cancers, with biochemical approaches (qRT–PCR, western blot and immunofluorescence studies) and analysed the cell behaviour in migration assays. Results: Specifically in melanoma, we revealed a strong reduction of AGO2 expression compared with primary melanocytes. The reduction of AGO2 expression was only found on protein level, whereas the mRNA level stayed unchanged hinting to post-transcriptional regulation. We could show that re-expression of AGO2 in melanoma leads to a strong improvement of regulatory effects due to increased functionality of small-interfering RNAs and short hairpin RNAs. Conclusion: We identified melanoma-specific downregulation of AGO2 and corresponding reduced RNAi efficiency. These findings will help to understand the molecular basis of malignant melanoma and can potentially lead to an improvement of therapeutic strategies. PMID:24169347

  11. Chromatin immunoprecipitation and multiplex sequencing (ChIP-Seq) to identify global transcription factor binding sites in the nematode Caenorhabditis elegans.

    PubMed

    Brdlik, Cathleen M; Niu, Wei; Snyder, Michael

    2014-01-01

    The global identification of transcription factor (TF) binding sites is a critical step in the elucidation of the functional elements of the genome. Several methods have been developed that map TF binding in human cells, yeast, and other model organisms. These methods make use of chromatin immunoprecipitation, or ChIP, and take advantage of the fact that formaldehyde fixation of living cells can be used to cross-link DNA sequences to the TFs that bind them in vivo. In ChIP, the cross-linked TF-DNA complexes are sheared by sonication, size fractionated, and incubated with antibody specific to the TF of interest to generate a library of TF-bound DNA sequences. ChIP-chip was the first technology developed to globally identify TF-bound DNA sequences and involves subsequent hybridization of the ChIP DNA to oligonucleotide microarrays. However, ChIP-chip proved to be costly, labor-intensive, and limited by the fixed number of probes available on the microarray chip. ChIP-Seq combines ChIP with massively parallel high-throughput sequencing (see Explanatory Chapter: Next Generation Sequencing) and has demonstrated vast improvement over ChIP-chip with respect to time and cost, signal-to-noise ratio, and resolution. In particular, multiplex sequencing can be used to achieve a higher throughput in ChIP-Seq analyses involving organisms with genomes of lower complexity than that of human (Lefrançois et al., 2009) and thereby reduce the cost and amount of time needed for each result. The multiplex ChIP-Seq method described in this section has been developed for Caenorhabditis elegans, but is easily adaptable for other organisms. © 2014 Elsevier Inc. All rights reserved.

  12. An Antiviral Defense Role of AGO2 in Plants

    PubMed Central

    Harvey, Jagger J. W.; Lewsey, Mathew G.; Patel, Kanu; Westwood, Jack; Heimstädt, Susanne; Carr, John P.; Baulcombe, David C.

    2011-01-01

    Background Argonaute (AGO) proteins bind to small-interfering (si)RNAs and micro (mi)RNAs to target RNA silencing against viruses, transgenes and in regulation of mRNAs. Plants encode multiple AGO proteins but, in Arabidopsis, only AGO1 is known to have an antiviral role. Methodology/Principal Findings To uncover the roles of specific AGOs in limiting virus accumulation we inoculated turnip crinkle virus (TCV) to Arabidopsis plants that were mutant for each of the ten AGO genes. The viral symptoms on most of the plants were the same as on wild type plants although the ago2 mutants were markedly hyper-susceptible to this virus. ago2 plants were also hyper-susceptible to cucumber mosaic virus (CMV), confirming that the antiviral role of AGO2 is not specific to a single virus. For both viruses, this phenotype was associated with transient increase in virus accumulation. In wild type plants the AGO2 protein was induced by TCV and CMV infection. Conclusions/Significance Based on these results we propose that there are multiple layers to RNA-mediated defense and counter-defense in the interactions between plants and their viruses. AGO1 represents a first layer. With some viruses, including TCV and CMV, this layer is overcome by viral suppressors of silencing that can target AGO1 and a second layer involving AGO2 limits virus accumulation. The second layer is activated when the first layer is suppressed because AGO2 is repressed by AGO1 via miR403. The activation of the second layer is therefore a direct consequence of the loss of the first layer of defense. PMID:21305057

  13. Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma

    PubMed Central

    Turchinovich, Andrey; Burwinkel, Barbara

    2012-01-01

    Studies of miRNA association with Argonaute (AGO) proteins in mammalian cells have indicated lack of bias toward particular AGO. However, to our knowledge, the use of quantitative methods for studying miRNA association with different AGOs has not been reported so far. In this work we compared the total miRNA content in AGO1 and AGO2 immunoprecipitates obtained from MCF7 adenocarcinoma cells using TaqMan Low Density miRNA Arrays and successfully verified selected miRNAs with qPCR. For most of the miRNA species AGO1 and AGO2 profiles were well correlated, however, some miRNAs demonstrated consistent biases toward one of the Argonautes. Furthermore, miRNAs which were predominantly AGO2-associated derived mostly from sense strands of the corresponding pre-miRNAs while the majority of AGO1 biased miRNAs originated from antisense strands of the pre-miRNAs. Additionally, we show that circulating miRNA in human blood plasma can be immunoprecipitated with both AGO1 and AGO2 antibody. However, unlike in cell lysates, AGO1 and AGO2 associated miRNA profiles in plasma did not correlate, indicating that many cell types contribute to circulating miRNA (given that expression of AGO proteins is tissue specific). Furthermore, AGO-specific miRNA profiles in blood cells differed significantly from miRNAs profiles in plasma indicating that most circulating miRNAs are likely to derive from non-blood cells. Since circulating miRNAs hold great promise as biomarkers for numerous cancers and other diseases, we hypothesize that AGO-specific miRNA profiles might add an additional dimension to circulating miRNA-based diagnostics. PMID:22858679

  14. Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity.

    PubMed

    van Eijl, Roderick A P M; van den Brand, Teun; Nguyen, Luan N; Mulder, Klaas W

    2017-08-04

    In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enrichment at enhancers and transcription start sites using ChIP-seq. However, antibody specificity issues can produce misleading results for ChIP, RNA-seq and Mass spectrometry. Therefore, we developed a CRISPR/Cas9 engineered AGO2(-/-) HEK293T cell line to validate our findings. ChIP-qPCR and immunoprecipitation combined with MS (IP-MS) showed that the 11A9 antibody associates with chromatin and SWI/SNF in the absence of AGO2. Furthermore, stoichiometry, IP-MS and co-IP analysis suggests a direct interaction of this antibody with SMARCC1, a component of the SWI/SNF complex. For this reason, particular care should be taken in performing and interpreting experiments in which the 11A9 antibody is used to study a nuclear role of AGO2.

  15. Digital Microfluidics for Immunoprecipitation.

    PubMed

    Seale, Brendon; Lam, Charis; Rackus, Darius G; Chamberlain, M Dean; Liu, Chang; Wheeler, Aaron R

    2016-10-04

    Immunoprecipitation (IP) is a common method for isolating a targeted protein from a complex sample such as blood, serum, or cell lysate. In particular, IP is often used as the primary means of target purification for the analysis by mass spectrometry of novel biologically derived pharmaceuticals, with particular utility for the identification of molecules bound to a protein target. Unfortunately, IP is a labor-intensive technique, is difficult to perform in parallel, and has limited options for automation. Furthermore, the technique is typically limited to large sample volumes, making the application of IP cleanup to precious samples nearly impossible. In recognition of these challenges, we introduce a method for performing microscale IP using magnetic particles and digital microfluidics (DMF-IP). The new method allows for 80% recovery of model proteins from approximately microliter volumes of serum in a sample-to-answer run time of approximately 25 min. Uniquely, analytes are eluted from these small samples in a format compatible with direct analysis by mass spectrometry. To extend the technique to be useful for large samples, we also developed a macro-to-microscale interface called preconcentration using liquid intake by paper (P-CLIP). This technique allows for efficient analysis of samples >100× larger than are typically processed on microfluidic devices. As described herein, DMF-IP and P-CLIP-DMF-IP are rapid, automated, and multiplexed methods that have the potential to reduce the time and effort required for IP sample preparations with applications in the fields of pharmacy, biomarker discovery, and protein biology.

  16. An Ago2-associated capped transcriptional start site small RNA suppressing adenovirus DNA replication.

    PubMed

    Kamel, Wael; Akusjärvi, Göran

    2017-08-24

    Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3'end. The MLP-TSS-sRNA is highly stable in cells and functionally active, downregulating complementary targets in a sequence and dose dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and pre-terminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and pre-terminal protein mRNA expression. As a consequence of this the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2.

    PubMed

    Frank, Filipp; Sonenberg, Nahum; Nagar, Bhushan

    2010-06-10

    MicroRNAs (miRNAs) mediate post-transcriptional gene regulation through association with Argonaute proteins (AGOs). Crystal structures of archaeal and bacterial homologues of AGOs have shown that the MID (middle) domain mediates the interaction with the phosphorylated 5' end of the miRNA guide strand and this interaction is thought to be independent of the identity of the 5' nucleotide in these systems. However, analysis of the known sequences of eukaryotic miRNAs and co-immunoprecipitation experiments indicate that there is a clear bias for U or A at the 5' position. Here we report the crystal structure of a MID domain from a eukaryotic AGO protein, human AGO2. The structure, in complex with nucleoside monophosphates (AMP, CMP, GMP, and UMP) mimicking the 5' end of miRNAs, shows that there are specific contacts made between the base of UMP or AMP and a rigid loop in the MID domain. Notably, the structure of the loop discriminates against CMP and GMP and dissociation constants calculated from NMR titration experiments confirm these results, showing that AMP (0.26 mM) and UMP (0.12 mM) bind with up to 30-fold higher affinity than either CMP (3.6 mM) or GMP (3.3 mM). This study provides structural evidence for nucleotide-specific interactions in the MID domain of eukaryotic AGO proteins and explains the observed preference for U or A at the 5' end of miRNAs.

  18. The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading.

    PubMed

    Tarallo, Roberta; Giurato, Giorgio; Bruno, Giuseppina; Ravo, Maria; Rizzo, Francesca; Salvati, Annamaria; Ricciardi, Luca; Marchese, Giovanna; Cordella, Angela; Rocco, Teresa; Gigantino, Valerio; Pierri, Biancamaria; Cimmino, Giovanni; Milanesi, Luciano; Ambrosino, Concetta; Nyman, Tuula A; Nassa, Giovanni; Weisz, Alessandro

    2017-10-06

    The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ - cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.

  19. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development

    PubMed Central

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael

    2016-01-01

    ABSTRACT Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at

  20. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis of the Penicillium chrysogenum Velvet Protein PcVelA Identifies Methyltransferase PcLlmA as a Novel Downstream Regulator of Fungal Development.

    PubMed

    Becker, Kordula; Ziemons, Sandra; Lentz, Katharina; Freitag, Michael; Kück, Ulrich

    2016-01-01

    Penicillium chrysogenum is the sole industrial producer of the β-lactam antibiotic penicillin, which is the most commonly used drug for treating bacterial infections. In P. chrysogenum and other filamentous fungi, secondary metabolism and morphogenesis are controlled by the highly conserved multisubunit velvet complex. Here we present the first chromatin immunoprecipitation next-generation sequencing (ChIP-seq) analysis of a fungal velvet protein, providing experimental evidence that a velvet homologue in P. chrysogenum (PcVelA) acts as a direct transcriptional regulator at the DNA level in addition to functioning as a regulator at the protein level in P. chrysogenum, which was previously described. We identified many target genes that are related to processes known to be dependent on PcVelA, e.g., secondary metabolism as well as asexual and sexual development. We also identified seven PcVelA target genes that encode putative methyltransferases. Yeast two-hybrid and bimolecular fluorescence complementation analyses showed that one of the putative methyltransferases, PcLlmA, directly interacts with PcVelA. Furthermore, functional characterization of PcLlmA demonstrated that this protein is involved in the regulation of conidiosporogenesis, pellet formation, and hyphal morphology, all traits with major biotechnological relevance. IMPORTANCE Filamentous fungi are of major interest for biotechnological and pharmaceutical applications. This is due mainly to their ability to produce a wide variety of secondary metabolites, many of which are relevant as antibiotics. One of the most prominent examples is penicillin, a β-lactam antibiotic that is produced on the industrial scale by fermentation of P. chrysogenum. In recent years, the multisubunit protein complex velvet has been identified as one of the key regulators of fungal secondary metabolism and development. However, until recently, only a little has been known about how velvet mediates regulation at the

  1. Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

    PubMed

    Teteloshvili, Nato; Smigielska-Czepiel, Katarzyna; Yuan, Ye; Seitz, Annika; de Jong, Debora; Rutgers, Bea; Jellema, Pytrick; van der Lei, Roelof Jan; Slezak-Prochazka, Izabella; Brouwer, Elisabeth; Boots, Annemieke M H; Kroesen, Bart-Jan; van den Berg, Anke; Kluiver, Joost

    2017-02-01

    MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

  2. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Inhibits RNA-Mediated Gene Silencing by Targeting Ago-2.

    PubMed

    Chen, Jing; Shi, Xibao; Zhang, Xiaozhuan; Wang, Li; Luo, Jun; Xing, Guangxu; Deng, Ruiguang; Yang, Hong; Li, Jinting; Wang, Aiping; Zhang, Gaiping

    2015-10-23

    Porcine reproductive and respiratory syndrome virus (PRRSV) infection strongly modulates the host's immune response. The RNA silencing pathway is an intracellular innate response to viral infections. However, it is unknown whether PRRSV interacts with cellular RNA silencing to facilitate the viral infection. Here, we report for the first time the interaction between PRRSV and RNA silencing in both the porcine macrophages and African green monkey kidney cell line (MARC-145) cell line, which were derived from African green monkey kidney cells and highly permissive for PRRSV infection. Our data demonstrated that PRRSV suppressed RNA silencing induced by short-hairpin (sh) RNA, double-strand (ds) RNA and microRNA (miRNA) and downregulated the expression of argonaute protein-2 (Ago-2), which is a key protein of the RNA silencing pathway in animal cells. Further, exogenous introduction of siRNA and shRNA downregulated Dicer or Ago-2 proteins of the cellular RNA silencing apparatus in MARC-145 cells and porcine macrophages, which, in turn, increased the viral replication and titers. The viral non-structure protein 1α (nsp-1α) and nsp11 of PRRSV were identified as the suppressors for cellular RNA silencing (RSSs) to downregulate the Ago-2 protein. Our results identify that PRRSV, through its nsp proteins, suppresses the cellular RNA silencing apparatus in favor of viral infection and supports a co-evolutionary process of the virus and the cellular RNA silencing process.

  3. Co-immunoprecipitation of Plant Receptor Kinases.

    PubMed

    Barberini, María Laura; Muschietti, Jorge P

    2017-01-01

    In order to comprehend the function of a particular protein, identification of the interacting protein partners is a useful approach. Co-immunoprecipitation (Co-IP) is employed to test physical interactions between proteins. Specific antibodies or antibodies against tagged versions can be used to immunoprecipitate the proteins. In this chapter, we describe a method to carry out Co-IP using recombinant membrane proteins expressed in yeast microsomal fractions.

  4. Structural analysis of 5'-mRNA-cap interactions with the human AGO2 MID domain.

    PubMed

    Frank, Filipp; Fabian, Marc R; Stepinski, Janusz; Jemielity, Jacek; Darzynkiewicz, Edward; Sonenberg, Nahum; Nagar, Bhushan

    2011-05-01

    In RNA silencing, microRNA (miRNA)-mediated translational repression occurs through mechanisms that do not invoke messenger-RNA (mRNA) target cleavage by Argonaute proteins. The nature of these mechanisms is unclear, but several recent studies have proposed that a direct interaction between the mRNA-cap and the middle (MID) domain of Argonautes is involved. Here, we present crystallographic and NMR data demonstrating that cap analogues do not bind significantly to the isolated MID domain of human Argonaute 2 (hAGO2) and are found in the miRNA 5'-nucleotide binding site in an implausible binding mode. Additionally, in vitro pull-down experiments with full-length hAGO2 indicate that the interaction with cap analogues is nonspecific.

  5. Stilbene derivatives promote Ago2-dependent tumour-suppressive microRNA activity

    PubMed Central

    Hagiwara, Keitaro; Kosaka, Nobuyoshi; Yoshioka, Yusuke; Takahashi, Ryou-u; Takeshita, Fumitaka; Ochiya, Takahiro

    2012-01-01

    It is well known that natural products are a rich source of compounds for applications in medicine, pharmacy, and biology. However, the exact molecular mechanisms of natural agents in human health have not been clearly defined. Here, we demonstrate for the first time that the polyphenolic phytoalexin resveratrol promotes expression and activity of Argonaute2 (Ago2), a central RNA interference (RNAi) component, which thereby inhibits breast cancer stem-like cell characteristics by increasing the expression of a number of tumour-suppressive miRNAs, including miR-16, -141, -143, and -200c. Most importantly, resveratrol-induced Ago2 resulted in a long-term gene silencing response. We also found that pterostilbene, which is a natural dimethylated resveratrol analogue, is capable of mediating Ago2-dependent anti-cancer activity in a manner mechanistically similar to that of resveratrol. These findings suggest that the dietary intake of natural products contributes to the prevention and treatment of diseases by regulating the RNAi pathway. PMID:22423322

  6. eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference

    NASA Astrophysics Data System (ADS)

    Yi, Tingfang; Arthanari, Haribabu; Akabayov, Barak; Song, Huaidong; Papadopoulos, Evangelos; Qi, Hank H.; Jedrychowski, Mark; Güttler, Thomas; Guo, Cuicui; Luna, Rafael E.; Gygi, Steven P.; Huang, Stephen A.; Wagner, Gerhard

    2015-05-01

    MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.

  7. Enrichment of methylated DNA by methyl-CpG immunoprecipitation.

    PubMed

    Sonnet, Miriam; Baer, Constance; Rehli, Michael; Weichenhan, Dieter; Plass, Christoph

    2013-01-01

    Normal DNA methylation is an epigenetic modification required for proper development. Aberrant DNA methylation, in contrast, is frequently observed in many different malignancies including leukemias and lymphomas. Global DNA methylation profiling addresses the methylated sequences (methylome) of patient genomes to identify disease-specific methylation patterns. Workload in methylome analyses can be considerably reduced by methylome enrichment using proteins or antibodies with high affinity to methylated DNA. Methyl-CpG Immunoprecipitation (MCIp) employs an immobilized recombinant human methyl-CpG binding domain protein 2, MBD2, which binds methylated CpGs in double-stranded DNA. Elution with increasing salt concentrations allows the fractionated enrichment of different degrees of methylation.

  8. Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages

    PubMed Central

    Wooden, Jessica; Ciborowski, Pawel

    2014-01-01

    The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a “one size fits all” rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM)a, we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10 minutes. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge. PMID:25220915

  9. Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

    PubMed

    Kandeel, Mahmoud; Al-Taher, Abdullah; Nakashima, Remi; Sakaguchi, Tomoya; Kandeel, Ali; Nagaya, Yuki; Kitamura, Yoshiaki; Kitade, Yukio

    2014-01-01

    Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

  10. Bioenergetics and Gene Silencing Approaches for Unraveling Nucleotide Recognition by the Human EIF2C2/Ago2 PAZ Domain

    PubMed Central

    Kandeel, Mahmoud; Al-Taher, Abdullah; Nakashima, Remi; Sakaguchi, Tomoya; Kandeel, Ali; Nagaya, Yuki; Kitamura, Yoshiaki; Kitade, Yukio

    2014-01-01

    Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3′-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3′-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain. PMID:24788663

  11. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  12. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

  13. Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

  14. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases.

  15. Taenia taeniaeformis: immunoprecipitation analysis of the protein antigens of oncospheres and larvae.

    PubMed

    Bowtell, D D; Mitchell, G F; Anders, R F; Lightowlers, M W; Rickard, M D

    1983-12-01

    Biosynthetically or exogenously labeled proteins and immunoprecipitated protein antigens of established 28-day-old larvae of Taenia taeniaeformis were compared with proteins and antigens of infective oncospheres using single and two-dimensional gel electrophoresis. Immunoprecipitation was carried out using sera from infected mice and mouse antisera raised to larvae or oncospheres, and emphasis was placed on identifying antigens common to both oncospheres and larvae. Two major larval antigens of Mr 40,000 and 200,000, designated Tt40 and Tt200, are common to somatic larval preparations and oncospheres. Additionally, two major oncosphere antigens of Mr 55,000 and 60,000, designated Tt55 and Tt60, are also present in larval excretory and secretory (i.e., ES or exoantigen) products. Information obtained from these immunoprecipitation analyses will facilitate isolation and production of common as well as stage-specific protein antigens in the development of defined-antigen vaccines in this model system of cysticercosis.

  16. RNA immunoprecipitation for determining RNA-protein associations in vivo.

    PubMed

    Gilbert, Chris; Svejstrup, Jesper Q

    2006-08-01

    Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. RNA sequences that cross-link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding steps for metazoan cells have not yet been worked out, it is likely that the yeast procedure can easily be adapted for use in other organisms.

  17. Activated platelets can deliver mRNA regulatory Ago2•microRNA complexes to endothelial cells via microparticles.

    PubMed

    Laffont, Benoit; Corduan, Aurélie; Plé, Hélène; Duchez, Anne-Claire; Cloutier, Nathalie; Boilard, Eric; Provost, Patrick

    2013-07-11

    Platelets play a crucial role in the maintenance of hemostasis, as well as in thrombosis. Upon activation, platelets release small membrane-bound microparticles (MPs) containing bioactive proteins and genetic materials from their parental cells that may be transferred to, and exert potent biological effects in, recipient cells of the circulatory system. Platelets have been shown to contain an abundant and diverse array of microRNAs, and platelet-derived MPs are the most abundant microvesicles in the circulation. Here we demonstrate that human platelets activated with thrombin preferentially release their miR-223 content in MPs. These MPs can be internalized by human umbilical vein endothelial cells (HUVEC), leading to the accumulation of platelet-derived miR-223. Platelet MPs contain functional Argonaute 2 (Ago2)•miR-223 complexes that are capable of regulating expression of a reporter gene in recipient HUVEC. Moreover, we demonstrate a role for platelet MP-derived miR-223 in the regulation of 2 endogenous endothelial genes, both at the messenger RNA and protein levels. Our results support a scenario by which platelet MPs may act as intercellular carriers of functional Ago2•microRNA complexes that may exert heterotypic regulation of gene expression in endothelial cells, and possibly other recipient cells of the circulatory system.

  18. Chromatin immunoprecipitation and microarray-based analysis of protein location

    PubMed Central

    Lee, Tong Ihn; Johnstone, Sarah E; Young, Richard A

    2010-01-01

    Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types. PMID:17406303

  19. Whole genome methylation profiling by immunoprecipitation of methylated DNA.

    PubMed

    Sharp, Andrew J

    2012-01-01

    I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.

  20. Analysis of in vivo transcription factor recruitment by chromatin immunoprecipitation of mouse embryonic kidney.

    PubMed

    Heliot, Claire; Cereghini, Silvia

    2012-01-01

    Chromatin immunoprecipitation (ChIP) is a powerful technique for examining transcription factor recruitment to chromatin, or histone modifications, at the level of specific genomic sequences. As such, it provides an invaluable tool for elucidating gene regulation at the molecular level. Combined with high-throughput methods such as second generation sequencing (ChIP-Seq), this technique is now commonly used for studying DNA-protein interactions at a genome-wide scale. The ChIP technique is based on covalent cross-linking of DNA and proteins with formaldehyde, followed by chromatin fragmentation, either enzymatic or by sonication, and immunoprecipitation of protein-DNA complexes using antibodies specific for the protein of interest. The immunoprecipitated DNA is then purified and the DNA sequences associated with the immunoprecipitated protein are identified by PCR (ChIP-PCR) or, alternatively, by direct sequencing (ChIP-Seq). Initially, the vast majority of ChIP experiments were performed on cultured cell lines. More recently, this technique has been adapted to a variety of tissues in different model organisms. We describe here a ChIP protocol on freshly isolated mouse embryonic kidneys for in vivo analysis of transcription factor recruitment on chromatin. This protocol has been easily adapted to other mouse embryonic tissues and has also been successfully scaled up to perform ChIP-Seq.

  1. Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.

    PubMed

    Emmott, Edward; Goodfellow, Ian

    2014-07-06

    Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

  2. Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells

    PubMed Central

    Pare, Justin M.; LaPointe, Paul; Hobman, Tom C.

    2013-01-01

    Argonaute proteins and small RNAs together form the RNA-induced silencing complex (RISC), the central effector of RNA interference (RNAi). The molecular chaperone Hsp90 is required for the critical step of loading small RNAs onto Argonaute proteins. Here we show that the Hsp90 cochaperones Cdc37, Aha1, FKBP4, and p23 are required for efficient RNAi. Whereas FKBP4 and p23 form a stable complex with hAgo2, the function of Cdc37 in RNAi appears to be indirect and may indicate that two or more Hsp90 complexes are involved. Our data also suggest that p23 and FKBP4 interact with hAgo2 before small RNA loading and that RISC loading takes place in the cytoplasm rather than in association with RNA granules. Given the requirement for p23 and FKBP4 for efficient RNAi and that these cochaperones bind to hAgo2, we predict that loading of hAgo2 is analogous to Hsp90-mediated steroid hormone receptor activation. To this end, we outline a model in which FKBP4, p23, and Aha1 cooperatively regulate the progression of hAgo2 through the chaperone cycle. Finally, we propose that hAgo2 and RNAi can serve as a robust model system for continued investigation into the Hsp90 chaperone cycle. PMID:23741051

  3. Perinatal protein malnutrition alters expression of miRNA biogenesis genes Xpo5 and Ago2 in mice brain.

    PubMed

    Berardino, Bruno G; Fesser, Estefanía A; Cánepa, Eduardo T

    2017-04-24

    Due to its widespread incidence, maternal malnutrition remains one of the major non-genetic factors affecting the development of newborn's brain. While all nutrients have certain influence on brain maturation, proteins appear to be the most critical for the development of neurological functions. An increasing number of studies point out that the effects of early-life nutritional inadequacy has long lasting effects on the brain and lead to permanent deficits in learning and behavior. Epigenetic mechanisms provide a potential link between the nutrition status during critical periods and changes in gene expression that may lead to disease phenotypes. Among those epigenetic mechanisms microRNAs (miRNAs) emerge as promising molecules for the link between nutrition and gene expression due to their relevance in many central nervous system functions. The objective of the current study was to evaluate the impact of perinatal protein malnutrition on the development of male and female mice offspring and to analyze the expression of the genes involved in the miRNA biogenesis pathway in different mouse brain structures. We demonstrated that early nutritional stress such as exposition to a protein-deficient diet during gestation and lactation reduced the hippocampal weight, delayed offspring's development and deregulated the expression of Xpo5 and Ago2 genes in hippocampus and hypothalamus of weanling mice. Moreover, an overall increase in mature miRNAs was consistent with the induction of Xpo5 mRNA. Altered miRNA biogenesis could modify the availability and functionality of miRNA becoming a causal factor of the adverse effects of protein malnutrition.

  4. Chromatin Immunoprecipitation (ChIP) in Schizosaccharomyces pombe.

    PubMed

    Cam, Hugh P; Whitehall, Simon

    2016-11-01

    Chromatin immunoprecipitation (ChIP), the cross-linking of chromatin followed by immunoprecipitation with antibodies against a chromatin target, is a key method for measuring association of proteins with a specific genomic region(s). As a negative control, a mock ChIP experiment in which no antibody is added to the immunoprecipitation reaction is included. Enriched DNA fragments from a ChIP experiment can be analyzed in a variety of ways. For semiquantitative analysis, a region of interest can be amplified using standard polymerase chain reaction (PCR) techniques. PCR products are analyzed on agarose (or polyacrylamide) gels and band intensity calculated with a standard imaging software. ChIP enrichment is usually calculated as the ratio of ChIP to input compared with a similar ratio for a reference region not expected to be enriched for that factor. For heterochromatin analysis, housekeeping genes such as act1(+) are good references. Real-time quantitative PCR (qPCR) can be used for a fully quantitative approach. If a factor is to be mapped across the genome, ChIP DNA can be amplified and labeled for microarray analysis or scrutinized on a next-generation DNA sequencing platform. © 2016 Cold Spring Harbor Laboratory Press.

  5. The RIPper case: identification of RNA-binding protein targets by RNA immunoprecipitation.

    PubMed

    Köster, Tino; Haas, Meike; Staiger, Dorothee

    2014-01-01

    Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

  6. Phosphorylation of Ago2 and Subsequent Inactivation of let-7a RNP-Specific MicroRNAs Control Differentiation of Mammalian Sympathetic Neurons

    PubMed Central

    Patranabis, Somi

    2016-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs that regulate gene expression posttranscriptionally by base pairing to the target mRNAs in animal cells. KRas, an oncogene known to be repressed by let-7a miRNAs, is expressed and needed for the differentiation of mammalian sympathetic neurons and PC12 cells. We documented a loss of let-7a activity during this differentiation process without any significant change in the cellular level of let-7a miRNA. However, the level of Ago2, an essential component that is associated with miRNAs to form RNP-specific miRNA (miRNP) complexes, shows an increase with neuronal differentiation. In this study, differentiation-induced phosphorylation and the subsequent loss of miRNA from Ago2 were noted, and these accounted for the loss of miRNA activity in differentiating neurons. Neuronal differentiation induces the phosphorylation of mitogen-activated protein kinase p38 and the downstream kinase mitogen- and stress-activated protein kinase 1 (MSK1). This in turn upregulates the phosphorylation of Ago2 and ensures the dissociation of miRNA from Ago2 in neuronal cells. MSK1-mediated miRNP inactivation is a prerequisite for the differentiation of neuronal cells, where let-7a miRNA gets unloaded from Ago2 to ensure the upregulation of KRas, a target of let-7a. We noted that the inactivation of let-7a is both necessary and sufficient for the differentiation of sympathetic neurons. PMID:26858302

  7. Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

    PubMed

    Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

    2017-01-31

    Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

  8. Temperature-Dependent Survival of Turnip Crinkle Virus-Infected Arabidopsis Plants Relies on an RNA Silencing-Based Defense That Requires DCL2, AGO2, and HEN1

    PubMed Central

    Zhang, Xiuchun; Zhang, Xiaofeng; Singh, Jasleen; Li, Dawei

    2012-01-01

    While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of Turnip crinkle virus (TCV) in Arabidopsis, plants containing loss-of-function mutations within the Dicer-like 2 (DCL2), Argonaute 2 (AGO2), and HEN1 RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction. PMID:22496240

  9. Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.

    PubMed

    Cheung, Hoi-Hung; Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee

    2012-01-01

    DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.

  10. Parent-of-origin effects of A1CF and AGO2 on testicular germ-cell tumors, testicular abnormalities, and fertilization bias

    PubMed Central

    Carouge, Delphine; Blanc, Valerie; Knoblaugh, Sue E.; Hunter, Robert J.; Davidson, Nicholas O.; Nadeau, Joseph H.

    2016-01-01

    Testicular tumors, the most common cancer in young men, arise from abnormalities in germ cells during fetal development. Unconventional inheritance for testicular germ cell tumor (TGCT) risk both in humans and mice implicates epigenetic mechanisms. Apolipoprotein B mRNA-editing enzyme complex 1 (APOBEC1) cytidine deaminase and Deadend-1, which are involved in C-to-U RNA editing and microRNA-dependent mRNA silencing, respectively, are potent epigenetic modifiers of TGCT susceptibility in the genetically predisposed 129/Sv inbred mouse strain. Here, we show that partial loss of either APOBEC1 complementation factor (A1CF), the RNA-binding cofactor of APOBEC1 in RNA editing, or Argonaute 2 (AGO2), a key factor in the biogenesis of certain noncoding RNAs, modulates risk for TGCTs and testicular abnormalities in both parent-of-origin and conventional genetic manners. In addition, non-Mendelian inheritance was found among progeny of A1cf and Ago2 mutant intercrosses but not in backcrosses and without fetal loss. Together these findings suggest nonrandom union of gametes rather than meiotic drive or preferential lethality. Finally, this survey also suggested that A1CF contributes to long-term reproductive performance. These results directly implicate the RNA-binding proteins A1CF and AGO2 in the epigenetic control of germ-cell fate, urogenital development, and gamete functions. PMID:27582469

  11. Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    PubMed Central

    Shang, Renfu; Zhang, Fengjuan; Xu, Beiying; Xi, Hairui; Zhang, Xue; Wang, Weihua; Wu, Ligang

    2015-01-01

    Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3′ end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3′ overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. PMID:26455506

  12. Microfluidics Technologies for Low Cell Number Chromatin Immunoprecipitation.

    PubMed

    Wu, Angela R; Quake, Stephen R

    2016-04-01

    Protein-DNA interactions are responsible for numerous critical cellular events: For example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site-specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologist's toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to 1 wk to complete, and the process is technically tricky, yet tedious. The ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies using rare or precious samples. For example, fluorescence-activated cell sorting (FACS) of cancer stem cells (CSCs) obtained from primary tumors, rarely yields more than ~100,000 CSCs per tumor. This protocol describes a microfluidics-based strategy for performing ChIP, which uses automation and scalability to reduce both total and hands-on assay time, and improve throughput. It allows whole fixed cells as input, and enables automated ChIP from as few as 2000 cells.

  13. DNA-enrichment microfluidic chip for chromatin immunoprecipitation.

    PubMed

    Oh, Hyun Jik; Park, Joong Yull; Park, Sung Eun; Lee, Bo Yun; Park, Jong Sung; Kim, Suel-Kee; Yoon, Tae Joong; Lee, Sang-Hoon

    2009-04-15

    Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting association of individual proteins with specific genomic regions; the technique requires several complex steps and is tedious. In this paper, we develop a microbead-packed microfluidic chip which eliminates most of the laborious, time-consuming, and skill-dependent processes of the ChIP procedure. A computational fluid dynamics model was established to analyze fluidic behavior in a microbead-packed microchannel. With the use of the new chip, a ChIP procedure was performed to purify the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene from human embryonic kidney cells (cell line 293). The ChIP capability of the microfluidic chip was evaluated and compared with that of a commercial assay kit; the precipitation performance of both methods was almost identical as shown by quantitative measurement of DNA. However, our chip offers the advantage of low resin volume, and the experimental time is greatly reduced. In addition, our method is less dependent on individual technical skill. Therefore, we expect that our microfluidic chip-based ChIP method will be widely used in DNA-, gene-, and protein-related research and will improve experimental efficiency.

  14. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles.

    PubMed

    Cejas, Paloma; Li, Lewyn; O'Neill, Nicholas K; Duarte, Melissa; Rao, Prakash; Bowden, Michaela; Zhou, Chensheng W; Mendiola, Marta; Burgos, Emilio; Feliu, Jaime; Moreno-Rubio, Juan; Guadalajara, Héctor; Moreno, Víctor; García-Olmo, Damián; Bellmunt, Joaquim; Mullane, Stephanie; Hirsch, Michelle; Sweeney, Christopher J; Richardson, Andrea; Liu, X Shirley; Brown, Myles; Shivdasani, Ramesh A; Long, Henry W

    2016-06-01

    Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.

  15. A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue.

    PubMed

    Haim, Yulia; Tarnovscki, Tanya; Bashari, Dana; Rudich, Assaf

    2013-11-01

    Chromatin immunoprecipitation (ChIP) has become a central method when studying in vivo protein-DNA interactions, with the major challenge being the hope to capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues including adipose tissue-derived cells, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Utilizing human paired omental and subcutaneous adipose tissue obtained during elective abdominal surgeries, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. We describe a complete working protocol for using ChIP on whole adipose tissue fragments. Specific steps required adaptation of the ChIP protocol to human whole adipose tissue. In particular, a cross-linking step was performed directly on fresh small tissue fragments. Nuclei were isolated before releasing chromatin, allowing better management of fat content; a sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. In conclusion, we describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.

  16. Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

    PubMed

    Komar, Dorota N; Mouriz, Alfonso; Jarillo, José A; Piñeiro, Manuel

    2016-01-14

    Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

  17. Identification of Transcribed Enhancers by Genome-Wide Chromatin Immunoprecipitation Sequencing.

    PubMed

    Blinka, Steven; Reimer, Michael H; Pulakanti, Kirthi; Pinello, Luca; Yuan, Guo-Cheng; Rao, Sridhar

    2017-01-01

    Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.

  18. Chromatin Immunoprecipitation Assay: Examining the Interaction of NFkB with the VEGF Promoter.

    PubMed

    Walton, Chad B; Matter, Michelle L

    2015-01-01

    The chromatin immunoprecipitation (ChIP) assay is a versatile technique used to evaluate the association of proteins with specific DNA regions both in vivo and in vitro. This assay can be used to identify proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. The ChIP assay can also be used to analyze binding of transcription factors, transcription cofactors, DNA replication factors, and DNA repair proteins. Here we describe a useful ChIP-qPCR protocol to examine the interaction of NFkB with the VEGF promoter in adult rat primary cardiomyocytes that have been mechanically stretched after attaching to the extracellular matrix protein laminin.

  19. Coimmunoprecipitation (co-IP) of Nuclear Proteins and Chromatin Immunoprecipitation (ChIP) from Arabidopsis.

    PubMed

    Fiil, Berthe Katrine; Qiu, Jin-Long; Petersen, Klaus; Petersen, Morten; Mundy, John

    2008-09-01

    INTRODUCTIONTranscriptional reprogramming occurs during development and in response to diverse stimuli and stresses. The isolation and characterization of nuclear proteins, particularly those binding to DNA and chromatin, are therefore important to understanding these processes. Two specific approaches to understanding the function of nuclear proteins involve the characterization of their protein-protein interactions, and of the transcriptional targets of specific transcription factors. Coimmunoprecipitation (co-IP) is a straightforward technique to study in vivo protein-protein interactions, and can identify interacting proteins or protein complexes present in cell extracts. Chromatin immunoprecipitation (ChIP) permits the identification of protein-DNA interactions in pull-down assays using specific antibodies against DNA-binding proteins, such as transcription factors or histone/chromatin-binding proteins. Here, we present detailed protocols for extraction of Arabidopsis seedlings, co-IP of nuclear proteins, and ChIP.

  20. Chromatin immunoprecipitation assay of brain tissues using Percoll gradient-purified nuclei.

    PubMed

    Ding, Baojin; Kilpatrick, Daniel L

    2013-01-01

    Protein-DNA interactions are critical to maintain genome stability, DNA replication, chromosome -segregation and to regulate gene expression. Chromatin immunoprecipitation (ChIP) is a powerful technique to study these interactions within living neurons and nervous tissue. In particular, ChIP analysis of chromatin in which protein-DNA interactions are first fixed in situ provides a valuable approach to identify specific transcription factor-DNA interactions and their regulation in the developing nervous system. Here we describe a procedure utilizing Percoll gradient purification of nuclei from fresh brain tissue pre-fixed with formaldehyde for ChIP analysis. This purification protocol provides an enrichment of neuronal nuclei in high yield. We also illustrate the suitability of chromatin prepared from Percoll-purified brain nuclei for ChIP analysis of regulated transcription factor interactions with neuronal gene promoters.

  1. Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry.

    PubMed

    Maccarrone, Giuseppina; Bonfiglio, Juan Jose; Silberstein, Susana; Turck, Christoph W; Martins-de-Souza, Daniel

    2017-01-01

    Identifying the partners of a given protein (the interactome) may provide leads about the protein's function and the molecular mechanisms in which it is involved. One of the alternative strategies used to characterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.

  2. Biochemical Analysis of Genome Functions Using Locus-Specific Chromatin Immunoprecipitation Technologies

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2016-01-01

    To isolate specific genomic regions that retain their molecular interactions, allowing direct identification of chromatin-bound molecules, we developed two locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies, insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP) using the clustered regularly interspaced short palindromic repeats (CRISPR) system or transcription activator-like (TAL) proteins. Essentially, a locus-specific ChIP consists of locus-tagging and affinity purification and can be combined with downstream analyses to identify molecules associated with the target genomic regions. In this review, we discuss the applications of locus-specific ChIP to analyze the genome functions, including transcription and epigenetic regulation. PMID:26819551

  3. Chromatin immunoprecipitation with fixed animal tissues and preparation for high-throughput sequencing.

    PubMed

    Cotney, Justin L; Noonan, James P

    2015-02-02

    Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful method used to identify genome-wide binding patterns of transcription factors and distribution of various histone modifications associated with different chromatin states. In most published studies, ChIP-Seq has been performed on cultured cells grown under controlled conditions, allowing generation of large amounts of material in a homogeneous biological state. Although such studies have provided great insight into the dynamic landscapes of animal genomes, they do not allow the examination of transcription factor binding and chromatin states in adult tissues, developing embryonic structures, or tumors. Such knowledge is critical to understanding the information required to create and maintain a complex biological tissue and to identify noncoding regions of the genome directly involved in tissues affected by complex diseases such as autism. Studying these tissue types with ChIP-Seq can be challenging due to the limited availability of tissues and the lack of complex biological states able to be achieved in culture. These inherent differences require alterations of standard cross-linking and chromatin extraction typically used in cell culture. Here we describe a general approach for using small amounts of animal tissue to perform ChIP-Seq directed at histone modifications and transcription factors. Tissue is homogenized before treatment with formaldehyde to ensure proper cross-linking, and a two-step nuclear isolation is performed to increase extraction of soluble chromatin. Small amounts of soluble chromatin are then used for immunoprecipitation (IP) and prepared for multiplexed high-throughput sequencing. © 2015 Cold Spring Harbor Laboratory Press.

  4. Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation

    PubMed Central

    Koziol, Magdalena J.; Bradshaw, Charles R.; Allen, George E.; Costa, Ana S.H.; Frezza, Christian

    2017-01-01

    dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses. Background This protocol was developed in order to identify regions in the genome that contain dA6m. It can be used to detect dA6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA6m in eukaryotic DNA. PMID:28180135

  5. Proteomics Analysis of Cellular Proteins Co-Immunoprecipitated with Nucleoprotein of Influenza A Virus (H7N9)

    PubMed Central

    Sun, Ningning; Sun, Wanchun; Li, Shuiming; Yang, Jingbo; Yang, Longfei; Quan, Guihua; Gao, Xiang; Wang, Zijian; Cheng, Xin; Li, Zehui; Peng, Qisheng; Liu, Ning

    2015-01-01

    Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities. PMID:26528969

  6. Eubacterial components similar to small nuclear ribonucleoproteins: identification of immunoprecipitable proteins and capped RNAs in a cyanobacterium and a gram-positive eubacterium.

    PubMed Central

    Kovacs, S A; O'Neil, J; Watcharapijarn, J; Moe-Kirvan, C; Vijay, S; Silva, V

    1993-01-01

    Small nuclear ribonucleoprotein (snRNP) particles play an important role in the processing of pre-mRNA. snRNPs have been identified immunologically in a variety of cells, but none have ever been observed in prokaryotic systems. This report provides the first evidence for the presence of snRNP-like components in two types of prokaryotic cells: those of the cyanobacterium Synechococcus leopoliensis and those of the gram-positive eubacterium Bacillus subtilis. These components consist of snRNP-immunoreactive proteins and RNAs, including some with the snRNP-unique 5' m2,2,7G (m3G) cap. Immunoreactivity was determined by immunoprecipitation procedures, with either antinuclear-antibody-positive (RNP- and Sm-monospecific) patient sera or a m3G monoclonal antibody, with radiolabelled cell extracts that were preadsorbed with antinuclear-antibody-negative sera. S. leopoliensis immunoprecipitates showed the presence of high-molecular-mass proteins (14 to 70 kDa) and RNAs (138 to 243 nucleotides) that are analogous in size to proteins and RNAs found in human (HEp-2) cell immunoprecipitates but absent in Escherichia coli immunoprecipitates. Thin-layer chromatography of S. leopoliensis immunoprecipitates confirmed the presence of a capped nucleotide similar to a capped nucleotide in HEp-2 immunoprecipitates; no such nucleotide was observed in E. coli immunoprecipitates. Immunoreactive RNAs (117-170 nucleotides) were identified in a second eubacterium, B. subtilis, as well. This work suggests that snRNPs or their evolutionary predecessors predate the emergence of eukaryotic cells. Images PMID:8458830

  7. miR-346 and miR-138 competitively regulate hTERT in GRSF1- and AGO2-dependent manners, respectively

    PubMed Central

    Song, Ge; Wang, Renjie; Guo, Junfei; Liu, Xuyuan; Wang, Fang; Qi, Ying; Wan, Haiying; Liu, Min; Li, Xin; Tang, Hua

    2015-01-01

    miRNAs typically downregulate the expression of target genes by binding to their 3′UTR, and dysregulation of miRNAs may contribute to tumorigenesis. Here, we found that miR-346 and miR-138 competitively bind to a common region in the 3′UTR of hTERT mRNA and have opposite effects on the expression and function of hTERT in human cervical cancer cells. Furthermore, G-rich RNA sequence binding factor 1 (GRSF1) mediates the miR-346-dependent upregulation of hTERT by binding to the miR-346 middle sequence motif (CCGCAU) which forms a “bulge loop” when miR-346 is bound to the hTERT 3′UTR, facilitating the recruitment of hTERT mRNA to ribosomes to promote translation in an AGO2-independent manner. Conversely, miR-138 suppresses hTERT expression in an AGO2-dependent manner. Interestingly, replacement of the miR-138 middle sequence with that of miR-346 results in an upregulation of hTERT expression in a GRSF1-dependent manner. Moreover, miR-346 depends on GRSF1 to upregulate another target gene, activin A receptor, type IIB (ACVR2B), in which miR-346 “CCGCAU” motif is essential. These findings reveal novel mechanisms of miRNA-mediated upregulation of target gene expression and describe the coordinated action of multiple miRNAs to control the fate of a single target mRNA through binding to its 3′UTR. PMID:26507454

  8. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans

    PubMed Central

    Mukhopadhyay, Arnab; Deplancke, Bart; Walhout, Albertha J M; Tissenbaum, Heidi A

    2009-01-01

    In order to determine how signaling pathways differentially regulate gene expression, it is necessary to identify the interactions between transcription factors (TFs) and their cognate cis-regulatory DNA elements. Here, we have outlined a chromatin immunoprecipitation (ChIP) protocol for use in whole Caenorhabditis elegans extracts. We discuss optimization of the procedure, including growth and harvesting of the worms, formaldehyde fixation, TF immunoprecipitation and analysis of bound sequences through real-time PCR. It takes ∼10–12 d to obtain the worm culture for ChIP; the ChIP procedure is spaced out over a period of 2.5 d with two overnight incubations. PMID:18388953

  9. A comparative analysis of shotgun-cloning and tagged-random amplification-cloning of chromatin immunoprecipitation-isolated genome fragments.

    PubMed

    White, Robert B; Ziman, Melanie R

    2006-07-28

    The cloning of transcription factor antibody-immunoprecipitated genomic fragments from chromatin immunoprecipitation (ChIP) experiments is a technically challenging procedure, especially when the input genomic DNA is isolated from whole tissues (in vivo) rather than cultured cells. Here we adapt a technique known as Tagged-Random PCR (T-PCR) to amplify ChIP-immunoprecipitated DNA from mouse embryonic tissue prior to cloning. Importantly, we then compare this technique with tandem shotgun-cloning experiments in terms of its capacity to identify target genes. We find that T-PCR dramatically increases the efficiency of cloning ChIP fragments without distortion of the relative location of cloned fragments to putative target genes. Thus, T-PCR is a simple procedure which greatly enhances the efficiency of cloning tissue-derived ChIP fragments.

  10. IMP-3 protects the mRNAs of cyclins D1 and D3 from GW182/AGO2-dependent translational repression.

    PubMed

    Deforzh, Evgeny; Vargas, Thaiz Rivera; Kropp, Jeremie; Vandamme, Marie; Pinna, Guillaume; Polesskaya, Anna

    2016-12-01

    IGF-2 mRNA binding protein 3 (IGF2BP3, IMP-3) is a well-known post-transcriptional regulatory factor of gene expression, mainly involved in embryonic development and oncogenesis. We have previously demonstrated that a subset of IMP-3 targets, such as the mRNAs of cyclins D1, D3 and G1, are positively regulated by IMP-3, and that this regulation depends on nuclear localization of IMP-3. In the present study, we show that as a first step following a knock-down of IMP-3, the protein levels of the cyclins rapidly decrease, while their mRNAs remain stable and associated with the polyribosomes, though not translated. We have elucidated the molecular mechanisms of this regulation, demonstrating that IMP-3 and its protein partners ILF3/NF90 and PTBP1 bind to the 3'UTRs of the cyclin mRNAs and protect them from the translational repression induced by miRNA-dependent recruitment of AGO2/GW182 complex in human cancer cells.

  11. Multiplex Sequential Immunoprecipitation of Insulin Secretory Granule Proteins from Radiolabeled Pancreatic Islets.

    PubMed

    Guest, Paul C

    2017-01-01

    Pulse radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Specific proteins can then be immunoprecipitated and analyzed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine, followed by multiplex sequential immunoprecipitation of insulin and three other secretory granule accessory proteins. This provided a means of distinguishing those pancreatic islet proteins with different biosynthetic rates in response to the media glucose concentrations.

  12. Important biological information uncovered in previously unaligned reads from chromatin immunoprecipitation experiments (ChIP-Seq)

    PubMed Central

    Ouma, Wilberforce Zachary; Mejia-Guerra, Maria Katherine; Yilmaz, Alper; Pareja-Tobes, Pablo; Li, Wei; Doseff, Andrea I.; Grotewold, Erich

    2015-01-01

    Establishing the architecture of gene regulatory networks (GRNs) relies on chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) methods that provide genome-wide transcription factor binding sites (TFBSs). ChIP-Seq furnishes millions of short reads that, after alignment, describe the genome-wide binding sites of a particular TF. However, in all organisms investigated an average of 40% of reads fail to align to the corresponding genome, with some datasets having as much as 80% of reads failing to align. We describe here the provenance of previously unaligned reads in ChIP-Seq experiments from animals and plants. We show that a substantial portion corresponds to sequences of bacterial and metazoan origin, irrespective of the ChIP-Seq chromatin source. Unforeseen was the finding that 30%–40% of unaligned reads were actually alignable. To validate these observations, we investigated the characteristics of the previously unaligned reads corresponding to TAL1, a human TF involved in lineage specification of hemopoietic cells. We show that, while unmapped ChIP-Seq read datasets contain foreign DNA sequences, additional TFBSs can be identified from the previously unaligned ChIP-Seq reads. Our results indicate that the re-evaluation of previously unaligned reads from ChIP-Seq experiments will significantly contribute to TF target identification and determination of emerging properties of GRNs. PMID:25727450

  13. Important biological information uncovered in previously unaligned reads from chromatin immunoprecipitation experiments (ChIP-Seq).

    PubMed

    Ouma, Wilberforce Zachary; Mejia-Guerra, Maria Katherine; Yilmaz, Alper; Pareja-Tobes, Pablo; Li, Wei; Doseff, Andrea I; Grotewold, Erich

    2015-03-02

    Establishing the architecture of gene regulatory networks (GRNs) relies on chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) methods that provide genome-wide transcription factor binding sites (TFBSs). ChIP-Seq furnishes millions of short reads that, after alignment, describe the genome-wide binding sites of a particular TF. However, in all organisms investigated an average of 40% of reads fail to align to the corresponding genome, with some datasets having as much as 80% of reads failing to align. We describe here the provenance of previously unaligned reads in ChIP-Seq experiments from animals and plants. We show that a substantial portion corresponds to sequences of bacterial and metazoan origin, irrespective of the ChIP-Seq chromatin source. Unforeseen was the finding that 30%-40% of unaligned reads were actually alignable. To validate these observations, we investigated the characteristics of the previously unaligned reads corresponding to TAL1, a human TF involved in lineage specification of hemopoietic cells. We show that, while unmapped ChIP-Seq read datasets contain foreign DNA sequences, additional TFBSs can be identified from the previously unaligned ChIP-Seq reads. Our results indicate that the re-evaluation of previously unaligned reads from ChIP-Seq experiments will significantly contribute to TF target identification and determination of emerging properties of GRNs.

  14. Immunoprecipitation and Mass Spectrometry Defines an Extensive RBM45 Protein-Protein Interaction Network

    PubMed Central

    Li, Yang; Collins, Mahlon; An, Jiyan; Geiser, Rachel; Tegeler, Tony; Tsantilas, Kristine; Garcia, Krystine; Pirrotte, Patrick; Bowser, Robert

    2016-01-01

    The pathological accumulation of RNA-binding proteins (RBPs) within inclusion bodies is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBP aggregation results in both toxic gain and loss of normal function. Determining the protein binding partners and normal functions of disease-associated RBPs is necessary to fully understand molecular mechanisms of RBPs in disease. Herein, we characterized the protein-protein interactions (PPIs) of RBM45, a RBP that localizes to inclusions in ALS/FTLD. Using immunoprecipitation coupled to mass spectrometry (IP-MS), we identified 132 proteins that specifically interact with RBM45 within HEK293 cells. Select PPIs were validated by immunoblot and immunocytochemistry, demonstrating that RBM45 associates with a number of other RBPs primarily via RNA-dependent interactions in the nucleus. Analysis of the biological processes and pathways associated with RBM45-interacting proteins indicates enrichment for nuclear RNA processing/splicing via association with hnRNP proteins and cytoplasmic RNA translation via eiF2 and eiF4 pathways. Moreover, several other ALS-linked RBPs, including TDP-43, FUS, Matrin-3, and hnRNP-A1, interact with RBM45, consistent with prior observations of these proteins within intracellular inclusions in ALS/FTLD. Taken together, our results define a PPI network for RBM45, suggest novel functions for this protein, and provide new insights into the contributions of RBM45 to neurodegeneration in ALS/FTLD. PMID:26979993

  15. Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation.

    PubMed

    Kondo, Yutaka; Shen, Lanlan; Yan, Pearlly S; Huang, Tim Hui-Ming; Issa, Jean-Pierre J

    2004-05-11

    Switching from acetylation to methylation at histone H3 lysine 9 (K9) has recently been shown to contribute to euchromatin gene silencing. To identify genes silenced by K9 modifications, we probed a human CpG island microarray with DNA obtained by chromatin immunoprecipitation (ChIP) in a cancer cell line using an anti-H3-K9 methylated antibody or an anti-H3-K9 acetylated antibody. Of the 27 clones with the highest signal ratio of K9 methylation over acetylation (Me/Ac), 13 contained repetitive sequences. Among 14 nonrepetitive clones, we identified 11 genes (seven known and four previously undescribed), one EST, and two unknown fragments. Using ChIP-PCR, all 18 examined clones showed higher ratios of H3-K9 Me/Ac than the active gene control, P21, thus confirming the microarray data. In addition, we found a strong correlation between the K9 Me/Ac ratio and CpG island DNA methylation (R = 0.92, P < 0.01), and five of seven genes examined (megalin, thrombospondin-4, KR18, latrophilin-3, and phosphatidylinositol-3-OH kinase P101 subunit) showed lack of expression by RT-PCR and reactivation by DNA methylation and/or histone deacetylase inhibition, suggesting that these genes are true targets of silencing through histone modifications. All five genes also showed significant DNA methylation in a cell line panel and in primary colon cancers. Our data suggest that CpG island microarray coupled with ChIP can identify novel targets of gene silencing in cancer. This unbiased approach confirms the tight coupling between DNA methylation and histone modifications in cancer and could be used to probe gene silencing in nonneoplastic conditions as well.

  16. Isolation of a thyroid hormone-responsive gene by immunoprecipitation of thyroid hormone receptor-DNA complexes.

    PubMed Central

    Bigler, J; Eisenman, R N

    1994-01-01

    Thyroid hormone (T3) receptor (TR) is a ligand-dependent transcription factor that acts through specific binding sites in the promoter region of target genes. In order to identify new genes that are regulated by T3, we used anti-TR antiserum to immunoprecipitate TR-DNA complexes from GH4 cell nuclei that had previously been treated with a restriction enzyme. Screening of the immunopurified, cloned DNA for TR binding sites by electrophoretic mobility shift assay yielded 53 positive clones. A subset of these clones was specifically immunoprecipitated with anti-TR antiserum and may therefore represent biologically significant binding sites. One of these clones, clone 122, was characterized in detail. It includes sequences highly related to the NICER long terminal repeat-like element and contains three TR binding sites as determined by DNase I footprinting. Two of the clone 122 TR binding sites are located upstream of the TATA box, and one is located downstream. The TR binding site downstream from the promoter was necessary and sufficient to confer T3-dependent regulation in transient transfection experiments. Expression of a reporter construct under the control of the clone 122 promoter region was activated by TR in the absence of ligand and returned to basal levels after T3 addition. Clone 122 sequences hybridize to at least two different mRNAs of approximately 6 and 10 kb from GH4 cells. The levels of both of these mRNAs increased upon removal of T3. Our studies suggest that specific immunoprecipitation of chromatin allows identification of binding sites and target genes for transcription factors. Images PMID:7935476

  17. A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.

    PubMed

    Baszczynski, C L

    1990-06-01

    Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.

  18. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes.

    PubMed

    Mohammed, Hisham; Taylor, Christopher; Brown, Gordon D; Papachristou, Evaggelia K; Carroll, Jason S; D'Santos, Clive S

    2016-02-01

    Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.

  19. Precise Identification of DNA-Binding Proteins Genomic Location by Exonuclease Coupled Chromatin Immunoprecipitation (ChIP-exo).

    PubMed

    Matteau, Dominick; Rodrigue, Sébastien

    2015-01-01

    DNA-binding proteins play a crucial role in all living organisms by interacting with various DNA sequences across the genome. While several methods have been used to study the interaction between DNA and proteins in vitro, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the standard technique for identifying the genome-wide location of DNA-binding proteins in vivo. However, the resolution of standard ChIP-seq methodology is limited by the DNA fragmentation process and presence of contaminating DNA. A significant improvement of the ChIP-seq technique results from the addition of an exonuclease treatment during the immunoprecipitation step (ChIP-exo) that lowers background noise and more importantly increases the identification of binding sites to a level near to single-base resolution by effectively footprinting DNA-bound proteins. By doing so, ChIP-exo offers new opportunities for a better characterization of the complex and fascinating architecture that resides in DNA-proteins interactions and provides new insights for the comprehension of important molecular mechanisms.

  20. Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

    PubMed Central

    Robson, K J; Chandra, T; MacGillivray, R T; Woo, S L

    1982-01-01

    The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans. Images PMID:6750607

  1. Detection of genes expressed in Bordetella bronchiseptica colonizing rat trachea by in vivo expressed-tag immunoprecipitation method.

    PubMed

    Abe, Hiroyuki; Kamitani, Shigeki; Fukui-Miyazaki, Aya; Shinzawa, Naoaki; Nakamura, Keiji; Horiguchi, Yasuhiko

    2015-05-01

    Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection.

  2. Methylated DNA immunoprecipitation (MeDIP) from low amounts of cells.

    PubMed

    Borgel, Julie; Guibert, Sylvain; Weber, Michael

    2012-01-01

    Methylated DNA immunoprecipitation (MeDIP) is an immunocapturing approach for unbiased enrichment of DNA that is methylated on cytosines. The principle is that genomic DNA is randomly sheared by sonication and immunoprecipitated with an antibody that specifically recognizes 5-methylcytidine (5mC), which can be combined with PCR or high-throughput analysis (microarrays, deep sequencing). The MeDIP technique has been originally used to generate DNA methylation profiles on a genome scale in mammals and plants. Here we provide an optimized version of the MeDIP protocol suitable for low amounts of DNA, which can be used to study DNA methylation in cellular populations available in small quantities.

  3. DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants.

    PubMed

    Posé, David; Yant, Levi

    2016-01-01

    Chromatin immunoprecipitation (ChIP) allows the precise identification of genomic loci that physically interact with a protein of interest, whether that protein is a transcription factor, a core polymerase, a histone, or other chromatin-associated protein. In short, tissue is first cross-linked to freeze a population of DNA-protein interactions at a stage of interest. Chromatin is then extracted, fragmented, and incubated with a specific antibody against the protein of interest. Next, the resultant DNA-protein complexes are immunoprecipitated and captured using beads that bind to the antibody constant region. Samples are finally reverse cross-linked to separate the bound fragments and the DNA is purified. This DNA is analyzed by quantitative PCR for enrichment of genomic regions expected to be bound by the protein under study. The protocol detailed in this chapter has been successfully applied in the identification of target genes for seven transcriptional regulators of diverse classes involved in Arabidopsis thaliana floral transition.

  4. Probing activation/deactivation of the BRASSINOSTEROID INSENSITIVE1 receptor kinase by immunoprecipitation

    PubMed Central

    Martins, Sara; Vert, Grégory; Jaillais, Yvon

    2017-01-01

    Summary Brassinosteroids are plant sterol-derived hormones that control plant growth and development. The BR receptor complex is encoded by the BRASSINOSTEROID INSENSITIVE1 (BRI1) and members of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE family. BR receptor complex activation and deactivation uses different post-translational modifications and recruitment of partner proteins. In this chapter, we describe optimized immunoprecipitation protocols and variants for biochemical analyses of BRI1 post-translational modification and protein-protein interaction. PMID:28124254

  5. Yo antibodies in ovarian and breast cancer patients detected by a sensitive immunoprecipitation technique.

    PubMed

    Monstad, S E; Storstein, A; Dørum, A; Knudsen, A; Lønning, P E; Salvesen, H B; Aarseth, J H; Vedeler, C A

    2006-04-01

    Onconeural antibodies are found in patients with cancer and are associated with paraneoplastic neurological syndromes (PNS). The objective of the present study was to assess the frequency of Yo antibodies in ovarian and breast cancer using a sensitive immunoprecipitation technique, and to look for any association of Yo antibodies with neurological symptoms and prognostic factors. A multiwell adapted fluid-phase immunoassay using radiolabelled recombinant cerebellar degeneration related protein (cdr2), produced by coupled in vitro transcription/translation was used for the detection of Yo antibodies. This technique combines high specificity and sensitivity with high sample analysing capacity for the antibody in question. Sera or EDTA-blood from 810 ovarian (n = 557) and breast cancer (n = 253) patients were analysed for Yo antibodies by immunoprecipitation, as well as immunofluorescence and immune blots. Two hundred healthy blood donors and sera from 17 patients with paraneoplastic cerebellar degeneration and Yo antibodies served as controls. Immunoprecipitation was more sensitive in detecting Yo antibodies than immunofluorescence and immune blots. The prevalence of Yo antibodies was 13/557 (2.3%) in ovarian cancer and 4/253 (1.6%) in breast cancer using immunoprecipitation. Yo antibodies were not correlated with specific histological subgroups. The Yo index of ovarian cancer patients in FIGO stage IV was higher compared to FIGO stage I-III. The prevalence of Yo antibodies was 3 times higher in patients with stage III breast cancer than in stage I and II. Only 2/17 (11.8%) patients with Yo antibodies detected during the screen of 810 cancer patients had PNS. The results show that the prevalence of Yo antibodies is low in ovarian and breast cancer. Yo antibodies may be associated with advanced cancer, but less often with PNS.

  6. Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we describe engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using the CRISPR system, for purification of specific genomic regions retaining molecular interactions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP combined with mass spectrometry (enChIP-MS), next-generation sequencing (enChIP-Seq), and RNA-Seq (enChIP-RNA-Seq) can identify proteins, other genomic regions, and RNA, respectively, that interact with the target genomic region.

  7. A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

    PubMed Central

    Down, Thomas A.; Rakyan, Vardhman K.; Turner, Daniel J.; Flicek, Paul; Li, Heng; Kulesha, Eugene; Gräf, Stefan; Johnson, Nathan; Herrero, Javier; Tomazou, Eleni M.; Thorne, Natalie P.; Bäckdahl, Liselotte; Herberth, Marlis; Howe, Kevin L.; Jackson, David K.; Miretti, Marcos M.; Marioni, John C.; Birney, Ewan; Hubbard, Tim J. P.; Durbin, Richard; Tavaré, Simon; Beck, Stephan

    2009-01-01

    DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation. PMID:18612301

  8. A Microfluidic Device with Integrated Sonication and Immunoprecipitation for Sensitive Epigenetic Assays

    PubMed Central

    2016-01-01

    Epigenetic studies increasingly require analysis of a small number of cells that are of one specific type and derived from patients or animals. In this report, we demonstrate a simple microfluidic device that integrates sonication and immunoprecipitation (IP) for epigenetic assays, such as chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP). By incorporating an ultrasonic transducer with a microfluidic chamber, we implemented microscale sonication for both shearing chromatin/DNA and mixing/washing of IP beads. Such integration allowed highly sensitive tests starting with 100 cross-linked cells for ChIP or 500 pg of genomic DNA for MeDIP (compared to 106–107 cells for ChIP and 1–10 μg of DNA for MeDIP in conventional assays). The entire on-chip process of sonication and IP took only 1 h. Our tool will be useful for highly sensitive epigenetic studies based on a small quantity of sample. PMID:26745449

  9. Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents.

    PubMed

    Howard, R J; Barnwell, J W

    1984-02-01

    Plasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor 125I-labelled antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200 000 and 180 000 were detected only after extraction with deoxycholate or SDS.

  10. Neuropilin-1 is a receptor for extracellular miRNA and AGO2/miRNA complexes and mediates the internalization of miRNAs that modulate cell function.

    PubMed

    Prud'homme, Gerald J; Glinka, Yelena; Lichner, Zsuzsanna; Yousef, George M

    2016-10-18

    Extracellular miRNAs are increasingly studied as markers for specific diseases. They are released in biological fluids in a remarkably stable form, and may play a role in intercellular communication. They are thought to be protected against degradation by either encapsulation within microparticles, or by binding to proteins (mostly AGO2). The particulate forms may be internalized by endocytosis or membrane fusion, but the protein-bound forms require a receptor mechanism for their uptake. A major question is whether there are natural cell-membrane receptors that capture and internalize protein-bound functional miRNAs. We examined neuropilin-1 (NRP1), in view of its properties as a receptor for many ligands, including growth factors such as vascular endothelial growth factor (VEGF), and efficiency at mediating ligand internalization. It is expressed by endothelial cells, many other normal cell types, and cancer cells. Here, we report that NRP1 binds miRNAs with high affinity, and promotes their entry into the cell. Furthermore, the internalized miRNAs remain functional, as they specifically regulate proliferation and migration of cancer cells, as well as tube formation by human endothelial cells. Anti-NRP1 antibodies or NRP1 siRNA knockdown block miRNA effects, further confirming NRP1-mediated uptake. VEGF does not compete with miRNAs for binding to NRP1. In addition, NRP1 binds extracellular AGO2 (carrying miRNA or not), and internalizes AGO2/miRNA complexes. Because miRNA bound to AGO2 appears to the most abundant form in body fluids, this may have important physiological and pathological effects.

  11. Neuropilin-1 is a receptor for extracellular miRNA and AGO2/miRNA complexes and mediates the internalization of miRNAs that modulate cell function

    PubMed Central

    Prud'homme, Gerald J.; Glinka, Yelena; Lichner, Zsuzsanna; Yousef, George M.

    2016-01-01

    Extracellular miRNAs are increasingly studied as markers for specific diseases. They are released in biological fluids in a remarkably stable form, and may play a role in intercellular communication. They are thought to be protected against degradation by either encapsulation within microparticles, or by binding to proteins (mostly AGO2). The particulate forms may be internalized by endocytosis or membrane fusion, but the protein-bound forms require a receptor mechanism for their uptake. A major question is whether there are natural cell-membrane receptors that capture and internalize protein-bound functional miRNAs. We examined neuropilin-1 (NRP1), in view of its properties as a receptor for many ligands, including growth factors such as vascular endothelial growth factor (VEGF), and efficiency at mediating ligand internalization. It is expressed by endothelial cells, many other normal cell types, and cancer cells. Here, we report that NRP1 binds miRNAs with high affinity, and promotes their entry into the cell. Furthermore, the internalized miRNAs remain functional, as they specifically regulate proliferation and migration of cancer cells, as well as tube formation by human endothelial cells. Anti-NRP1 antibodies or NRP1 siRNA knockdown block miRNA effects, further confirming NRP1-mediated uptake. VEGF does not compete with miRNAs for binding to NRP1. In addition, NRP1 binds extracellular AGO2 (carrying miRNA or not), and internalizes AGO2/miRNA complexes. Because miRNA bound to AGO2 appears to the most abundant form in body fluids, this may have important physiological and pathological effects. PMID:27486976

  12. Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

    NASA Technical Reports Server (NTRS)

    Biermann, B. J.; Pao, L. I.; Feldman, L. J.

    1994-01-01

    Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

  13. Pr-specific phytochrome phosphorylation in vitro by a protein kinase present in anti-phytochrome maize immunoprecipitates

    NASA Technical Reports Server (NTRS)

    Biermann, B. J.; Pao, L. I.; Feldman, L. J.

    1994-01-01

    Protein kinase activity has repeatedly been found to co-purify with the plant photoreceptor phytochrome, suggesting that light signals received by phytochrome may be transduced or modulated through protein phosphorylation. In this study immunoprecipitation techniques were used to characterize protein kinase activity associated with phytochrome from maize (Zea mays L.). A protein kinase that specifically phosphorylated phytochrome was present in washed anti-phytochrome immunoprecipitates of etiolated coleoptile proteins. No other substrate tested was phosphorylated by this kinase. Adding salts or detergents to disrupt low-affinity protein interactions reduced background phosphorylation in immunoprecipitates without affecting phytochrome phosphorylation, indicating that the protein kinase catalytic activity is either intrinsic to the phytochrome molecule or associated with it by high-affinity interactions. Red irradiation (of coleoptiles or extracts) sufficient to approach photoconversion saturation reduced phosphorylation of immunoprecipitated phytochrome. Subsequent far-red irradiation reversed the red-light effect. Phytochrome phosphorylation was stimulated about 10-fold by a co-immunoprecipitated factor. The stimulatory factor was highest in immunoprecipitates when Mg2+ was present in immunoprecipitation reactions but remained in the supernatant in the absence of Mg2+. These observations provide strong support for the hypothesis that phytochrome-associated protein kinase modulates light responses in vivo. Since only phytochrome was found to be phosphorylated, the co-immunoprecipitated protein kinase may function to regulate receptor activity.

  14. Methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) using low amounts of genomic DNA.

    PubMed

    Zhao, Ming-Tao; Whyte, Jeffrey J; Hopkins, Garrett M; Kirk, Mark D; Prather, Randall S

    2014-06-01

    DNA modifications, such as methylation and hydroxymethylation, are pivotal players in modulating gene expression, genomic imprinting, X-chromosome inactivation, and silencing repetitive sequences during embryonic development. Aberrant DNA modifications lead to embryonic and postnatal abnormalities and serious human diseases, such as cancer. Comprehensive genome-wide DNA methylation and hydroxymethylation studies provide a way to thoroughly understand normal development and to identify potential epigenetic mutations in human diseases. Here we established a working protocol for methylated DNA immunoprecipitation combined with next-generation sequencing [methylated DNA immunoprecipitation (MeDIP)-seq] for low starting amounts of genomic DNA. By using spike-in control DNA sets with standard cytosine, 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC), we demonstrate the preferential binding of antibodies to 5mC and 5hmC, respectively. MeDIP-PCRs successfully targeted highly methylated genomic loci with starting genomic DNA as low as 1 ng. The enrichment efficiency declined for constant spiked-in controls but increased for endogenous methylated regions. A MeDIP-seq library was constructed starting with 1 ng of DNA, with the majority of fragments between 250 bp and 600 bp. The MeDIP-seq reads showed higher quality than the Input control. However, after being preprocessed by Cutadapt, MeDIP (97.53%) and Input (94.98%) reads showed comparable alignment rates. SeqMonk visualization tools indicated MeDIP-seq reads were less uniformly distributed across the genome than Input reads. Several commonly known unmethylated and methylated genomic loci showed consistent methylation patterns in the MeDIP-seq data. Thus, we provide proof-of-principle that MeDIP-seq technology is feasible to profile genome-wide DNA methylation in minute DNA samples, such as oocytes, early embryos, and human biopsies.

  15. Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence.

    PubMed

    Neubauer, Heidi A; Pitson, Stuart M

    2016-01-01

    Sphingosine kinase 2 (SK2) is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs). Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout ( Sphk2(-/-)) MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

  16. Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

    PubMed Central

    Ebersole, Brittany; Petko, Jessica; Levenson, Robert

    2014-01-01

    We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay utilizes 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a GPCR responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins including most GPCRs. PMID:24463015

  17. Sequential Co-immunoprecipitation and Immunoblot Approach to Determine Oligomerisation of G-Protein-Coupled Receptors.

    PubMed

    Guest, Paul C

    2017-01-01

    G-protein-coupled receptors (GPCRs) play a major role in psychiatric disorders and are the targets of several current therapeutic approaches in this field. A number of studies have now shown that GPCRs can assemble as high molecular weight homo- and hetero-oligomers, which could affect ligand binding, intracellular signalling or trafficking. This information could be critical in design of new drugs to treat neurological and psychiatric disorders. This chapter describes a sequential co-immunoprecipitation and immunoblot protocol for determining oligomerisation of the 5-hydroxytryptamine (HT)1A receptor with other GPCRs in co-transfected HEK-293 cells.

  18. Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

    SciTech Connect

    Semelr, B.L.; Anderson, C.W.; Hanecak, R.; Dorner, L.F.; Wimmer, E.

    1982-02-01

    A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNA sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.

  19. Fragment complementation and co-immunoprecipitation assays for understanding R protein structure and function.

    PubMed

    Moffett, Peter

    2011-01-01

    Plant disease resistance (R) proteins confer protection against specific pathogens or pathogen isolates. R proteins function by recognizing pathogen-encoded avirulence (Avr) proteins and translating this recognition event into an initiation of downstream signaling pathways. Key to understanding this process is the study of the protein-protein interactions involving R proteins. Recognition and signaling mechanisms are mediated by both intramolecular interactions that take place between different domains of R proteins as well as intermolecular interactions between R proteins and additional plant proteins. These processes have been studied in part by using Agrobacterium-mediated transient expression of R protein fragments in Nicotiana benthamiana which allows for the rapid assessment of functionality. Furthermore, pairs of proteins or protein fragments can be transiently expressed as fusions with different epitope tags. One putative protein partner is subjected to immunoprecipitation. Subsequent immunoblotting is performed to determine whether the second protein has remained associated (or co-immunoprecipitated) with the first, indicating a protein-protein interaction. This technique has contributed substantially to structure-function analyses of R proteins and to the characterization of interactions between R proteins and other plant proteins.

  20. Immunoprecipitation of Serum Albumin with Protein A-Sepharose: A Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bohinski, Robert C.

    2000-11-01

    An exercise has been designed and optimized to acquaint students with the simple yet powerful technique of immunoprecipitation. Protein A-Sepharose (PA-S) is used as a solid-phase precipitant to recover bovine serum albumin (BSA, the antigen) recognized by anti-BSA antibody (Ab). The high degree of binding specificity between antigen and antibody is illustrated by recovery of BSA from a complex mixture of proteins obtained from wheat germ and chicken breast. Various controls are included for a thorough data analysis. The solid phase of Ag/Ab/PA-S is recovered by centrifugation, thoroughly washed, and treated to dissociate the BSA antigen. Samples are examined by discontinuous denaturing gel electrophoresis (SDS-PAGE) with Coomassie blue staining. The supernatants, containing proteins that are not precipitated, are also analyzed. Antigenic cross-reactivity, ranging from strong to none, is demonstrated in a second part by using serum albumins from seven different sources. Systems can be set up, shaken, and prepared for electrophoresis in a single lab period with time for laboratory lecture and discussion about antibody structure and function, antibody-based methods in general, and immunoprecipitation in particular.

  1. Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications

    EPA Science Inventory

    Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and the response to environmental stimuli. More than 130 unique histone modifications have been described to date and chromatin immunoprecipitation (ChIP) allows for the explorat...

  2. Improving chromatin immunoprecipitation (ChIP) by suppression of method-induced DNA-damage signaling.

    PubMed

    Beneke, Sascha

    2015-01-01

    Genomic DNA is always associated with proteins that modulate the accessibility of the genetic information. This chromatin is the essential structure in which all nuclear activity from regulation to replication, transcription, and repair takes place. This dynamic structure can be most efficiently analyzed by using the method of chromatin immunoprecipitation (ChIP), where application of cell-permeable cross-linkers to living cells induces covalent bridging between proteins and adjacent DNA in the nucleus. After fragmentation of the DNA, the complexed proteins are isolated by binding to specific antibodies. The attached DNA is isolated and can be analyzed. This method has been improved multiple times and adjusted to different experimental needs. This chapter describes a further advance based on the observation that the current standard method itself induces alterations in the chromatin.

  3. Mapping genomic targets of DNA helicases by chromatin immunoprecipitation in Saccharomyces cerevisiae.

    PubMed

    Cobb, Jennifer; van Attikum, Haico

    2010-01-01

    DNA helicases utilize the energy of nucleotide hydrolysis to unwind the two annealed strands of the DNA helix and are involved in many aspects of DNA metabolism such as replication, recombination, and repair. Chromatin immunoprecipitation (ChIP) has been instrumental in determining the genomic targets of many DNA helicases and DNA helicase-containing complexes including the minichromosome maintenance (Mcm) proteins 2-7, the RecQ helicase Sgs1 as well as the Rvb1 and Rvb2 helicase-containing INO80 and SWR1 chromatin remodeling complexes. Here we describe a ChIP method that has been successfully used to map these proteins at chromosomal double-strand breaks and replication forks in the model organism Saccharomyces cerevisiae.

  4. Generation of high quality chromatin immunoprecipitation DNA template for high-throughput sequencing (ChIP-seq).

    PubMed

    Deliard, Sandra; Zhao, Jianhua; Xia, Qianghua; Grant, Struan F A

    2013-04-19

    ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions. For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers. Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

  5. Trout red blood cell arrestin (TRCarr), a novel member of the arrestin family: cloning, immunoprecipitation and expression of recombinant TRCarr.

    PubMed

    Jahns, R; Borgese, F; Lindenthal, S; Straub, A; Motais, R; Fiévet, B

    1996-06-01

    Arrestins are cytosolic proteins involved in the desensitization of G-protein-coupled receptors. We report the cloning of trout red blood cell arrestin which shows 76, 82 and 52% identity with bovine beta-arrestin1, beta-arrestin2 and retinal arrestin respectively. Antibodies were generated against the C-terminus of trout red blood cell arrestin. These antibodies detected arrestin in erythrocyte cytosol and were able to precipitate the native protein. The Na+/H+ antiporter of trout red blood cell is activated by beta-adrenergic stimulation and is then desensitized whereas the transmembrane signalling pathway is not. To investigate the subcellular distribution of arrestin on beta-adrenergic activation and desensitization of the antiporter, precipitation experiments were carried out on trout erythrocytes. A desensitization-dependent shift in cytosolic arrestin to the membranes could not be detected using the immunoprecipitation technique but we cannot exclude the possibility that a small number of cytosolic arrestins might be involved in the regulation of membrane proteins in trout erythrocyte. Recombinant trout arrestin was produced in a protease-deficient Escherichia coli strain and its functionality was tested in a reconstituted rhodopsin assay. The recombinant protein provides a suitable tool for investigating the target for arrestin in trout red blood cell, which still remains to be identified.

  6. Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

    PubMed Central

    Miranda, E I; Garrido-Guerrero, E; Garcia-Carranca, A; Gariglio, P

    1992-01-01

    Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin. Images PMID:1311833

  7. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*

    PubMed Central

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca

    2016-01-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  8. Prion disease blood test using immunoprecipitation and improved quaking-induced conversion.

    PubMed

    Orrú, Christina D; Wilham, Jason M; Raymond, Lynne D; Kuhn, Franziska; Schroeder, Björn; Raeber, Alex J; Caughey, Byron

    2011-01-01

    A key challenge in managing transmissible spongiform encephalopathies (TSEs) or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at or below infectious levels. Of particular interest are tests capable of detecting prions in blood components such as plasma, but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests. One of the latter tests is quaking-induced conversion (QuIC), which can be as sensitive as in vivo bioassays, but much more rapid, higher throughput, and less expensive. Now we have integrated antibody 15B3-based immunoprecipitation with QuIC reactions to increase sensitivity and isolate prions from inhibitors such as those in plasma samples. Coupling of immunoprecipitation and an improved real-time QuIC reaction dramatically enhanced detection of variant Creutzfeldt-Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10(14)-fold, containing ~2 attogram (ag) per ml of proteinase K-resistant prion protein, were readily detected, indicating ~10,000-fold greater sensitivity for vCJD brain than has previously been reported. We also discriminated between plasma and serum samples from scrapie-infected and uninfected hamsters, even in early preclinical stages. This combined assay, which we call "enhanced QuIC" (eQuIC), markedly improves prospects for routine detection of low levels of prions in tissues, fluids, or environmental samples. Transmissible spongiform encephalopathies (TSEs) are largely untreatable and are difficult to diagnose definitively prior to irreversible clinical decline or death. The transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity. A few sufficiently sensitive in vitro methods have been reported, but most have major limitations that would preclude their use in routine diagnostic or screening applications

  9. Allele-specific chromatin immunoprecipitation studies show genetic influence on chromatin state in human genome.

    PubMed

    Kadota, Mitsutaka; Yang, Howard H; Hu, Nan; Wang, Chaoyu; Hu, Ying; Taylor, Philip R; Buetow, Kenneth H; Lee, Maxwell P

    2007-05-18

    Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d'Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d'Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena.

  10. Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

    PubMed

    Rehimi, Rizwan; Bartusel, Michaela; Solinas, Francesca; Altmüller, Janine; Rada-Iglesias, Alvaro

    2017-08-29

    Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10(4) - 5 x 10(5) cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

  11. Protocol: Chromatin immunoprecipitation (ChIP) methodology to investigate histone modifications in two model diatom species

    PubMed Central

    2012-01-01

    In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies. PMID:23217141

  12. Chromatin immunoprecipitation and gene expression analysis of neuronal subtypes after fluorescence activated cell sorting

    PubMed Central

    Finegersh, Andrey; Homanics, Gregg E.

    2016-01-01

    Background With advances in cell capture, gene expression can now be studied in neuronal subtypes and single cells; however, studying epigenetic mechanisms that underlie these changes presents challenges. Moreover, chromatin immunoprecipitation (ChIP) protocols optimized for low cell number do not adequately address technical issues and cell loss while preparing tissue for fluorescence activated cell sorting (FACS). Developing a reliable FACS-ChIP protocol without the need for pooling tissue from multiple animals would enable study of epigenetic mechanisms in neuronal subtypes. Methods FACS was used to isolate dopamine 1 receptor (D1R) expressing cells from the nucleus accumbens (NAc) of a commercially available BAC transgenic mouse strain. D1R+ cells were used to study gene expression as well as histone modifications at gene promoters using a novel native ChIP protocol. Results Isolated cells had enrichment of the dopamine 1 receptor (D1R) mRNA and nearly undetectable levels of GFAP and D2R mRNA. ChIP analysis demonstrated the association of activating or repressive histone modifications with highly expressed or silent gene promoters, respectively. Comparison with existing methods: The ChIP protocol developed in this paper enables characterization of histone modifications from ~30,000 FAC-sorted neurons. Conclusions We describe a one day FACS-ChIP protocol that can be applied to epigenetic studies of neuronal subtypes without pooling tissue. PMID:26868730

  13. Searching for Biomarkers: Humoral Response Profiling with Luciferase Immunoprecipitation Systems (LIPS)

    PubMed Central

    Burbelo, Peter D.; Ching, Kathryn H.; Bren, Kathleen E.; Iadarola, Michael J.

    2013-01-01

    B-cell mediated humoral responses are triggered in many human diseases including autoimmune, cancer, neurologic, and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire, for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed Luciferase immunoprecipitation systems (LIPS) that harnesses light emitting proteins to generate high definition antibody profiles optimal for both diagnostics and biomarker discovery. Here we describe the results and implications from a range of LIPS antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis, and provide a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring. PMID:21679112

  14. Chromatin immunoprecipitation to investigate origin association of replication factors in mammalian cells.

    PubMed

    Leman, Adam R; Noguchi, Eishi

    2014-01-01

    A variety of DNA-binding proteins regulate DNA transactions including DNA replication and DNA damage response. To initiate DNA replication in S phase of the cell cycle, numerous replication proteins must be recruited to the replication origin in order to unwind and synthesize DNA. Some replication factors stay at the origin, while replisome components move with the replication fork. When the replisome encounters DNA damage or other issues during DNA replication, the replication fork stalls and accumulates single-stranded DNA that triggers the ATR-dependent replication checkpoint, in order to slow down S phase and arrest the cell cycle at the G2-M transition. It is also possible that replication forks collapse, leading to double-strand breaks that recruit various DNA damage response proteins to activate cell cycle checkpoints and DNA repair pathways. Therefore, defining the localization of DNA transaction factors during the cell cycle should provide important insights into mechanistic understanding of DNA replication and its related processes. In this chapter, we describe a chromatin immunoprecipitation method to locate replisome components at replication origins in human cells.

  15. A Fast Carrier Chromatin Immunoprecipitation Method Applicable to Microdissected Tissue Samples

    PubMed Central

    Hao, Haiping; Liu, Hester; Gonye, Gregory; Schwaber, James S.

    2008-01-01

    Transcriptional regulation studies of CNS neurons are complicated by both cellular diversity and plasticity. Microdissection of specific functionally related populations of neurons can greatly reduce these issues, but typically excludes the use of many technologies due to tissue requirements, such as Chromatin Immunoprecipitation (ChIP), a powerful tool for studying in vivo protein-DNA interactions. We have developed a fast carrier ChIP (Fast CChIP) method for analyzing specific in vivo transcription factor-DNA interactions in as little as 0.2 mm3 brain tissue. Using an antibody against phosphorylated cyclic-AMP response element binding (CREB) protein, we confirmed phospho-CREB (pCREB) binding at the c-fos gene promoter. Then we further demonstrated the applicability of Fast CChIP in determining hypertension-induced pCREB binding at the c-fos gene promoter in the rat nucleus tractus solitarius (NTS), confirming CREB’s role in mediating hypertension-induced c-fos expression. This method will be broadly applicable to individual brain nucleus and biopsy/surgical samples. PMID:18502516

  16. Keratin proteins in human lung carcinomas. Combined use of morphology, keratin immunocytochemistry, and keratin immunoprecipitation.

    PubMed Central

    Banks-Schlegel, S. P.; McDowell, E. M.; Wilson, T. S.; Trump, B. F.; Harris, C. C.

    1984-01-01

    Light-microscopic immunocytochemistry and electron microscopy demonstrated that adenocarcinomas (AC) and squamous cell (epidermoid) carcinomas (SCCs) of human lung contained keratin proteins in the form of tonofilament bundles. However, moderately differentiated (md) SCCs contained abundant keratin, whereas poorly differentiated (pd) SCCs and all ACs contained lesser amounts. Lung tumors with the diagnosis of AC or SCC, as defined by WHO criteria, were also analyzed by immunoprecipitation techniques for the presence of keratin proteins. Regardless of the degree of tumor differentiation, SCCs contained a 44 kd keratin which was lacking in ACs. Interestingly, normal bronchial epithelium also contained the same 44 kd keratin. In addition, as SCCs became more differentiated, they exhibited even greater differences in the profile of synthesized keratins. Specifically, the relative abundance of the intermediate-sized keratins (57 and 59 kd) was increased in the md SCCs. Although keratin protein patterns appear to be a valuable adjunct in distinguishing AC from SCC, their usefulness as a diagnostic tool will require survey of a larger number of poorly differentiated tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:6198920

  17. Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

    PubMed

    Soyer, Jessica L; Möller, Mareike; Schotanus, Klaas; Connolly, Lanelle R; Galazka, Jonathan M; Freitag, Michael; Stukenbrock, Eva H

    2015-06-01

    The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. A systematic immunoprecipitation approach reinforces the concept of common conformational alterations in amyotrophic lateral sclerosis-linked SOD1 mutants.

    PubMed

    Fujisawa, Takao; Yamaguchi, Namiko; Kadowaki, Hisae; Tsukamoto, Yuka; Tsuburaya, Naomi; Tsubota, Atsushi; Takahashi, Hiromitsu; Naguro, Isao; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji; Nishitoh, Hideki; Homma, Kengo; Ichijo, Hidenori

    2015-10-01

    Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1(mut)) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1(mut), the DBR-neighboring region [SOD1(30-40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30-40). We found that MS27 could distinguish SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785-MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS. Copyright © 2015. Published by Elsevier Inc.

  19. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2013-09-13

    Isolation of specific genomic regions retaining molecular interactions is necessary for their biochemical analysis. Here, we established a novel method, engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP), for purification of specific genomic regions retaining molecular interactions. We showed that enChIP using the CRISPR system efficiently isolates specific genomic regions. In this form of enChIP, specific genomic regions are immunoprecipitated with antibody against a tag(s), which is fused to a catalytically inactive form of Cas9 (dCas9), which is co-expressed with a guide RNA (gRNA) and recognizes endogenous DNA sequence in the genomic regions of interest. enChIP-mass spectrometry (enChIP-MS) targeting endogenous loci identified associated proteins. enChIP using the CRISPR system would be a convenient and useful tool for dissecting chromatin structure of genomic regions of interest. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Accounting for immunoprecipitation efficiencies in the statistical analysis of ChIP-seq data

    PubMed Central

    2013-01-01

    Background ImmunoPrecipitation (IP) efficiencies may vary largely between different antibodies and between repeated experiments with the same antibody. These differences have a large impact on the quality of ChIP-seq data: a more efficient experiment will necessarily lead to a higher signal to background ratio, and therefore to an apparent larger number of enriched regions, compared to a less efficient experiment. In this paper, we show how IP efficiencies can be explicitly accounted for in the joint statistical modelling of ChIP-seq data. Results We fit a latent mixture model to eight experiments on two proteins, from two laboratories where different antibodies are used for the two proteins. We use the model parameters to estimate the efficiencies of individual experiments, and find that these are clearly different for the different laboratories, and amongst technical replicates from the same lab. When we account for ChIP efficiency, we find more regions bound in the more efficient experiments than in the less efficient ones, at the same false discovery rate. A priori knowledge of the same number of binding sites across experiments can also be included in the model for a more robust detection of differentially bound regions among two different proteins. Conclusions We propose a statistical model for the detection of enriched and differentially bound regions from multiple ChIP-seq data sets. The framework that we present accounts explicitly for IP efficiencies in ChIP-seq data, and allows to model jointly, rather than individually, replicates and experiments from different proteins, leading to more robust biological conclusions. PMID:23721376

  1. RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases.

    PubMed

    Halász, László; Karányi, Zsolt; Boros-Oláh, Beáta; Rózsa, Tímea; Sipos, Éva; Nagy, Éva; Mosolygó-L, Ágnes; Mázló, Anett; Rajnavölgyi, Éva; Halmos, Gábor; Székvölgyi, Lóránt

    2017-03-24

    The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection, and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised the mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection.

  2. Detection of residual toxin in tissues of ricin-poisoned mice by sandwich enzyme-linked immunosorbent assay and immunoprecipitation.

    PubMed

    Men, Jinshuan; Lang, Liwei; Wang, Chenyu; Wu, Junhua; Zhao, Yu; Jia, Pei-Yuan; Wei, Wenqing; Wang, Yuxia

    2010-06-15

    This work aimed to evaluate a method to detect the residual ricin in animal tissues. Immunoprecipitation and sandwich enzyme-linked immunosorbent assay (ELISA) were used to detect ricin in the tissues of intoxicated mice. The monoclonal antibodies (Mabs) 4C13 and 3D74 were used to assay the whole ricin molecules via sandwich ELISA. Mab 4C13 was conjugated with Sepharose 4B to capture ricin or ricin A chain by immunoprecipitation. Mice injected intravenously with ricin at the dosage of 5 microg/mouse were killed at different time points after intoxication. The serum, liver, kidney, lung, and intestine were harvested. High levels of ricin were found in serum and liver samples at each poisoning time point by sandwich ELISA, suggesting the possibility of determining ricin intoxication by detecting residual ricin in the serum. However, this method turned out to be ineffective for examining ricin in the kidney, lung, and intestine of poisoned mice. Although the same tissue samples of intoxicated mice were analyzed by immunoprecipitation, positive bands were found. This indicated that some components in the kidney, lung, and intestine could bind with ricin and interfere in its binding activity with the coated antibody. Immunoprecipitation could be used to measure the existence of ricin in these samples. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  3. Racial Differences in Expression Levels of miRNA Machinery-Related Genes, Dicer, Drosha, DGCR8, and AGO2, in Asian Korean Papillary Thyroid Carcinoma and Comparative Validation Using the Cancer Genome Atlas

    PubMed Central

    Kim, Jaegil; Park, Woo-Jae; Jeong, Kwang-Joon; Kang, Sun Hee; Kwon, Sun Young

    2017-01-01

    Aberrant regulation of microRNA (miRNA) machinery components is associated with various human cancers, including papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer, and a higher prevalent female malignancy. The purpose of this study is to investigate racial differences in mRNA expression levels of four miRNA machinery components, Dicer, Drosha, DGCR8, and AGO2, and their correlations with clinicopathological characteristics. Forty PTC samples from female Asian Korean PTC patients were enrolled. Using qPCR, we examined mRNA expression levels of the components and next validated our results by comparison with results of female white American in the TCGA PTC project. Interestingly, mRNA expression levels of the selected factors were altered in the TCGA PTC samples. However, only Drosha showed a significantly lower expression level in Asian Korean PTC samples. Furthermore, the mRNA expression levels of the four components showed no association with clinicopathological characteristics in both groups. On the other hand, positive correlations were observed between altered mRNA expression levels of Dicer and Drosha and DGCR8 and Drosha in TCGA PTC samples. These findings collectively revealed that altered mRNA expression levels of miRNA machinery components might be responsible for racial differences in the carcinogenesis of PTC. PMID:28352639

  4. Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

    PubMed Central

    Brigidi, G. Stefano; Bamji, Shernaz X

    2013-01-01

    Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons. Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment. Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly

  5. Immunoprecipitation of native botulinum neurotoxin complexes from Clostridium botulinum subtype A strains.

    PubMed

    Lin, Guangyun; Tepp, William H; Bradshaw, Marite; Fredrick, Chase M; Johnson, Eric A

    2015-01-01

    Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected.

  6. D-Isonucleotide (isoNA) incorporation around cleavage site of passenger strand promotes the vibration of Ago2-PAZ domain and enhances in vitro potency of siRNA.

    PubMed

    Huang, Ye; Tian, Miao; Zhang, Yichao; Sheng, Gang; Chen, Zhuo; Ma, Yuan; Chen, Yue; Peng, Yihong; Zhao, Yi-Lei; Wang, Yanli; Zhang, Lihe; Yang, Zhenjun

    2015-11-28

    It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.

  7. Determination of glutamic acid decarboxylase antibodies (GADA) IgG subclasses – comparison of three immunoprecipitation assays (IPAs)

    PubMed Central

    Hillman, M; Törn, C; Landin-Olsson, M

    2007-01-01

    IgG subclasses of glutamic acid decarboxylase (GAD65) antibodies (GADA) may reflect the immunological state in the pancreas of GADA-positive patients with autoimmune diabetes. The use of biotin-conjugated antibodies and streptavidin Sepharose are used commonly in immunoprecipitation assays (IPA) based on 125I- or 35S-labelled antigens to capture IgG subclasses directed against IA-2 or GAD65. We have compared three different immunoprecipitation assays for the determination of GADA IgG subclasses. Two of the assays were based on the biotin and streptavidin systems provided in a solid (immobilized) or liquid (mobilized) phase binding environment. The third assay was based on N-hydroxysuccinimide (immobilized) interaction with primary amines (i.e. lysine residues) on the antibody. We found the liquid phase binding assay (LPBA) to be the most stable assay, with a comparatively low coefficient of variation and background. PMID:17666094

  8. Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield.

    PubMed

    Payvar, F; Schimke, R T

    1979-11-01

    We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non-specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (E) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.

  9. Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

    PubMed

    Burlibașa, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

  10. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  11. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  12. RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription

    PubMed Central

    Sandoval, Juan; Rodríguez, José L.; Tur, Gema; Serviddio, Gaetano; Pereda, Javier; Boukaba, Abdelhalim; Sastre, Juan; Torres, Luis; Franco, Luis; López-Rodas, Gerardo

    2004-01-01

    We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to those of other procedures. However, in genes whose mRNA is more stable (e.g. the hemopexin, hpx, gene) RNAPol-ChIP informs on real-time transcription with results comparable to those of methods such as nuclear run-on or run-off, which require the isolation of highly purified nuclei. Moreover, RNAPol-ChIP advantageously compares with methods based on the analysis of steady-state mRNA (northern blot or RT–PCR). Additional advantages of RNAPol-ChIP, such as the possibility of combining it with classical ChIP analysis to study transcription-associated changes in chromatin are discussed. PMID:15247321

  13. Urb-RIP – An Adaptable and Efficient Approach for Immunoprecipitation of RNAs and Associated RNAs/Proteins

    PubMed Central

    Cottrell, Kyle A.; Djuranovic, Sergej

    2016-01-01

    Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb—RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the “resurrected” snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein. PMID:27930710

  14. Identification of novel 14-3-3ζ interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK).

    PubMed

    Ge, Feng; Li, Wen-Liang; Bi, Li-Jun; Tao, Sheng-Ce; Zhang, Zhi-Ping; Zhang, Xian-En

    2010-11-05

    The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. To gain insight into the molecular action of 14-3-3ζ in multiple myeloma (MM), we performed a systematic proteomic analysis of 14-3-3ζ-associated proteins. This analysis, recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrates RNAi, SILAC, immunoprecipitation, and quantitative MS technologies. Quantitative mass spectrometry analysis allowed us to distinguish 14-3-3ζ-interacting proteins from background proteins, resulting in the identification of 292 proteins in total with 95 novel interactions. Three 14-3-3ζ-interacting proteins-BAX, HSP70, and BAG3-were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. Our results therefore not only uncover a large number of novel 14-3-3ζ-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of 14-3-3ζ. This study also demonstrated that QUICK is a useful approach to detect specific protein-protein interactions with very high confidence and may have a wide range of applications in the investigation of protein complex interaction networks.

  15. AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation

    PubMed Central

    2013-01-01

    ChIP-seq is an established manually-performed method for identifying DNA-protein interactions genome-wide. Here, we describe a protocol for automated high-throughput (AHT) ChIP-seq. To demonstrate the quality of data obtained using AHT-ChIP-seq, we applied it to five proteins in mouse livers using a single 96-well plate, demonstrating an extremely high degree of qualitative and quantitative reproducibility among biological and technical replicates. We estimated the optimum and minimum recommended cell numbers required to perform AHT-ChIP-seq by running an additional plate using HepG2 and MCF7 cells. With this protocol, commercially available robotics can perform four hundred experiments in five days. PMID:24200198

  16. Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo.

    PubMed

    Schuch, Ronaldo; Agelopoulos, Konstantin; Neumann, Anna; Brandt, Burkhard; Bürger, Horst; Korsching, Eberhard

    2012-02-20

    Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs. The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue. The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme

  17. A ChIP on the shoulder? Chromatin immunoprecipitation and validation strategies for ChIP antibodies.

    PubMed

    Wardle, Fiona C; Tan, Haihan

    2015-01-01

    Chromatin immunoprecipitation (ChIP) is a technique widely used in the study of epigenetics and transcriptional regulation of gene expression. However, its antibody-centric nature exposes it to similar challenges faced by other antibody-based procedures, of which the most prominent are issues of specificity and affinity in antigen recognition. As with other techniques that make use of antibodies, recent studies have shown the need for validation of ChIP antibodies in order to be sure they recognize the advertised protein or epitope. We summarize here the issues surrounding ChIP antibody usage, and highlight the toolkit of validation methods that can be employed by investigators looking to appraise these reagents.

  18. The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus.

    PubMed

    Qiu, Jing; Kleineidam, Anna; Gouraud, Sabine; Yao, Song Tieng; Greenwood, Mingkwan; Hoe, See Ziau; Hindmarch, Charles; Murphy, David

    2014-11-01

    The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

  19. RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA

    PubMed Central

    Alspach, Elise; Stewart, Sheila A.

    2016-01-01

    Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014). PMID:27453911

  20. The relative infrequency and low levels of neutralising and immunoprecipitating antibody to herpes simplex viruses types 1 and 2 in patients with a history of recurrent herpes genitalis.

    PubMed

    Woodman, C B; Stocker, D; Sugrue, D; Desberbasques, M; Hartley, C E; Fuller, A; Buchan, A; Skinner, G R

    1983-01-01

    Twenty-seven per cent of 70 patients with a history of recurrent herpes genitalis but no concomitant history of recurrent oral or peri-genital disease, had no detectable neutralising antibody against type 1 or type 2 herpes simplex virus; the prevalence and levels of neutralising antibody were similar to 53 patients with no history of herpetic disease and significantly lower than 67 patients with a history of recurrent herpes genitalis in association with oral or peri-genital disease all of whom had neutralising antibody against both virus types. There were similar differences between groups for immunoprecipitating antibody where 80% of patients were herpes genitalis alone had no detectable immunoprecipitating antibody. The results indicate that the failure to detect immunising and immunoprecipitating antibody in an individual's serum is compatible with a long and even severe history of recurrent herpes genitalis and consequently that the development of neutralising antibody does not necessarily indicate an episode of primary herpetic disease.

  1. Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

    NASA Astrophysics Data System (ADS)

    Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

    2012-06-01

    There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

  2. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

    PubMed Central

    Statham, Aaron L.; Robinson, Mark D.; Song, Jenny Z.; Coolen, Marcel W.; Stirzaker, Clare; Clark, Susan J.

    2012-01-01

    The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq), which can directly interrogate genetic and epigenetic processes that occur in normal and diseased cells. Unlike most previous reports based on correlative techniques, we found using direct bisulfite sequencing of Polycomb H3K27me3-enriched DNA from normal and prostate cancer cells that DNA methylation and H3K27me3-marked histones are not always mutually exclusive, but can co-occur in a genomic region-dependent manner. Notably, in cancer, the co-dependency of marks is largely redistributed with an increase of the dual repressive marks at CpG islands and transcription start sites of silent genes. In contrast, there is a loss of DNA methylation in intergenic H3K27me3-marked regions. Allele-specific methylation status derived from the BisChIP-seq data clearly showed that both methylated and unmethylated alleles can simultaneously be associated with H3K27me3 histones, highlighting that DNA methylation status in these regions is not dependent on Polycomb chromatin status. BisChIP-seq is a novel approach that can be widely applied to directly interrogate the genomic relationship between allele-specific DNA methylation, histone modification, or other important epigenetic regulators. PMID:22466171

  3. Specific detection of peste des petits ruminants virus antibodies in sheep and goat sera by the luciferase immunoprecipitation system.

    PubMed

    Berguido, Francisco J; Bodjo, Sanne Charles; Loitsch, Angelika; Diallo, Adama

    2016-01-01

    Peste des petits ruminants (PPR) is a contagious and often fatal transboundary animal disease affecting mostly sheep, goats and wild small ruminants. This disease is endemic in most of Africa, the Middle, Near East, and large parts of Asia. The causal agent is peste des petits ruminants virus (PPRV), which belongs to the genus Morbillivirus in the family Paramyxoviridae. This genus also includes measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). All are closely related viruses with serological cross reactivity. In this study, we have developed a Luciferase Immunoprecipitation System (LIPS) for the rapid detection of antibodies against PPRV in serum samples and for specific differentiation from antibodies against RPV. PPR and rinderpest (RP) serum samples were assayed by PPR-LIPS and two commercially available PPR cELISA tests. The PPR-LIPS showed high sensitivity and specificity for the samples tested and showed no cross reactivity with RPV unlike the commercial PPR cELISA tests which did cross react with RPV. Based on the results shown in this study, PPR-LIPS is presented as a good candidate for the specific serosurveillance of PPR.

  4. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    PubMed

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  5. Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

    PubMed Central

    Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun; Kim, Tackhoon; Chung, Chaeuk; Ryu, Ji Young; Lee, Hanki; Park, Kihyun; Lee, Sangkyu; Jones, Walton D.; Lim, Dae-Sik; Hyeon, Changbong; Do Heo, Won; Yoon, Tae-Young

    2013-01-01

    Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level. PMID:23422673

  6. Multifunctionalization of cetuximab with bioorthogonal chemistries and parallel EGFR profiling of cell-lines using imaging, FACS and immunoprecipitation approaches.

    PubMed

    Reschke, Melanie L; Uprety, Rajendra; Bodhinayake, Imithri; Banu, Matei; Boockvar, John A; Sauve, Anthony A

    2014-12-01

    The ability to derivatize antibodies is currently limited by the chemical structure of antibodies as polypeptides. Modern methods of bioorthogonal and biocompatible chemical modifications could make antibody functionalization more predictable and easier, without compromising the functions of the antibody. To explore this concept, we modified the well-known anti-epidermal growth factor receptor (EGFR) drug, cetuximab (Erbitux®), with 5-azido-2-nitro-benzoyl (ANB) modifications by optimization of an acylation protocol. We then show that the resulting ANB-cetuximab can be reliably modified with dyes (TAMRA and carboxyrhodamine) or a novel synthesized cyclooctyne modified biotin. The resulting dye- and biotin-modified cetuximabs were then tested across several assay platforms with several cell lines including U87, LN229, F98EGFR, F98WT and HEK293 cells. The assay platforms included fluorescence microscopy, FACS and biotin-avidin based immunoprecipitation methods. The modified antibody performs consistently in all of these assay platforms, reliably determining relative abundances of EGFR expression on EGFR expressing cells (LN229 and F98EGFR) and failing to cross react with weak to negative EGFR expressing cells (U87, F98WT and HEK293). The ease of achieving diverse and assay relevant functionalizations as well as the consequent rapid construction of highly correlated antigen expression data sets highlights the power of bioorthogonal and biocompatible methods to conjugate macromolecules. These data provide a proof of concept for a multifunctionalization strategy that leverages the biochemical versatility and antigen specificity of antibodies.

  7. Clinical and public health research using methylated DNA Immunoprecipitation (MeDIP): A comparison of commercially available kits to examine differential DNA methylation across the genome

    PubMed Central

    Brebi-Mieville, Priscilla; Ili-Gangas, Carmen; Leal-Rojas, Pamela; Noordhuis, Maartje; Soudry, Ethan; Perez, Jimena; Roa, Juan Carlos; Sidransky, David; Guerrero-Preston, Rafael

    2012-01-01

    The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp™ Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies. PMID:22207357

  8. Nanobody®-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites

    PubMed Central

    Nguyen-Duc, Trong; Peeters, Eveline; Muyldermans, Serge; Charlier, Daniel; Hassanzadeh-Ghassabeh, Gholamreza

    2013-01-01

    Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid–protein and protein–protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA–protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation. PMID:23275538

  9. Analysis of antibody responses to Hymenolepis nana infection in mice by the enzyme-linked immunosorbent assay and immunoprecipitation.

    PubMed

    Ito, A; Honey, R D; Scanlon, T; Lightowlers, M W; Rickard, M D

    1988-05-01

    Serum antibody responses in two strains of mice infected with embryonated eggs of Hymenolepis nana were analysed by the enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) using sodium deoxycholate (DOC)-solubilized antigens prepared from embryonated eggs (eggs), mouse-derived cysticercoids (cysts) and adult tapeworms with immature segments only (adults). Highly susceptible dd mice, which harbour mature tapeworms for a long period (greater than 70 days), produced high levels of antibodies to all three different stages of H. nana. BALB/c mice, almost all of which expel adult tapeworms by 30 days after infection, produced high levels of antibody against egg antigens only. The high antibody titres to cyst and adult antigens in dd mice did not lead to expulsion of the worms. However, worms are rejected early in BALB/c mice when there is little or no detectable serum antibody. The antibody responses to eggs seen in BALB/c mice which had long since shed their adult worms were probably due to ingestion of eggs from faeces of other infected mice. Antibodies to eggs were not detected in BALB/c mice which were initially inoculated with eggs (day 0) and then treated with praziquantel on day 6 after the tissue phase of infection only. The different antibody responses to egg antigens and the other two antigens (cyst and adult) in BALB/c mice suggest a difference in antigen specificity between eggs and both cysts and adults. A major antigen component with Mr 32,000 appears to be specific to the egg (or oncosphere) stage of H. nana. Antibody to this major component of eggs was absorbed only with intact eggs, but not with intact cysts nor adults with immature segments only, so that the antigen appears to be on the surface of the oncosphere.

  10. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging.

    PubMed

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M; Johnson, Aaron M; Ren, Xiaojun

    2015-11-20

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.

  11. Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging*♦

    PubMed Central

    Tatavosian, Roubina; Zhen, Chao Yu; Duc, Huy Nguyen; Balas, Maggie M.; Johnson, Aaron M.; Ren, Xiaojun

    2015-01-01

    Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes. PMID:26381410

  12. Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter

    PubMed Central

    Rogge, George A; Shen, Li-Ling; Kuhar, Michael J.

    2010-01-01

    Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. PMID:20451507

  13. A Laboratory Practical Illustrating the Use of the ChIP-qPCR Method in a Robust Model: Estrogen Receptor Alpha Immunoprecipitation Using MCF-7 Culture Cells

    ERIC Educational Resources Information Center

    Lacazette, Eric

    2017-01-01

    Chromatin immunoprecipitation followed by qPCR analysis (ChIP-qPCR) is a widely used technique to study gene expression. A large number of students in molecular biology and more generally in life sciences will be confronted with the use of this technique, which is quite difficult to set up and can lead to misinterpretation if not carefully…

  14. Functional analysis of neuronal microRNAs in Caenorhabditis elegans dauer formation by combinational genetics and Neuronal miRISC immunoprecipitation.

    PubMed

    Than, Minh T; Kudlow, Brian A; Han, Min

    2013-06-01

    Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic "enhancer" approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It

  15. Functional Analysis of Neuronal MicroRNAs in Caenorhabditis elegans Dauer Formation by Combinational Genetics and Neuronal miRISC Immunoprecipitation

    PubMed Central

    Than, Minh T.; Kudlow, Brian A.; Han, Min

    2013-01-01

    Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic “enhancer” approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It

  16. Heterotrimeric Galphaq11 co-immunoprecipitates with surface-anchored GRP78 from plasma membranes of alpha2M*-stimulated macrophages.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2008-05-01

    We have previously shown that a fraction of newly expressed GRP78 is translocated to the cell surface in association with the co-chaperone MTJ-1. Proteinase and methylamine-activated alpha(2)M (alpha(2)M*) bind to cell surface-associated GRP78 activating phosphoinositide-specific phospholipase C coupled to a pertussis toxin-insensitive heterotrimeric G protein, generating IP(3)/calcium signaling. We have now studied the association of pertussis toxin-insensitive Galphaq11, with GRP78/MTJ-1 complexes in the plasma membranes of alpha(2)M*-stimulated macrophages. When GRP78 was immunoprecipitated from plasma membranes of macrophages stimulated with alpha(2)M*, Galphaq11, and MTJ-1 were co-precipitated. Likewise Galphaq11 and GRP78 co-immunoprecipitated with MTJ-1 while GRP78 and MTJ-1 co-immunoprecipitated with Galphaq11. Silencing GRP78 expression with GRP78 dsRNA or MTJ-1 with MTJ-1 dsRNA greatly reduced the levels of Galphaq11 co-precipitated with GRP78 or MTJ-1. In conclusion, we show here that plasma membrane-associated GRP78 is coupled to pertussis toxin-insensitive Galphaq11 and forms a ternary signaling complex with MTJ-1.

  17. [Immunoprecipitation mapping of the TRX-associated chromosome elements in the fork head gene promoter in the Drosophila melanogaster salivary gland cells].

    PubMed

    Riakhovskiĭ, A A; Tillib, S V

    2007-09-01

    Using the method of immunoprecipitation of the in vivo crosslinked and sheared by sonication chromatin, mapping of potential trithorax-associated regulatory elements within the extended (9 kb) promoter region of the fork head gene (fkh) in the Drosophila melanogaster salivary gland cells was performed. Relative homogeneity of the salivary gland cells, along with the parallel use of the antibodies to different domains of the same trithorax protein (TRX), and the introduction of cross-hybridization steps for additional specific enrichment of initial DNA libraries, provided improvement of the method effectiveness and identification of one major and two less expressed potential TRX-binding sites.

  18. Performance of Competitive and Indirect Enzyme-Linked Immunosorbent Assays, Gel Immunoprecipitation with Native Hapten Polysaccharide, and Standard Serological Tests in Diagnosis of Sheep Brucellosis

    PubMed Central

    Marín, C. M.; Moreno, E.; Moriyón, I.; Díaz, R.; Blasco, J. M.

    1999-01-01

    Competitive and standard enzyme-linked immunosorbent assays (ELISAs), rose bengal (RB), complement fixation, and agar gel immunoprecipitation with native hapten (AGID-NH) were compared by using sera from Brucella-free, Brucella melitensis-infected, and B. melitensis Rev1-vaccinated sheep. The most sensitive tests were indirect ELISA and RB, and the most specific tests were AGID-NH and competitive ELISA. We show that RB followed by AGID-NH is a simple and effective system for diagnosing sheep brucellosis. PMID:10066666

  19. Identification of miR-215 mediated targets/pathways via translational immunoprecipitation expression analysis (TrIP-chip)

    PubMed Central

    Fesler, Andrew; Xu, Xiao; Zheng, Xiao; Li, Xiaodong; Jiang, Jingting; Russo, James J.; Ju, Jingfang

    2015-01-01

    Steady state mRNA expression profiling can identify the majority of miRNA targets. However, some translationally repressed miRNA targets are missed and thus not considered for functional validation. Therefore, analysis of mRNA translation can enhance miRNA target identification for functional studies. We have applied a unique approach to identify miRNA targets in a small number of cells. Actively translating mRNAs are associated with polyribosomes and newly synthesized peptide chains are associated with molecular chaperones such as HSP70s. Affinity capture beads were used to capture HSP70 chaperones associated with polyribosome complexes. The isolated actively translating mRNAs were used for high throughput expression profiling analysis. miR-215 is an important miRNA in colorectal cancer and loss of miR-215 is significantly associated with prognosis of this disease. miR-215 suppresses the expression of several key targets. We utilized the affinity capture approach to isolate miR-215 mediated mRNA target transcripts. This approach provides a unique way to identify targets regulated by non-coding RNAs and RNA binding proteins from a small number of cells. PMID:26287603

  20. Detection of anti-U3-RNP/fibrillarin IgG antibodies by line immunoblot assay has comparable clinical significance to immunoprecipitation testing in systemic sclerosis.

    PubMed

    Peterson, Lisa K; Jaskowski, Troy D; Mayes, Maureen D; Tebo, Anne E

    2016-04-01

    The aim of this study was to evaluate the performance and clinical relevance of a commercially available line immunoblot assay (LIA) for detecting anti-U3-RNP/fibrillarin (anti-U3-RNP), against immunoprecipitation (gold standard). This study involved a multi-ethnic cohort of 1000 American systemic sclerosis (SSc) patients and 50 healthy controls. Antinuclear antibodies and centromere antibodies were detected by indirect immunofluorescent antibody test, anti-topo I by immunodiffusion and anti-RNAP III by ELISA. The presence of anti-U3-RNP in select serum samples was detected by immunoprecipitation (IP) and LIA. By IP, U3-RNP antibody was detected in 75 (7.5 %) patients with SSc. Overall agreement between LIA and IP was very good (κ = 0.966). Analytic sensitivity and specificity of the U3-RNP LIA was 100 and 94.7 %, respectively. Clinical features associated with positivity for the anti-U3-RNP antibody include diffuse cutaneous SSc and increased prevalence of renal crisis, consistent with previous studies that used IP. Testing for U3-RNP antibodies is only performed by a small number of laboratories due to the complexity of both performance and interpretation of the IP. LIA is faster and less complex than IP. Excellent agreement between IP and LIA demonstrates that LIA is an acceptable and attractive alternative to IP for anti-U3-RNP detection.

  1. Binding of lac repressor-GFP fusion protein to lac operator sites inserted in the tobacco chloroplast genome examined by chromatin immunoprecipitation.

    PubMed

    Newell, Christine A; Gray, John C

    2010-08-01

    Chromatin immunoprecipitation (ChIP) has been used to detect binding of DNA-binding proteins to sites in nuclear and mitochondrial genomes. Here, we describe a method for detecting protein-binding sites on chloroplast DNA, using modifications to the nuclear ChIP procedures. The method was developed using the lac operator (lacO)/lac repressor (LacI) system from Escherichia coli. The lacO sequences were integrated into a single site between the rbcL and accD genes in tobacco plastid DNA and homoplasmic transplastomic plants were crossed with transgenic tobacco plants expressing a nuclear-encoded plastid-targeted GFP-LacI fusion protein. In the progeny, the GFP-LacI fusion protein could be visualized in living tissues using confocal microscopy, and was found to co-localize with plastid nucleoids. Isolated chloroplasts from the lacO/GFP-LacI plants were lysed, treated with micrococcal nuclease to digest the DNA to fragments of approximately 600 bp and incubated with antibodies to GFP and protein A-Sepharose. PCR analysis on DNA extracted from the immunoprecipitate demonstrated IPTG (isopropylthiogalactoside)-sensitive binding of GFP-LacI to lacO. Binding of GFP-LacI to endogenous sites in plastid DNA showing sequence similarity to lacO was also detected, but required reversible cross-linking with formaldehyde. This may provide a general method for the detection of binding sites on plastid DNA for specific proteins.

  2. Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: demonstration by monoclonal antibody binding and immunoprecipitation.

    PubMed Central

    Pober, J S; Gimbrone, M A

    1982-01-01

    The expression of Ia-like antigens by cultured human endothelial cells has been investigated by means of monoclonal antibody binding to intact cells and by immunoprecipitation of radioiodinated membrane proteins. Primary growing and confluent cultures of human umbilical vein endothelium express little, if any, detectable Ia-like antigens under standard culture conditions. However, treatment of primary cultures with the lectin phytohemagglutinin induces the expression of Ia-like antigens. This action of the lectin uniformly affects all the endothelial cells in a culture, does not depend on cell division, and is associated with a cell shape change. The data presented in this report provide unequivocal serological and biochemical demonstration of Ia-like antigens on human vascular endothelial cells. The fact that the expression of Ia-like antigens by endothelium can be induced may have important implications for organ transplantation and for regulation of the immunological response. Images PMID:6815654

  3. Equivalence of Visual Immunoprecipitate Assay (VIP) for Salmonella for the detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study.

    PubMed

    Feldsine, P T; Mui, L A; Forgey, R L; Kerr, D E

    2000-01-01

    Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Visual Immunoprecipitate Assay (VIP) for Salmonella and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the VIP method and one by the AOAC culture method. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between VIP for Salmonella interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The method was adopted Official First Action status by AOAC INTERNATIONAL.

  4. Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

    PubMed Central

    Sharov, Alexei A; Masui, Shinji; Sharova, Lioudmila V; Piao, Yulan; Aiba, Kazuhiro; Matoba, Ryo; Xin, Li; Niwa, Hitoshi; Ko, Minoru SH

    2008-01-01

    Background Target genes of a transcription factor (TF) Pou5f1 (Oct3/4 or Oct4), which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES) cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP)-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR) criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR < 0.2) to a compendium of published and new microarray data (3, 6, 12, and 24 hr after Pou5f1 suppression) and published ChIP data, we identified 420 tentative target genes (TTGs) for Pou5f1. The majority of TTGs (372) were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion We have identified the most reliable sets of direct target genes for key pluripotency genes – Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly. PMID:18522731

  5. Identifying Hazards

    EPA Pesticide Factsheets

    The federal government has established a system of labeling hazardous materials to help identify the type of material and threat posed. Summaries of information on over 300 chemicals are maintained in the Envirofacts Master Chemical Integrator.

  6. Detection of Antibodies to Varicella-Zoster Virus in Recipients of the Varicella Vaccine by Using a Luciferase Immunoprecipitation System Assay

    PubMed Central

    Ali, Mir A.; Bayat, Ahmad; Steinberg, Sharon P.; Park, Hosun; Gershon, Anne A.; Burbelo, Peter D.

    2014-01-01

    A high-throughput test to detect varicella-zoster virus (VZV) antibodies in varicella vaccine recipients is not currently available. One of the most sensitive tests for detecting VZV antibodies after vaccination is the fluorescent antibody to membrane antigen (FAMA) test. Unfortunately, this test is labor-intensive, somewhat subjective to read, and not commercially available. Therefore, we developed a highly quantitative and high-throughput luciferase immunoprecipitation system (LIPS) assay to detect antibody to VZV glycoprotein E (gE). Tests of children who received the varicella vaccine showed that the gE LIPS assay had 90% sensitivity and 70% specificity, a viral capsid antigen enzyme-linked immunosorbent assay (ELISA) had 67% and 87% specificity, and a glycoprotein ELISA (not commercially available in the United States) had 94% sensitivity and 74% specificity compared with the FAMA test. The rates of antibody detection by the gE LIPS and glycoprotein ELISA were not statistically different. Therefore, the gE LIPS assay may be useful for detecting VZV antibodies in varicella vaccine recipients. (This study has been registered at ClinicalTrials.gov under registration no. NCT00921999.) PMID:24990909

  7. Transcriptome-wide Identification of RNA-binding Protein Binding Sites Using Photoactivatable-Ribonucleoside-Enhanced Crosslinking Immunoprecipitation (PAR-CLIP).

    PubMed

    Maatz, Henrike; Kolinski, Marcin; Hubner, Norbert; Landthaler, Markus

    2017-04-03

    RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA-protein crosslinking efficiency in standard CLIP-seq, photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) has been developed. Here, living cells or whole organisms are fed photo-activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV-induced mutations that mark the crosslinking nucleotide and allow transcriptome-wide identification of RBP binding sites at single-nucleotide resolution. © by 2017 John Wiley & Sons, Inc.

  8. Chromatin immunoprecipitation analysis of the tobacco PR-1a- and the truncated CaMV 35S promoter reveals differences in salicylic acid-dependent TGA factor binding and histone acetylation.

    PubMed

    Butterbrodt, Thomas; Thurow, Corinna; Gatz, Christiane

    2006-07-01

    Salicylic acid (SA) is a plant signalling molecule needed for the induction of defence responses upon attack by a variety of pathogens. Truncation of the Cauliflower Mosaic Virus (CaMV) 35S promoter down to 90 bp has identified activation sequence-1 (as-1) as an autonomous SA-responsive cis element. The as-1-like elements are found in a number of SA-inducible promoters like e.g. the tobacco PR-1a promoter. They are recognized by basic/leucine zipper (bZIP) transcription factors of the TGA family. In tobacco leaves, TGA2.2 is the most abundant TGA factor. TGA2.2 is required for the expression of as-1-containing promoters. Here we unravel clear differences between the "truncated" CaMV 35S and the PR-1a promoter with respect to in vivo TGA binding and histone acetylation. Chromatin immunoprecipitation (ChIP) analysis revealed SA-inducible recruitment of tobacco TGA2.2 as well as SA-inducible histone acetylation at the PR-1a promoter. In contrast, no influence of SA on TGA2.2 binding and histone acetylation was detectable at the "truncated" CaMV 35S promoter. The finding of SA-independent TGA factor binding in the absence of additional flanking regulatory sequences suggests that transcriptional activation is not necessarily mediated by inducible DNA binding of TGA factors. Plants with severely reduced TGA2.2 protein levels also showed SA-induced histone acetylation at the PR-1a promoter indicating that regulatory events independent from TGA2.2 function are initiated at the PR-1a promoter.

  9. Occurrence and significance of antibody to liver-specific membrane lipoprotein by double-antibody immunoprecipitation method in sera of patients with acute and chronic liver diseases.

    PubMed

    Kakumu, S; Arakawa, Y; Goji, H; Kashio, T; Yata, K

    1979-04-01

    A double-antibody immunoprecipitation method was developed for detecting antibody to liver-specific membrane lipoprotein (anti-LSP) in sera of patients with various liver diseases and primary nonhepatic autoimmune diseases. Liver-specific membrane lipoprotein prepared from normal rat livers was labeled with 125I (chloramine-T) and monospecific antibody raised in rabbits. Cross-reactivity and absorption studies demonstrated that the assay used was highly specific. The frequency and titer of anti-LSP were similar for HBsAg-positive and -negative patients with both acute and chronic liver diseases. Patients with chronic active hepatitis had the highest frequenzy (25 of 44 cases, 57%) when compared with those with chronic persistent hepatitis (5 of 23 cases, 22%) and nonalcoholic cirrhosis (8 of 21 cases, 38%). Of the anti-LSP positive cases, the mean titer in patients with chronic active hepatitis tended to be the highest. In patients recovered from acute viral hepatitis, anti-LSP was transiently positive (7 of 20 cases, 35%) in the acute phase. In those who progressed to chronic hepatitis, a late rise as well as an early rise occurred in 6 of 10 patients before the diagnosis was made. Two of 6 patients with primary biliary cirrhosis had anti-LSP, but none of 41 patients with other nonviral liver diseases and none of 60 patients with primary nonhepatic autoimmune diseases. These data indicate that an autoimmune reaction directed against LSP can be initiated during the acute phase of viral hepatitis and it may persist in chronic hepatitis in both HBsAg-positive and -negative cases.

  10. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

    PubMed

    Peach, Sally E; Rudomin, Emily L; Udeshi, Namrata D; Carr, Steven A; Jaffe, Jacob D

    2012-05-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

  11. Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture.

    PubMed

    Nguyen, Binh Cao Quan; Be Tu, Pham Thi; Tawata, Shinkichi; Maruta, Hiroshi

    2015-08-01

    Cucurbitacin I (CBI) is a triterpene from a bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 and the G protein RAC, leading to the inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, a propolis produced in New Zealand, contains CAPE (caffeic acid phenethyl ester) as the major anti-cancer ingredient which directly down-regulates RAC, leading to the inactivation of PAK1. Since PAK1 is essential for the growth of RAS cancer cells such as A549 cell line which carry an oncogenic K-RAS mutant, and the melanogenesis in skin cells, here using these PAK1-blockers as model compounds, we introduce a new approach to the quick assessment of PAK1-blockers in cell culture. First, combining the immuno-precipitation (IP) of PAK1 from cell lysate and the in vitro ATP_Glo kinase assay kit (called "Macaroni-Western" assay), we confirmed that both CBI and Bio 30 inactivate PAK1 in A549 lung cancer cells in 24 h, and inhibit their PAK1-dependent growth in 72 h. Furthermore, we verified that CBI inhibits the PAK1/PAK4-dependent melanogenesis in melanoma cells by far more than 50%, while Bio 30 inhibits the melanogenesis only by 50%, with only a merginal effect on their growth per se. Since the "Macaroni-Western" kinase assay and melanogenesis are both rather simple and quick, the combination of these two cell culture assays would be highly useful for selecting both "potent" (highly cell-permeable) and "safe" (non-toxic) natural or synthetic PAK1-blockers.

  12. Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing

    PubMed Central

    2010-01-01

    Background Cancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized. Results Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation. Conclusions This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes. PMID:20181289

  13. Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology.

    PubMed

    Sun, Lei; Ii, Adlai L Pappy; Pham, Tiffany T; Shanley, Thomas P

    2015-06-29

    Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces "immune tolerance" of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαl°xl°x;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαl°xl°x;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

  14. Genome-Wide Mapping of the Distribution of CarD, RNAP σ(A), and RNAP β on the Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation Sequencing.

    PubMed

    Landick, Robert; Krek, Azra; Glickman, Michael S; Socci, Nicholas D; Stallings, Christina L

    2014-12-01

    CarD is an essential mycobacterial protein that binds the RNA polymerase (RNAP) and affects the transcriptional profile of Mycobacterium smegmatis and Mycobacterium tuberculosis (6). We predicted that CarD was directly regulating RNAP function but our prior experiments had not determined at what stage of transcription CarD was functioning and at which genes CarD interacted with the RNAP. To begin to address these open questions, we performed Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. The distribution of RNAP subunits β and σ(A) were also profiled. We expected that RNAP β would be present throughout transcribed regions and RNAP σ(A) would be predominantly enriched at promoters based on work in Escherichia coli (3), however this had yet to be determined in mycobacteria. The ChIP-seq analyses revealed that CarD was never present on the genome in the absence of RNAP, was primarily associated with promoter regions, and was highly correlated with the distribution of RNAP σ(A). The colocalization of σ(A) and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. Here we describe in detail the data from the ChIP-seq experiments associated with the study published by Srivastava and colleagues in the Proceedings of the National Academy of Science in 2013 (5) as well as discuss the findings from this dataset in relation to both CarD and mycobacterial transcription as a whole. The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE48164).

  15. Transformation-Related Antigens Identified by Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Strand, Mette

    1980-06-01

    Tumor-cell proteins that were antigenic in a syngeneic animal were identified by immunoprecipitation with monoclonal antibodies. Spleen cells of BALB/c mice immunized with plasma membranes of Kirsten RNA sarcoma virus-transformed BALB/3T3 cells were fused with NS-l myeloma cells. Antibodies secreted into the culture fluid from these hybridomas were distinguished by their reactivity against proteins of different target cells. A total of 191 cultures were established; 143 produced antibodies that bound to BALB/3T3 cells transformed by the RNA sarcoma virus, of which antibodies from 82 bound to BALB/3T3 transformed with simian virus 40, and antibodies from 56 bound to BALB/3T3 cells. Thus, more than 50% of the cultures produced antibodies that possibly were specific to antigens of the transformed cell. Twenty different hybridomas have been cloned, and antibodies from eight of these were found to immunoprecipitate five different proteins. A protein of approximately 32,000 daltons was precipitated from BALB/3T3 cells transformed by the RNA sarcoma virus, simian virus 40, or methylcholanthrene but not from untransformed BALB/3T3 cells. A protein of about 300,000 daltons was precipitated from all four cell lines; precipitation was enhanced in the viral transformed cells. Proteins of approximately 57,000, 54,000, and 8500 daltons were immunoprecipitated from all four cell lines.

  16. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    PubMed Central

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the “histone code.” We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to “canonical” chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living

  17. Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src.

    PubMed Central

    Modderman, P W; von dem Borne, A E; Sonnenberg, A

    1994-01-01

    P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src. Images Figure 1 Figure 2 Figure 3

  18. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes.

  19. PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins

    PubMed Central

    Spitzer, Jessica; Hafner, Markus; Landthaler, Markus; Ascano, Manuel; Farazi, Thalia; Wardle, Greg; Nusbaum, Jeff; Khorshid, Mohsen; Burger, Lukas; Zavolan, Mihaela; Tuschl, Thomas

    2014-01-01

    We recently developed a protocol for the transcriptome-wide isolation of RNA recognition elements readily applicable to any protein or ribonucleoprotein complex directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner et al., 2010). Briefly, immunoprecipitation of the RNA-binding protein of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonu-clease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa technology (see Explanatory Chapter: Next Generation Sequencing). By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs. PMID:24581442

  20. Constitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells. Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer.

    PubMed

    Gazi, Lucien; López-Giménez, Juan F; Rüdiger, Martin P; Strange, Philip G

    2003-10-01

    Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.

  1. Identifying true protein complex constituents in interaction proteomics: the example of the DMXL2 protein complex.

    PubMed

    Li, Ka Wan; Chen, Ning; Klemmer, Patricia; Koopmans, Frank; Karupothula, Ramesh; Smit, August B

    2012-08-01

    A typical high-sensitivity antibody affinity purification-mass spectrometry experiment easily identifies hundreds of protein interactors. However, most of these are non-valid resulting from multiple causes other than interaction with the bait protein. To discriminate true interactors from off-target recognition, we propose to differentially include an (peptide) antigen during the antibody incubation in the immuno-precipitation experiment. This contrasts the specific antibody-bait protein interactions, versus all other off-target protein interactions. To exemplify the power of the approach, we studied the DMXL2 interactome. From the initial six immuno-precipitations, we identified about 600 proteins. When filtering for interactors present in all anti-DMXL2 antibody immuno-precipitation experiments, absent in the bead controls, and competed off by the peptide antigen, this hit list is reduced to ten proteins, including known and novel interactors of DMXL2. Together, our approach enables the use of a wide range of available antibodies in large-scale protein interaction proteomics, while gaining specificity of the interactions. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Materials Data on Mn3(AgO2)4 (SG:152) by Materials Project

    SciTech Connect

    Kristin Persson

    2016-04-14

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  3. Using Quantitative Seroproteomics to Identify Antibody Biomarkers in Pancreatic Cancer

    PubMed Central

    Jhaveri, Darshil T.; Kim, Min-Sik; Thompson, Elizabeth D.; Huang, Lanqing; Sharma, Rajni; Klein, Alison P.; Zheng, Lei; Le, Dung T.; Laheru, Daniel A.; Pandey, Akhilesh; Jaffee, Elizabeth M.; Anders, Robert A.

    2016-01-01

    Pancreatic cancer is the fourth leading cause of cancer deaths in the US. Less than 6% of patients survive beyond the fifth year due to inadequate early diagnostics and ineffective treatment options. Our lab has developed an allogeneic, granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting pancreatic cancer vaccine (GVAX) that has been tested in phase II clinical trials. Here, we employed a novel Serum Antibodies based SILAC-Immunoprecipitation (SASI) approach to identify proteins that elicit an antibody response after vaccination. The SASI approach utilizes immunoprecipitation with patient derived antibodies that is coupled to quantitative stable isotope–labeled amino acids in cell culture (SILAC). Using mass spectrometric analysis, we identified more than 150 different proteins that induce an antibody response after vaccination. The regulatory subunit 12A of protein phosphatase 1 (MYPT1 or PPP1R12A), regulatory subunit 8 of the 26S proteasome (PSMC5), and the transferrin receptor (TFRC) were shown to be pancreatic cancer associated antigens recognized by post-vaccination antibodies in the sera of patients with favorable disease-free survival after GVAX therapy. We further interrogated these proteins in over 80 GVAX-treated patients’ pancreases and uniformly found a significant increase in the expression of MYPT1, PSMC5, and TFRC in neoplastic compared to non-neoplastic pancreatic ductal epithelium. We show that the novel SASI approach can identify antibody targets specifically expressed in patients with improved disease-free survival after cancer vaccine therapy. These targets need further validation to be considered as possible pancreatic cancer biomarkers. PMID:26842750

  4. Using Quantitative Seroproteomics to Identify Antibody Biomarkers in Pancreatic Cancer.

    PubMed

    Jhaveri, Darshil T; Kim, Min-Sik; Thompson, Elizabeth D; Huang, Lanqing; Sharma, Rajni; Klein, Alison P; Zheng, Lei; Le, Dung T; Laheru, Daniel A; Pandey, Akhilesh; Jaffee, Elizabeth M; Anders, Robert A

    2016-03-01

    Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Less than 6% of patients survive beyond the fifth year due to inadequate early diagnostics and ineffective treatment options. Our laboratory has developed an allogeneic, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting pancreatic cancer vaccine (GVAX) that has been tested in phase II clinical trials. Here, we employed a serum antibodies-based SILAC immunoprecipitation (SASI) approach to identify proteins that elicit an antibody response after vaccination. The SASI approach uses immunoprecipitation with patient-derived antibodies that is coupled to quantitative stable isotope-labeled amino acids in cell culture (SILAC). Using mass spectrometric analysis, we identified more than 150 different proteins that induce an antibody response after vaccination. The regulatory subunit 12A of protein phosphatase 1 (MYPT1 or PPP1R12A), regulatory subunit 8 of the 26S proteasome (PSMC5), and the transferrin receptor (TFRC) were shown to be pancreatic cancer-associated antigens recognized by postvaccination antibodies in the sera of patients with favorable disease-free survival after GVAX therapy. We further interrogated these proteins in over 80 GVAX-treated patients' pancreases and uniformly found a significant increase in the expression of MYPT1, PSMC5, and TFRC in neoplastic compared with non-neoplastic pancreatic ductal epithelium. We show that the novel SASI approach can identify antibody targets specifically expressed in patients with improved disease-free survival after cancer vaccine therapy. These targets need further validation to be considered as possible pancreatic cancer biomarkers. ©2016 American Association for Cancer Research.

  5. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay*

    PubMed Central

    AbuSamra, Dina B.; Al-Kilani, Alia; Hamdan, Samir M.; Sakashita, Kosuke; Gadhoum, Samah Z.; Merzaban, Jasmeen S.

    2015-01-01

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. PMID:26124272

  6. Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions.

    PubMed

    Atwood, Blake L; Woolnough, Jessica L; Lefevre, Gaelle M; Saint Just Ribeiro, Mariana; Felsenfeld, Gary; Giles, Keith E

    2016-08-19

    The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA cross-linking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.

  7. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

    PubMed Central

    Chandra, Govind; Bibb, Maureen J.; Findlay, Kim C.; Buttner, Mark J.

    2016-01-01

    ABSTRACT WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) in Streptomyces venezuelae. In the first demonstration of in vivo genome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiA in vivo. PMID:27094333

  8. Identifying Development Needs

    ERIC Educational Resources Information Center

    Clark, R. J.; Turner, T. S.

    1976-01-01

    To identify management development needs some organizations have adopted the assessment center method, using simulations of work situations performed before and evaluated by specially trained observers (assessors). (MF)

  9. Gang Identifiers and Terminology.

    ERIC Educational Resources Information Center

    Cantrell, Mary Lynn

    1992-01-01

    Provides lists of gang identifiers and terminology. Suggests that, to find out names and associated identifiers of local gangs, readers should talk to their local police. Included in listing are descriptions of gang-related symbols, physical signals, graffiti, slogans, right-left rules, colors, clothing, jewelry, hair styles, and fingernails. Also…

  10. 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

    2015-09-02

    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.

  11. Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients

    PubMed Central

    Lahner, Edith; Brigatti, Cristina; Marzinotto, Ilaria; Carabotti, Marilia; Scalese, Giulia; Davidson, Howard W; Wenzlau, Janet M; Bosi, Emanuele; Piemonti, Lorenzo; Annibale, Bruno; Lampasona, Vito

    2017-01-01

    Objectives: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls. Methods: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies. Results: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (P<0.0001). The area under the receiver-operating characteristic curve was 0.98 (95% CI 0.965–0.996) for ATP4A, and 0.99 (95% CI 0.979–1.000) for ATP4B, both higher as compared with that of EIA: 0.86 (95% CI 0.809–0.896), P<0.0001. Sensitivity-specificity were 100–89% for ATP4A and 100–90% for ATP4B assay. Compared with LIPS, EIA for parietal cell autoantibodies showed a lower sensitivity (72%, P<0.0001) at a similar specificity (92%, P=0.558). Conclusions: Positivity to both, ATP4A and ATP4B autoantibodies is closely associated with atrophic body gastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa. PMID:28102858

  12. Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces.

    PubMed

    Bush, Matthew J; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Buttner, Mark J

    2016-04-19

    WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmentally controlled cell division that leads to sporulation. The biochemical function of Wbl proteins has been controversial; here, we set out to determine unambiguously if WhiB functions as a transcription factor using chromatin immunoprecipitation sequencing (ChIP-seq) in Streptomyces venezuelae. In the first demonstration of in vivo genome-wide Wbl binding, we showed that WhiB regulates the expression of key genes required for sporulation by binding upstream of ~240 transcription units. Strikingly, the WhiB regulon is identical to the previously characterized WhiA regulon, providing an explanation for the identical phenotypes of whiA and whiB mutants. Using ChIP-seq, we demonstrated that in vivo DNA binding by WhiA depends on WhiB and vice versa, showing that WhiA and WhiB function cooperatively to control expression of a common set of WhiAB target genes. Finally, we show that mutation of the cysteine residues that coordinate the [4Fe-4S] cluster in WhiB prevents DNA binding by both WhiB and WhiA in vivo. Despite the central importance of WhiB-like (Wbl) proteins in actinomycete biology, a conclusive demonstration of their biochemical function has been elusive, and they have been difficult to study, particularly in vitro, largely because they carry an oxygen-sensitive [4Fe-4S] cluster. Here we used genome-wide ChIP-seq to investigate the function of Streptomyces WhiB, the founding member of the Wbl family. The advantage of this approach is that the oxygen sensitivity of the [4Fe-4S] cluster becomes irrelevant once the protein has been cross-linked to DNA in vivo. Our data provide the most compelling in vivo evidence to date that Whi

  13. Adenosine A1( )receptors are selectively coupled to Gα(i-3) in postmortem human brain cortex: Guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding/immunoprecipitation study.

    PubMed

    Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; García-Sevilla, Jesús A

    2015-10-05

    By means of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay combined with immunoprecipitation using anti-Gα subunit antibody, we recently reported 5-HT2A receptor- and M1 muscarinic acetylcholine receptor-mediated Gαq activation in rat cerebral cortical membranes (Odagaki et al., 2014). In the present study, this method has been applied to postmortem human brains, with focusing on adenosine receptor-mediated G-protein activation. In the exploratory experiments using a series of agonists and the antibodies specific to each Gα subtypes in the presence of low (10 nM) or high (50 μM) concentration of GDP, the most prominent increases in specific [(35)S]GTPγS binding in the membranes prepared from human prefrontal cortex were obtained for the combinations of adenosine (1mM)/anti-Gαi-3 in the presence of 50 μM GDP as well as 5-HT (100 μM)/anti-Gαq and carbachol (1mM)/anti-Gαq in the presence of 10nM GDP. Adenosine-induced activation of Gαi-3 emerged only when GDP concentrations were increased higher than 10 μM, and the following experiments were performed in the presence of 300 μM GDP. Adenosine increased specific [(35)S]GTPγS binding to Gαi-3 in a concentration-dependent manner to 251.4% of the basal unstimulated binding, with an EC50 of 1.77 μM. The involvement of adenosine A1 receptor was verified by the experiments using selective agonists and antagonists at adenosine A1 or A3 receptor. Among the α subunits of Gi/o class (Gαi-1, Gαi-2, Gαi-3, and Gαo.), only Gαi-3 was activated by 1mM adenosine, indicating that human brain adenosine A1 receptor is coupled preferentially, if not exclusively, to Gαi-3. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. CLIP Identifies Nova-Regulated RNA Networks in the Brain

    NASA Astrophysics Data System (ADS)

    Ule, Jernej; Jensen, Kirk B.; Ruggiu, Matteo; Mele, Aldo; Ule, Aljaž; Darnell, Robert B.

    2003-11-01

    Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition. Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA. To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP). Thirty-four transcripts were identified multiple times by Nova CLIP. Three-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibition. Splicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory γ-aminobutyric acid and glycine receptors, two previously identified Nova splicing targets. Thus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.

  15. Metal alloy identifier

    DOEpatents

    Riley, William D.; Brown, Jr., Robert D.

    1987-01-01

    To identify the composition of a metal alloy, sparks generated from the alloy are optically observed and spectrographically analyzed. The spectrographic data, in the form of a full-spectrum plot of intensity versus wavelength, provide the "signature" of the metal alloy. This signature can be compared with similar plots for alloys of known composition to establish the unknown composition by a positive match with a known alloy. An alternative method is to form intensity ratios for pairs of predetermined wavelengths within the observed spectrum and to then compare the values of such ratios with similar values for known alloy compositions, thereby to positively identify the unknown alloy composition.

  16. Identifying learning styles.

    PubMed

    Hughes, Grace

    2016-12-14

    What was the nature of the CPD activity, practice-related feedback and/or event and/or experience in your practice? The article explored different learning styles and outlined some of the models that can be used to identify them. It discussed the limitations of these models, indicating that although they can be helpful in identifying a student's preferred learning style, this is not 'fixed' and might change over time. Learning is also influenced by other factors, such as culture and age.

  17. Identifying Technical Vocabulary

    ERIC Educational Resources Information Center

    Chung, Teresa Mihwa; Nation, Paul

    2004-01-01

    This study compared four different approaches to identifying technical words in an anatomy text. The first approach used a four step rating scale, and was used as the comparison for evaluating the other three approaches. It had a high degree of reliability. The least successful approach was that using clues provided by the writer such as labels in…

  18. Identifying Early School Leaving

    ERIC Educational Resources Information Center

    Poole, Millicent E.

    1978-01-01

    Using a matched sample of students who stay in school and those who drop out, or leave early, an attempt was made, via multiple discriminant analysis, to identify the significant characteristics which distinguish "drop outs" from their peers who remain at school. Results indicated that both types of students have similar value orientations and…

  19. Understanding and identifying variability

    USDA-ARS?s Scientific Manuscript database

    Variability is the quality of being uneven or lacking uniformity. Understanding the causes, magnitude, and strategies to manage variability within crop production settings can be used to directly improve agricultural production systems. Identifying when the magnitude of variability is high enough to...

  20. Identifying Nursing's Future Leaders.

    ERIC Educational Resources Information Center

    Gunning, Carolyn S.; Hawken, Patty L.

    1990-01-01

    A study determined that encouraging and supporting students in professional activities while they were still in school would lead those students to participate in professional nursing organizations after they graduated. Organized nursing needs to identify the factors that influence nurses to join organizations and concentrate on these factors to…

  1. Identifying Marine Phytoplankton

    NASA Astrophysics Data System (ADS)

    Hargraves, Paul E.

    Until recently, anyone who needed to accurately identify marine phytoplankton had one of four choices: use the outdated Englishlanguage volumes by E. E. Cupp and N. I. Hendey plus the more recent book by J. Dodge, acquire a working knowledge of German and use the old volumes by Schiller and Hustedt, spend huge amounts of time in an exceedingly well-equipped marine science library trying in vain to keep up with the rapidly evolving field of phytoplankton systematics and taxonomy, or track down one of the rarest of endangered species—a phytoplankton taxonomist—and beg for help.To these unfortunate choices is added one considerably more hopeful: Identifying Marine Phytoplankton. This volume, which has seven contributing authors, contains most of the taxonomic groups that make up the planktonic autotrophs and some heterotrophs of the seas, coasts, and estuaries of the world (missing are cyanobacteria and some of the picoplankton groups).

  2. Oil Shale Identifier

    NASA Astrophysics Data System (ADS)

    Rocha, J.; Santacruz, J.; Randall, S.; McRae Alvarado, B.

    2016-12-01

    The main objective of our project was to identify which type of rock and where certain resources were positioned based on their properties, from well log data. During our research, we created best estimates of ranges for selected sedimentary rocks, by researching the properties of every rock. Although there are over a 1,000 different types of rocks and materials, each one has its own classifications, unique to it. When unearthing for resources, it is important to have knowledge of what type it is you are going for. These classifications are determined from logging tools, and are stored in charts called "Well Log Data". These Well Log Data charts are used to identify and determine what rock is underground in the reservoirs. Knowing what kind of rock is underneath would make the process of oil drilling both time and cost efficient. This project is a plug-in created in Petrel and Ocean to be able to identify the type of rock or mineral, specifically oil shale, based on its qualifications.

  3. On identified predictive control

    NASA Technical Reports Server (NTRS)

    Bialasiewicz, Jan T.

    1993-01-01

    Self-tuning control algorithms are potential successors to manually tuned PID controllers traditionally used in process control applications. A very attractive design method for self-tuning controllers, which has been developed over recent years, is the long-range predictive control (LRPC). The success of LRPC is due to its effectiveness with plants of unknown order and dead-time which may be simultaneously nonminimum phase and unstable or have multiple lightly damped poles (as in the case of flexible structures or flexible robot arms). LRPC is a receding horizon strategy and can be, in general terms, summarized as follows. Using assumed long-range (or multi-step) cost function the optimal control law is found in terms of unknown parameters of the predictor model of the process, current input-output sequence, and future reference signal sequence. The common approach is to assume that the input-output process model is known or separately identified and then to find the parameters of the predictor model. Once these are known, the optimal control law determines control signal at the current time t which is applied at the process input and the whole procedure is repeated at the next time instant. Most of the recent research in this field is apparently centered around the LRPC formulation developed by Clarke et al., known as generalized predictive control (GPC). GPC uses ARIMAX/CARIMA model of the process in its input-output formulation. In this paper, the GPC formulation is used but the process predictor model is derived from the state space formulation of the ARIMAX model and is directly identified over the receding horizon, i.e., using current input-output sequence. The underlying technique in the design of identified predictive control (IPC) algorithm is the identification algorithm of observer/Kalman filter Markov parameters developed by Juang et al. at NASA Langley Research Center and successfully applied to identification of flexible structures.

  4. Identifying Distant AGNs

    NASA Astrophysics Data System (ADS)

    Trouille, Laura; Barger, Amy; Tremonti, Christy

    2014-07-01

    The Baldwin, Phillips, and Terlevich emission-line ratio diagnostic ([OIII]/Hβ versus [NII]/Hα, hereafter BPT diagram) efficiently separates galaxies whose signal is dominated by star formation (BPT-SF) from those dominated by AGN activity (BPT-AGN). Yet the BPT diagram is limited to z<0.5, the redshift at which [NII]λ6584 leaves the optical spectral window. Using the Sloan Digital Sky Survey (SDSS), we construct a new diagnostic, or TBT diagram, that is based on rest-frame g-z color, [NeIII]λ3869, and [OII]λλ3726+3729 and can be used for galaxies out to z<1.4. The TBT diagram identifies 98.7% of the SDSS BPT-AGN as TBT-AGN and 97% of the SDSS BPT-SF as TBT-SF. Furthermore, it identifies 97% of the OPTX Chandra X-ray selected AGNs as TBT-AGN. This is in contrast to the BPT diagram, which misidentifies 20% of X-ray selected AGNs as BPT-SF.

  5. Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein.

    PubMed

    Sugimoto, Yoichiro; Chakrabarti, Anob M; Luscombe, Nicholas M; Ule, Jernej

    2017-03-01

    The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing. Mapping of the sequenced arms to a reference transcriptome identifies the exact locations of duplexes. hiCLIP data can directly identify all types of RNA duplexes bound by RBPs, including those that are challenging to predict computationally, such as intermolecular and long-range intramolecular duplexes. Moreover, the use of an adaptor that links the two arms of the RNA duplex permits hiCLIP to unambiguously identify the duplexes. Here we describe in detail the procedure for a hiCLIP experiment and the subsequent streamlined data analysis with an R package, 'hiclipr' (https://github.com/luslab/hiclipr/). Preparation of the library for high-throughput DNA sequencing takes ∼7 d and the basic bioinformatic pipeline takes 1 d.

  6. Encrypting personal identifiers.

    PubMed Central

    Meux, E

    1994-01-01

    STUDY SETTING. A statewide patient discharge database contained only one unique identifier: the social security number (SSN). A method was developed to transform (encrypt) the SSN so that it could be made publicly available, for purposes of linking discharge records, without revealing the SSN itself. The method of encrypting the SSN into a Record Linkage Number (RLN) is described. PRINCIPAL FINDINGS. The same RLN will always result from the same SSN; it is highly improbable that the same RLN would be produced by two different SSNs; the SSN cannot be derived from the RLN, even given access to the encryption program; the encryption method cannot be determined through knowledge of a number of SSN/RLN combinations; and the method can be described, evaluated, and adapted for use by other researchers without compromising confidentiality of the RLNs resulting from the method. PMID:8005792

  7. List identifies threatened ecosystems

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2012-09-01

    The International Union for Conservation of Nature (IUCN) announced on 9 September that it will develop a new Red List of Ecosystems that will identify which ecosystems are vulnerable or endangered. The list, which is modeled on the group's Red List of Threatened Species™, could help to guide conservation activities and influence policy processes such as the Convention on Biological Diversity, according to the group. “We will assess the status of marine, terrestrial, freshwater, and subterranean ecosystems at local, regional, and global levels,” stated Jon Paul Rodriguez, leader of IUCN's Ecosystems Red List Thematic Group. “The assessment can then form the basis for concerted implementation action so that we can manage them sustainably if their risk of collapse is low or restore them if they are threatened and then monitor their recovery.”

  8. Identifying potential academic leaders

    PubMed Central

    White, David; Krueger, Paul; Meaney, Christopher; Antao, Viola; Kim, Florence; Kwong, Jeffrey C.

    2016-01-01

    Objective To identify variables associated with willingness to undertake leadership roles among academic family medicine faculty. Design Web-based survey. Bivariate and multivariable analyses (logistic regression) were used to identify variables associated with willingness to undertake leadership roles. Setting Department of Family and Community Medicine at the University of Toronto in Ontario. Participants A total of 687 faculty members. Main outcome measures Variables related to respondents’ willingness to take on various academic leadership roles. Results Of all 1029 faculty members invited to participate in the survey, 687 (66.8%) members responded. Of the respondents, 596 (86.8%) indicated their level of willingness to take on various academic leadership roles. Multivariable analysis revealed that the predictors associated with willingness to take on leadership roles were as follows: pursuit of professional development opportunities (odds ratio [OR] 3.79, 95% CI 2.29 to 6.27); currently holding at least 1 leadership role (OR 5.37, 95% CI 3.38 to 8.53); a history of leadership training (OR 1.86, 95% CI 1.25 to 2.78); the perception that mentorship is important for one’s current role (OR 2.25, 95% CI 1.40 to 3.60); and younger age (OR 0.97, 95% CI 0.95 to 0.99). Conclusion Willingness to undertake new or additional leadership roles was associated with 2 variables related to leadership experiences, 2 variables related to perceptions of mentorship and professional development, and 1 demographic variable (younger age). Interventions that support opportunities in these areas might expand the pool and strengthen the academic leadership potential of faculty members. PMID:27331226

  9. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR/sub 2/), also identifies a 72Kd secreted fragment of CR/sub 2/ that contains the C3d-binding site

    SciTech Connect

    Myones, B.L.; Ross, G.D.

    1986-03-05

    CR/sub 2/ is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR/sub 2/, blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR/sub 2/, does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR/sub 2/ because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of /sup 125/I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from /sup 125/I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence /sup 3/H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of /sup 125/I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR/sub 2/ molecule, also identifies a 72Kd shed fragment of CR/sub 2/ isolated from Raji cell media, which contains the C3d-binding site.

  10. Identifying Phronotypes in Psychiatry

    PubMed Central

    Kozel, F. Andrew

    2010-01-01

    Refinements in the methods of diagnosis for psychiatric disorders are critically needed. These new methods should be based on objectively measured brain characteristics that provide clinically useful information. Studying the brain with respect to psychiatric disorders, however, faces numerous challenges. Utilizing techniques learned in other areas of medicine to deal with symptoms that lead to complex disorders can provide insight into improving diagnostic models in psychiatry. Specifically, many areas of medicine use objective measures of an organ's function or characteristic to guide clinical management of particular subjective complaints. In psychiatry, an objectively measured brain characteristic that provides clinically useful information is proposed to be that person's “phronotype.” Important requirements to developing phronotypes are discussed. Identifying phronotypes in psychiatry will require a specific investigative approach that must be grounded in rigorous scientific methodology. Successfully developing such markers will have a profound impact on clinical care, clinical research, basic science research, and most importantly the lives of those suffering from these illnesses. PMID:21423450

  11. Identifying Adolescent Sleep Problems

    PubMed Central

    Short, Michelle A.; Gradisar, Michael; Gill, Jason; Camfferman, Danny

    2013-01-01

    Objectives To examine the efficacy of self-report and parental report of adolescent sleep problems and compare these findings to the incidence of adolescents who fulfill clinical criteria for a sleep problem. Sleep and daytime functioning factors that predict adolescents’ self-identification of a sleep problem will also be examined. Method 308 adolescents (aged 13–17 years) from eight socioeconomically diverse South Australian high schools participated in this study. Participants completed a survey battery during class time, followed by a 7-day Sleep Diary and the Flinders Fatigue Scale completed on the final day of the study. Parents completed a Sleep, Medical, Education and Family History Survey. Results The percentage of adolescents fulfilling one or more of the criteria for a sleep problem was inordinately high at 66%. Adolescent self-reporting a sleep problem was significantly lower than the adolescents who had one or more of the clinical criteria for a sleep problem (23.1% vs. 66.6%; χ2 = 17.46, p<.001). Parental report of their adolescent having a sleep problem was significantly lower than adolescent self-report (14.3% vs. 21.1%, p<.001). Adolescents who reported unrefreshing sleep were 4.81 times more likely to report a sleep problem. For every hour that bedtime was delayed, the odds of self-reporting a sleep problem increased by 1.91 times, while each additional 10 minutes taken to fall asleep increased the odds 1.40 times. Conclusion While many adolescents were found to have sleep patterns indicative of a sleep problem, only a third of this number self-identify having a sleep problem, while only a sixth of this number are indicated by parental report. This study highlights important features to target in future sleep education and intervention strategies for both adolescents and parents. PMID:24086501

  12. Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria

    PubMed Central

    Mantel, Pierre-Yves; Hjelmqvist, Daisy; Walch, Michael; Kharoubi-Hess, Solange; Nilsson, Sandra; Ravel, Deepali; Ribeiro, Marina; Grüring, Christof; Ma, Siyuan; Padmanabhan, Prasad; Trachtenberg, Alexander; Ankarklev, Johan; Brancucci, Nicolas M.; Huttenhower, Curtis; Duraisingh, Manoj T.; Ghiran, Ionita; Kuo, Winston P.; Filgueira, Luis; Martinelli, Roberta; Marti, Matthias

    2016-01-01

    Malaria remains one of the greatest public health challenges worldwide, particularly in sub-Saharan Africa. The clinical outcome of individuals infected with Plasmodium falciparum parasites depends on many factors including host systemic inflammatory responses, parasite sequestration in tissues and vascular dysfunction. Production of pro-inflammatory cytokines and chemokines promotes endothelial activation as well as recruitment and infiltration of inflammatory cells, which in turn triggers further endothelial cell activation and parasite sequestration. Inflammatory responses are triggered in part by bioactive parasite products such as hemozoin and infected red blood cell-derived extracellular vesicles (iRBC-derived EVs). Here we demonstrate that such EVs contain functional miRNA-Argonaute 2 complexes that are derived from the host RBC. Moreover, we show that EVs are efficiently internalized by endothelial cells, where the miRNA-Argonaute 2 complexes modulate target gene expression and barrier properties. Altogether, these findings provide a mechanistic link between EVs and vascular dysfunction during malaria infection. PMID:27721445

  13. Blood pressure loci identified with a gene-centric array.

    PubMed

    Johnson, Toby; Gaunt, Tom R; Newhouse, Stephen J; Padmanabhan, Sandosh; Tomaszewski, Maciej; Kumari, Meena; Morris, Richard W; Tzoulaki, Ioanna; O'Brien, Eoin T; Poulter, Neil R; Sever, Peter; Shields, Denis C; Thom, Simon; Wannamethee, Sasiwarang G; Whincup, Peter H; Brown, Morris J; Connell, John M; Dobson, Richard J; Howard, Philip J; Mein, Charles A; Onipinla, Abiodun; Shaw-Hawkins, Sue; Zhang, Yun; Davey Smith, George; Day, Ian N M; Lawlor, Debbie A; Goodall, Alison H; Fowkes, F Gerald; Abecasis, Gonçalo R; Elliott, Paul; Gateva, Vesela; Braund, Peter S; Burton, Paul R; Nelson, Christopher P; Tobin, Martin D; van der Harst, Pim; Glorioso, Nicola; Neuvrith, Hani; Salvi, Erika; Staessen, Jan A; Stucchi, Andrea; Devos, Nabila; Jeunemaitre, Xavier; Plouin, Pierre-François; Tichet, Jean; Juhanson, Peeter; Org, Elin; Putku, Margus; Sõber, Siim; Veldre, Gudrun; Viigimaa, Margus; Levinsson, Anna; Rosengren, Annika; Thelle, Dag S; Hastie, Claire E; Hedner, Thomas; Lee, Wai K; Melander, Olle; Wahlstrand, Björn; Hardy, Rebecca; Wong, Andrew; Cooper, Jackie A; Palmen, Jutta; Chen, Li; Stewart, Alexandre F R; Wells, George A; Westra, Harm-Jan; Wolfs, Marcel G M; Clarke, Robert; Franzosi, Maria Grazia; Goel, Anuj; Hamsten, Anders; Lathrop, Mark; Peden, John F; Seedorf, Udo; Watkins, Hugh; Ouwehand, Willem H; Sambrook, Jennifer; Stephens, Jonathan; Casas, Juan-Pablo; Drenos, Fotios; Holmes, Michael V; Kivimaki, Mika; Shah, Sonia; Shah, Tina; Talmud, Philippa J; Whittaker, John; Wallace, Chris; Delles, Christian; Laan, Maris; Kuh, Diana; Humphries, Steve E; Nyberg, Fredrik; Cusi, Daniele; Roberts, Robert; Newton-Cheh, Christopher; Franke, Lude; Stanton, Alice V; Dominiczak, Anna F; Farrall, Martin; Hingorani, Aroon D; Samani, Nilesh J; Caulfield, Mark J; Munroe, Patricia B

    2011-12-09

    Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 × 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery and follow-up data identified SNPs significantly associated with BP at p < 8.56 × 10(-7) at four further loci (NPR3, HFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.

  14. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.

    PubMed

    Wang, Liangyan; Lu, Huizhi; Wang, Yunguang; Yang, Su; Xu, Hong; Cheng, Kaiying; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2017-03-01

    Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

  15. Machine Learning Helps Identify CHRONO as a Circadian Clock Component

    PubMed Central

    Venkataraman, Anand; Ramanathan, Chidambaram; Kavakli, Ibrahim H.; Hughes, Michael E.; Baggs, Julie E.; Growe, Jacqueline; Liu, Andrew C.; Kim, Junhyong; Hogenesch, John B.

    2014-01-01

    Over the last decades, researchers have characterized a set of “clock genes” that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics. PMID:24737000

  16. Autoantibodies to Argonaute 2 (Su antigen).

    PubMed

    Satoh, Minoru; Chan, Jason Y F; Ceribelli, Angela; Vazquez del-Mercado, Monica; Chan, Edward K L

    2013-01-01

    Like many other classical autoantibodies in systemic rheumatic diseases, anti-Su antibodies were originally defined by the double immunodiffusion assay in the early 80s. However, despite its high prevalence, only a few reports on anti-Su were published in the following years and the progress in characterizing the target antigens and clinical significance was slow, probably due to its inconsistent or poor reactivity in other standard immunoassays. In 2006 the target antigen was identified as the microRNA (miRNA)-binding protein Argonaute 2 (Ago2). Ago2 is a key component of the RNA-induced silencing complex enriched in cytoplasmic foci called GW bodies. Due to preferential reactivity of human autoantibodies with native antigens, immunoprecipitation is the only method to reliably detect anti-Su/Ago2 antibodies. Anti-Su/Ago2 does not appear to have disease specificity since it is found in 10-20% of patients with various rheumatic diseases, including systemic lupus erythematosus, scleroderma, polymyositis/dermatomyositis, and Sjögren's syndrome, as well as apparently healthy individuals at lower prevalence. The clinical significance and the mechanism of production of anti-Su/Ago2 remains to be clarified.

  17. Quantitative BrdU immunoprecipitation method demonstrates that Fkh1 and Fkh2 are rate-limiting activators of replication origins that reprogram replication timing in G1 phase.

    PubMed

    Peace, Jared M; Villwock, Sandra K; Zeytounian, John L; Gan, Yan; Aparicio, Oscar M

    2016-03-01

    The Saccharomyces cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. We hypothesized that, as stimulators of early origin activation, Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, are not well-suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advances the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase.

  18. Identifying combinatorial regulation of transcription factors and binding motifs

    PubMed Central

    Kato, Mamoru; Hata, Naoya; Banerjee, Nilanjana; Futcher, Bruce; Zhang, Michael Q

    2004-01-01

    Background Combinatorial interaction of transcription factors (TFs) is important for gene regulation. Although various genomic datasets are relevant to this issue, each dataset provides relatively weak evidence on its own. Developing methods that can integrate different sequence, expression and localization data have become important. Results Here we use a novel method that integrates chromatin immunoprecipitation (ChIP) data with microarray expression data and with combinatorial TF-motif analysis. We systematically identify combinations of transcription factors and of motifs. The various combinations of TFs involved multiple binding mechanisms. We reconstruct a new combinatorial regulatory map of the yeast cell cycle in which cell-cycle regulation can be drawn as a chain of extended TF modules. We find that the pairwise combination of a TF for an early cell-cycle phase and a TF for a later phase is often used to control gene expression at intermediate times. Thus the number of distinct times of gene expression is greater than the number of transcription factors. We also see that some TF modules control branch points (cell-cycle entry and exit), and in the presence of appropriate signals they can allow progress along alternative pathways. Conclusions Combining different data sources can increase statistical power as demonstrated by detecting TF interactions and composite TF-binding motifs. The original picture of a chain of simple cell-cycle regulators can be extended to a chain of composite regulatory modules: different modules may share a common TF component in the same pathway or a TF component cross-talking to other pathways. PMID:15287978

  19. Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

    PubMed Central

    Heimer, Susan R.; Mobley, Harry L. T.

    2001-01-01

    Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins. PMID:11157956

  20. Stochastic control system parameter identifiability

    NASA Technical Reports Server (NTRS)

    Lee, C. H.; Herget, C. J.

    1975-01-01

    The parameter identification problem of general discrete time, nonlinear, multiple input/multiple output dynamic systems with Gaussian white distributed measurement errors is considered. The knowledge of the system parameterization was assumed to be known. Concepts of local parameter identifiability and local constrained maximum likelihood parameter identifiability were established. A set of sufficient conditions for the existence of a region of parameter identifiability was derived. A computation procedure employing interval arithmetic was provided for finding the regions of parameter identifiability. If the vector of the true parameters is locally constrained maximum likelihood (CML) identifiable, then with probability one, the vector of true parameters is a unique maximal point of the maximum likelihood function in the region of parameter identifiability and the constrained maximum likelihood estimation sequence will converge to the vector of true parameters.

  1. Mass Spectrometry to Identify New Biomarkers of Nerve Agent Exposure

    DTIC Science & Technology

    2010-04-01

    channels , receptors, and scaffoldingproteins to and from synaptic sites. Synapses are highly vulnerable to impair- ments in transport. Perturbations to...Petrucelli, 2009). Microtubules serve as tracks for transport of mito- chondria, synaptic vesicles, components of ion channels , receptors, and scaffolding...Y.K., 2005. Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal

  2. Identifying the Emergency Management Profession.

    ERIC Educational Resources Information Center

    Chiesa, Adele M.

    1987-01-01

    In building an emergency management library collection within a training institution, technical data become secondary to identifying common goals, methods, and systems found at the federal, state, and local levels for responding to and planning for disasters and crises. These goals help identify emergency management skills public officials should…

  3. Towards an immuno-precipitated neurodevelopmental animal model of schizophrenia.

    PubMed

    Meyer, Urs; Feldon, Joram; Schedlowski, Manfred; Yee, Benjamin K

    2005-01-01

    Epidemiological studies have indicated an association between maternal bacterial and viral infections during pregnancy and the higher incidence of schizophrenia in the resultant offspring post-puberty. One hypothesis asserts that the reported epidemiological link is mediated by prenatal activation of the foetal immune system in response to the elevation of maternal cytokine level due to infection. Here, we report that pregnant mouse dams receiving a single exposure to the cytokine-releasing agent, polyriboinosinic-polyribocytidilic acid (PolyI:C; at 2.5, 5.0, or 10.0 mg/kg) on gestation day 9 produced offspring that subsequently exhibited multiple schizophrenia-related behavioural deficits in adulthood, in comparison to offspring from vehicle injected or non-injected control dams. The efficacy of the PolyI:C challenge to induce cytokine responses in naïve non-pregnant adult female mice and in foetal brain tissue when injected to pregnant mice were further ascertained in separate subjects: (i) a dose-dependent elevation of interleukin-10 was detected in the adult female mice at 1 and 6h post-injection, (ii) 12 h following prenatal PolyI:C challenge, the foetal levels of interleukin-1beta were elevated. The spectrum of abnormalities included impairments in exploratory behaviour, prepulse inhibition, latent inhibition, the US-pre-exposure effect, spatial working memory; and enhancement in the locomotor response to systemic amphetamine (2.5 mg/kg, i.p.) as well as in discrimination reversal learning. The neuropsychological parallels between prenatal PolyI:C treatment in mice and psychosis in humans, demonstrated here, leads us to conclude that prenatal PolyI:C treatment represents one of the most powerful environmental-developmental models of schizophrenia to date. The uniqueness of this model lies in its epidemiological and immunological relevance. It is, sui generis, ideally suited for the investigation of the neuropsychoimmunological mechanisms implicated in the developmental aetiology and disease processes of schizophrenia.

  4. Identifying Context Variables in Research.

    ERIC Educational Resources Information Center

    Piazza, Carolyn L.

    1987-01-01

    Identifies context variables in written composition from theoretical perspectives in cognitive psychology, sociology, and anthropology. Considers how multiple views of context from across the disciplines can build toward a broader definition of writing. (JD)

  5. Identifying Occupationally Specific Affective Behaviors.

    ERIC Educational Resources Information Center

    Pucel, David J.

    1993-01-01

    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  6. Identifying Context Variables in Research.

    ERIC Educational Resources Information Center

    Piazza, Carolyn L.

    1987-01-01

    Identifies context variables in written composition from theoretical perspectives in cognitive psychology, sociology, and anthropology. Considers how multiple views of context from across the disciplines can build toward a broader definition of writing. (JD)

  7. Methods for identifying translational researchers.

    PubMed

    Feeney, Mary K; Johnson, Timothy; Welch, Eric W

    2014-03-01

    There is currently no generally accepted method for identifying the community of translational researchers when evaluating Clinical and Translational Science Centers. We use data from the multiyear evaluation of the University of Illinois at Chicago Center for Clinical and Translational Science (CCTS) to investigate the complexities of reliably identifying translational researchers. We use three methods to identify translational researchers: (1) participating in CCTS services and programs; (2) self-identifying as a translational researcher; and (3) engaging in activities that are characteristic of translational science. We find little overlap of these differently defined research groups. We conclude with a discussion of how the findings suggest challenges for evaluating translational science programs and the need for better definition, communication, and demonstration of translational science for scientists and evaluators.

  8. Identifying Occupationally Specific Affective Behaviors.

    ERIC Educational Resources Information Center

    Pucel, David J.

    1993-01-01

    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  9. Identifying Clients Predisposed To Failure

    ERIC Educational Resources Information Center

    Carnes, G. D.

    1973-01-01

    Studies are reviewed that report the prediction of rehabilitation failure from personality measures. Related research is discussed that suggest the dynamics underlying a key concept, the "hypochondriacally organized personality" which is identifiable from the Rorschach anatomy response percentage. (Author)

  10. Identifying discharge practice training needs.

    PubMed

    Lees, L; Emmerson, K

    A training needs analysis tool was developed to identify nurses' discharge training needs and to improve discharge practice. The tool includes 49 elements of discharge practice subdivided into four areas: corporate, operational, clinical and nurse-led discharge. The tool was disseminated to 15 wards on two hospital sites with assistance from the practice development team. Analysis of discharge training is important to assess discharge training needs and to identify staff who may assist with training.

  11. Genome-wide analysis of histone modifications by ChIP-chip to identify silenced genes in gastric cancer.

    PubMed

    Zhu, Xinjiang; Liu, Jian; Xu, Xiaoyang; Zhang, Chundong; Dai, Dongqiu

    2015-05-01

    The present study aimed to identify novel histone modification markers in gastric cancer (GC) by chromatin immunoprecipitation microarray (ChIP-chip) analysis and to determine whether these markers were able to discriminate between normal and GC cells. We also tested for correlations with DNA methylation. We probed a human CpG island microarray with DNA from a GC cell line (MKN45) by chromatin immunoprecipitation (ChIP). ChIP-reverse-transcriptase quantitative polymerase chain reaction PCR (RT-qPCR) was used to validate the microarray results. Additionally, mRNA expression levels and the DNA methylation of potential target genes were evaluated by RT-qPCR and methylation-specific PCR (MSP). The moults showed that 134 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over acetylation and 46 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over H3-K4 trimethylation in MKN45 cells. The ChIP-qPCR results agreed with those obtained from the ChIP-chip analysis. Aberrant DNA methylation status and mRNA expression levels were also identified for selected genes (PSD, SMARCC1 and Vps37A) in the GC cell lines. The results suggest that CpG island microarray coupled with ChIP (ChIP-chip) can identify novel targets of gene silencing in GC. Additionally, ChIP-chip is the best approach for assessing the genome-wide status of epigenetic regulation, which may allow for a broader genomic understanding compared to the knowledge that has been accumulated from single-gene studies.

  12. Individual Identifiability Predicts Population Identifiability in Forensic Microsatellite Markers.

    PubMed

    Algee-Hewitt, Bridget F B; Edge, Michael D; Kim, Jaehee; Li, Jun Z; Rosenberg, Noah A

    2016-04-04

    Highly polymorphic genetic markers with significant potential for distinguishing individual identity are used as a standard tool in forensic testing [1, 2]. At the same time, population-genetic studies have suggested that genetically diverse markers with high individual identifiability also confer information about genetic ancestry [3-6]. The dual influence of polymorphism levels on ancestry inference and forensic desirability suggests that forensically useful marker sets with high levels of individual identifiability might also possess substantial ancestry information. We study a standard forensic marker set-the 13 CODIS loci used in the United States and elsewhere [2, 7-9]-together with 779 additional microsatellites [10], using direct population structure inference to test whether markers with substantial individual identifiability also produce considerable information about ancestry. Despite having been selected for individual identification and not for ancestry inference [11], the CODIS markers generate nontrivial model-based clustering patterns similar to those of other sets of 13 tetranucleotide microsatellites. Although the CODIS markers have relatively low values of the F(ST) divergence statistic, their high heterozygosities produce greater ancestry inference potential than is possessed by less heterozygous marker sets. More generally, we observe that marker sets with greater individual identifiability also tend toward greater population identifiability. We conclude that population identifiability regularly follows as a byproduct of the use of highly polymorphic forensic markers. Our findings have implications for the design of new forensic marker sets and for evaluations of the extent to which individual characteristics beyond identification might be predicted from current and future forensic data.

  13. Identifying tier one key suppliers.

    PubMed

    Wicks, Steve

    2013-01-01

    In today's global marketplace, businesses are becoming increasingly reliant on suppliers for the provision of key processes, activities, products and services in support of their strategic business goals. The result is that now, more than ever, the failure of a key supplier has potential to damage reputation, productivity, compliance and financial performance seriously. Yet despite this, there is no recognised standard or guidance for identifying a tier one key supplier base and, up to now, there has been little or no research on how to do so effectively. This paper outlines the key findings of a BCI-sponsored research project to investigate good practice in identifying tier one key suppliers, and suggests a scalable framework process model and risk matrix tool to help businesses effectively identify their tier one key supplier base.

  14. Persistent Identifiers Implementation in EOSDIS

    NASA Technical Reports Server (NTRS)

    Ramapriyan, H. K. " Rama"

    2016-01-01

    This presentation provides the motivation for and status of implementation of persistent identifiers in NASA's Earth Observation System Data and Information System (EOSDIS). The motivation is provided from the point of view of long-term preservation of datasets such that a number of questions raised by current and future users can be answered easily and precisely. A number of artifacts need to be preserved along with datasets to make this possible, especially when the authors of datasets are no longer available to address users questions. The artifacts and datasets need to be uniquely and persistently identified and linked with each other for full traceability, understandability and scientific reproducibility. Current work in the Earth Science Data and Information System (ESDIS) Project and the Distributed Active Archive Centers (DAACs) in assigning Digital Object Identifiers (DOI) is discussed as well as challenges that remain to be addressed in the future.

  15. [Advances in recently identified coronaviruses].

    PubMed

    Geng, He-Yuan; Tan, Wen-Jie

    2013-01-01

    Coronaviruses are a large family of viruses which include viruses that cause the common cold and severe acute respiratory syndrome (SARS) in humans and other diseases in animals. There are considerable genetic diversities within coronaviruses due to their wide rang hosts and their special gene replication and transcription mechanisms. During this process, gene recombinations often occur, resulting in novel subtype or coronavirus emerge constantly. Of note are SARS-like-CoVs and novel HCoV-EMC identified in 2012. This minireview summarized major advances of recently identified coronaviruses, focusing on the genome structures and interspecies jumping mechanism of coronavirus.

  16. Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

    SciTech Connect

    Fardini, Yann; Perez-Cervera, Yobana; Camoin, Luc; Pagesy, Patrick; Lefebvre, Tony; Issad, Tarik

    2015-06-26

    O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified. - Highlights: • We mutate four previously identified O-GlcNAcylation sites on Foxo1. • Unexpectedly, these mutations do not reduce Foxo1 O-GlcNAcylation. • These mutation do not reduce Foxo1 transcriptional activity. • We identify a new O-GlcNAcylation site on Foxo1 by mass spectrometry. • Mutation of this site increases Foxo1 transcriptional activity.

  17. Testing times: identifying puberty in an identified skeletal sample.

    PubMed

    Henderson, Charlotte Y; Padez, Cristina

    2017-06-01

    Identifying the onset of puberty in skeletal remains can provide evidence of social changes associated with the onset of adulthood. This paper presents the first test of a skeletal method for identifying stages of development associated with the onset of puberty in a skeletal sample of known age and cause of death. Skeletal methods for assessing skeletal development associated with changes associated with puberty were recorded in the identified skeletal collection in Coimbra, Portugal. Historical data on the onset of menarche in this country are used to test the method. As expected, females mature faster than their male counterparts. There is some side asymmetry in development. Menarche was found to have been achieved by an average age of 15. Asymmetry must be taken into account when dealing with partially preserved skeletons. Age of menarche is consistent, although marginally higher, than the age expected based on historical data for this time and location. Skeletal development in males could not be tested against historical data, due to the lack of counterpart historical data. The ill health known to be present in this prematurely deceased population may have delayed skeletal development and the onset of puberty.

  18. Identifying Pilots with Parkinson's Disease.

    PubMed

    Clem, Peter A; Navathe, Pooshan D; Drane, Michael A

    2016-06-01

    In 2012 the Australian Institute of Health and Welfare produced a report titled 'Dementia in Australia.'(2) The report noted that the number of people with dementia in Australia would reach almost 400,000 by 2020. Australia is a jurisdiction which does not impose a mandatory retirement age for pilots. With an aging population it was hypothesized that conditions such as Parkinson's disease (PD) were likely to be seen more commonly by the Civil Aviation Safety Authority (CASA). It was decided that this was an appropriate time to retrospectively study the data held by CASA. An interrogation of CASA databases was undertaken. Data was produced comparing percentage of Class 1 certificate holders over 60 yr of age against time. A cohort of pilots and controllers with PD was identified. The history of the cases was reviewed. The study confirms that the pilot population is aging in line with population trends. Over a period from 1992 to 2012, 22 cases of pilots and controllers with PD were identified. The study confirmed that PD will be of increased relevance over the next decade. Gaps between policy and practice managing past cases were identified. Updated guidelines have been published aiming to address the deficiencies identified in the study. Historically pilots and controllers have been able to maintain certification for an average of 3.75 yr. This information should be of benefit to clinicians, pilots, and controllers when considering occupation and treatment options.

  19. "Geriatricizing" Hospitalists: Identifying Educational Opportunities

    ERIC Educational Resources Information Center

    Friedman, Susan M.; Gillespie, Suzanne M.; Medina-Walpole, Annette M.; Caprio, Thomas V.; Karuza, Jurgis; McCann, Robert M.

    2013-01-01

    The objective of this study was to identify differences between geriatricians and hospitalists in caring for hospitalized older adults, so as to inform faculty development programs that have the goal of improving older patient care. Eleven hospitalists and 13 geriatricians were surveyed regarding knowledge, confidence, and practice patterns in…

  20. SOCIODEMOGRAPHIC DATA USED FOR IDENTIFYING ...

    EPA Pesticide Factsheets

    Due to unique social and demographic characteristics, various segments of the population may experience exposures different from those of the general population, which, in many cases, may be greater. When risk assessments do not characterize subsets of the general population, the populations that may experience the greatest risk remain unidentified. When such populations are not identified, the social and demographic data relevant to these populations is not considered when preparing exposure estimates, which can underestimate exposure and risk estimates for at-risk populations. Thus, it is necessary for risk or exposure assessors characterizing a diverse population, to first identify and then enumerate certain groups within the general population who are at risk for greater contaminant exposures. The document entitled Sociodemographic Data Used for Identifying Potentially Highly Exposed Populations (also referred to as the Highly Exposed Populations document), assists assessors in identifying and enumerating potentially highly exposed populations. This document presents data relating to factors which potentially impact an individual or group's exposure to environmental contaminants based on activity patterns (how time is spent), microenvironments (locations where time is spent), and other socio-demographic data such as age, gender, race and economic status. Populations potentially more exposed to various chemicals of concern, relative to the general population

  1. "Geriatricizing" Hospitalists: Identifying Educational Opportunities

    ERIC Educational Resources Information Center

    Friedman, Susan M.; Gillespie, Suzanne M.; Medina-Walpole, Annette M.; Caprio, Thomas V.; Karuza, Jurgis; McCann, Robert M.

    2013-01-01

    The objective of this study was to identify differences between geriatricians and hospitalists in caring for hospitalized older adults, so as to inform faculty development programs that have the goal of improving older patient care. Eleven hospitalists and 13 geriatricians were surveyed regarding knowledge, confidence, and practice patterns in…

  2. Identifying the Gifted Child Humorist.

    ERIC Educational Resources Information Center

    Fern, Tami L.

    1991-01-01

    This study attempted to identify gifted child humorists among 1,204 children in grades 3-6. Final identification of 13 gifted child humorists was determined through application of such criteria as funniness, originality, and exemplary performance or product. The influence of intelligence, development, social factors, sex differences, family…

  3. Identifying Innovative Agricultural Education Programs

    ERIC Educational Resources Information Center

    Rayfield, John; Murphy, Tim; Briers, Gary; Lewis, Lauren

    2012-01-01

    Researchers identified innovative agricultural education programs across the United States. A Delphi study was conducted with the teachers in innovative programs. According to the teachers, innovative programs in 2020 will use hands-on activities and will be run by highly motivated teachers. The purpose of innovative programs in the future will be…

  4. Identifiability, exchangeability and confounding revisited

    PubMed Central

    Greenland, Sander; Robins, James M

    2009-01-01

    In 1986 the International Journal of Epidemiology published "Identifiability, Exchangeability and Epidemiological Confounding". We review the article from the perspective of a quarter century after it was first drafted and relate it to subsequent developments on confounding, ignorability, and collapsibility. PMID:19732410

  5. Methods of Identifying Marketing Competencies

    ERIC Educational Resources Information Center

    Crawford, Lucy C.; Ertel, Kenneth A.

    1970-01-01

    The competency pattern and the task analysis are two approaches used to identify content that affects the distributive education curriculum. These and other research tools provide essential information about capabilities required for entrance into and persistence in careers at prescribed levels of distributive jobs. (Author)

  6. Identifying predictors of treatment response.

    PubMed

    Yoder, Paul; Compton, Don

    2004-01-01

    This article provides a rationale for considering predictors of growth in a treatment group as inadequate to identifying predictors of treatment response. When we interpret predictors of growth in a treatment group as synonymous with predictors of treatment response, we implicitly attribute all of the treated children's growth to the treatment, an untenable assumption under most conditions. We also contend that the use of standard scores in predictors of growth studies does not allow us to differentiate growth from treatment, from growth from other factors. We present two research methodologies that are appropriate methods of identifying predictors of treatment response: (a) single-subject experimental logic utilized to identify the specific participants in which treatment responses (not just growth) were found, combined with follow-up group comparison logic to identify the characteristics on which responders and nonresponders differ, and (b) statistical interactions among child/family/context characteristics and randomly assigned group membership. Principles for selecting potential predictors of treatment response are provided.

  7. Identified Sins in Teaching Reading.

    ERIC Educational Resources Information Center

    Ediger, Marlow

    This paper cites as selected taboos in teaching reading (identified by educators very frequently) the following: (1) homogeneous grouping; (2) round robin reading; (3) use of textbooks and workbooks in the curriculum; (4) individual endeavors in school work; (5) memorization of content; and (6) the controlled vocabulary in reading. The paper…

  8. Metadata, Identifiers, and Physical Samples

    NASA Astrophysics Data System (ADS)

    Arctur, D. K.; Lenhardt, W. C.; Hills, D. J.; Jenkyns, R.; Stroker, K. J.; Todd, N. S.; Dassie, E. P.; Bowring, J. F.

    2016-12-01

    Physical samples are integral to much of the research conducted by geoscientists. The samples used in this research are often obtained at significant cost and represent an important investment for future research. However, making information about samples - whether considered data or metadata - available for researchers to enable discovery is difficult: a number of key elements related to samples are difficult to characterize in common ways, such as classification, location, sample type, sampling method, repository information, subsample distribution, and instrumentation, because these differ from one domain to the next. Unifying these elements or developing metadata crosswalks is needed. The iSamples (Internet of Samples) NSF-funded Research Coordination Network (RCN) is investigating ways to develop these types of interoperability and crosswalks. Within the iSamples RCN, one of its working groups, WG1, has focused on the metadata related to physical samples. This includes identifying existing metadata standards and systems, and how they might interoperate with the International Geo Sample Number (IGSN) schema (schema.igsn.org) in order to help inform leading practices for metadata. For example, we are examining lifecycle metadata beyond the IGSN `birth certificate.' As a first step, this working group is developing a list of relevant standards and comparing their various attributes. In addition, the working group is looking toward technical solutions to facilitate developing a linked set of registries to build the web of samples. Finally, the group is also developing a comparison of sample identifiers and locators. This paper will provide an overview and comparison of the standards identified thus far, as well as an update on the technical solutions examined for integration. We will discuss how various sample identifiers might work in complementary fashion with the IGSN to more completely describe samples, facilitate retrieval of contextual information, and

  9. Identifying the potential rural optometrist.

    PubMed

    Kegel-Flom, P

    1976-09-01

    Rural optometrists were found to differ from urban optometrists in background, environmental attitude, and interest patterns. Attitude toward the urban environment and place of origin were the best predictors of an optometrist's practice location. When "urbanism" and "origin" were scaled and placed in a multiple regression equation to predict practice location, identification of an optometrist's location as rural or urban was highly accurate. Most importantly, scores on the equation were predictive of optometry students' future practice locations. A single cut-off point on the equation correctly identified 79% of students who entered rural or isolated small city practice and 81% of those who entered urban practice. The findings suggest that optometry students most likely to enter rural (or indeed urban) practice can be objectively identified early in, or even prior to, training. Such identification may assist educators in selecting and training optometrists who will deliver vision care to people in areas of greatest need.

  10. Identifying and managing problem drinkers.

    PubMed Central

    Kahan, M.

    1996-01-01

    Problem drinking is far more common than severe alcohol dependence and is associated with considerable morbidity and health care costs. Whereas patients with alcohol dependence respond best to intensive treatment, one or more brief sessions of physician advice and counseling reduces alcohol consumption among problem drinkers. The two most useful tools to identify problem drinkers are the CAGE and the drinking problem question. PMID:8653034

  11. Identifying Targets from Filtering Effects

    DTIC Science & Technology

    2012-10-24

    Introduction Considering a radar (or sonar) target as more than a simple point scatterer brings up the possibility of identifying the target based on the...2000. [8] M. Vespe, C. J. Baker, and H. D. Griffiths , "Automatic target regognition using multi-diversity radar," Radar, Sonar \\& Navigation, IET, vol...A. Taflove and S. C. Hagness, Computational Electrodynamics : The Finite Difference Time Domain Method. Norwood, MA: Artech House, 2005.

  12. A Genome-Wide Regulatory Framework Identifies Maize Pericarp Color1 Controlled Genes[C][W

    PubMed Central

    Morohashi, Kengo; Casas, María Isabel; Ferreyra, Lorena Falcone; Mejía-Guerra, María Katherine; Pourcel, Lucille; Yilmaz, Alper; Feller, Antje; Carvalho, Bruna; Emiliani, Julia; Rodriguez, Eduardo; Pellegrinet, Silvina; McMullen, Michael; Casati, Paula; Grotewold, Erich

    2012-01-01

    Pericarp Color1 (P1) encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize (Zea mays) silks and red phlobaphene pigments in pericarps and other floral tissues, which makes P1 an important visual marker. Using genome-wide expression analyses (RNA sequencing) in pericarps and silks of plants with contrasting P1 alleles combined with chromatin immunoprecipitation coupled with high-throughput sequencing, we show here that the regulatory functions of P1 are much broader than the activation of genes corresponding to enzymes in a branch of flavonoid biosynthesis. P1 modulates the expression of several thousand genes, and ∼1500 of them were identified as putative direct targets of P1. Among them, we identified F2H1, corresponding to a P450 enzyme that converts naringenin into 2-hydroxynaringenin, a key branch point in the P1-controlled pathway and the first step in the formation of insecticidal C-glycosyl flavones. Unexpectedly, the binding of P1 to gene regulatory regions can result in both gene activation and repression. Our results indicate that P1 is the major regulator for a set of genes involved in flavonoid biosynthesis and a minor modulator of the expression of a much larger gene set that includes genes involved in primary metabolism and production of other specialized compounds. PMID:22822204

  13. Molecular profiling of activated olfactory neurons identifies odorant receptors for odors in vivo

    PubMed Central

    Jiang, Yue; Gong, Naihua Natalie; Hu, Xiaoyang Serene; Ni, Mengjue Jessica; Pasi, Radhika

    2015-01-01

    The mammalian olfactory system uses a large family of odorant receptors to detect and discriminate amongst a myriad of volatile odor molecules. Understanding odor coding requires comprehensive mapping between odorant receptors and corresponding odors. Here we present high–throughput in vivo identification of odorant receptor repertoires responding to odorants, using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor–stimulated mice followed by RNA–Seq. This approach screens the endogenously expressed odorant receptors against an odor in one set of experiments, using awake and freely behaving mice. In combination with validations in a heterologous system, we identify sets of odorant receptors for two odorants, acetophenone and 2,5–dihydro–2,4,5–trimethylthiazoline (TMT), encompassing 69 odorant receptor–odorant pairs. We also identified shared amino acid residues specific to the acetophenone or TMT receptors, and developed models to predict receptor activation by acetophenone. This study provides a means to understand the combinatorial coding of odors in vivo. PMID:26322927

  14. The coilin interactome identifies hundreds of small noncoding RNAs that traffic through Cajal bodies.

    PubMed

    Machyna, Martin; Kehr, Stephanie; Straube, Korinna; Kappei, Dennis; Buchholz, Frank; Butter, Falk; Ule, Jernej; Hertel, Jana; Stadler, Peter F; Neugebauer, Karla M

    2014-11-06

    Coilin protein scaffolds Cajal bodies (CBs)-subnuclear compartments enriched in small nuclear RNAs (snRNAs)-and promotes efficient spliceosomal snRNP assembly. The molecular function of coilin, which is intrinsically disordered with no defined motifs, is poorly understood. We use UV crosslinking and immunoprecipitation (iCLIP) to determine whether mammalian coilin binds RNA in vivo and to identify targets. Robust detection of snRNA transcripts correlated with coilin ChIP-seq peaks on snRNA genes, indicating that coilin binding to nascent snRNAs is a site-specific CB nucleator. Surprisingly, several hundred small nucleolar RNAs (snoRNAs) were identified as coilin interactors, including numerous unannotated mouse and human snoRNAs. We show that all classes of snoRNAs concentrate in CBs. Moreover, snoRNAs lacking specific CB retention signals traffic through CBs en route to nucleoli, consistent with the role of CBs in small RNP assembly. Thus, coilin couples snRNA and snoRNA biogenesis, making CBs the cellular hub of small ncRNA metabolism.

  15. Identifying Network Perturbation in Cancer

    PubMed Central

    Logsdon, Benjamin A.; Gentles, Andrew J.; Lee, Su-In

    2016-01-01

    We present a computational framework, called DISCERN (DIfferential SparsE Regulatory Network), to identify informative topological changes in gene-regulator dependence networks inferred on the basis of mRNA expression datasets within distinct biological states. DISCERN takes two expression datasets as input: an expression dataset of diseased tissues from patients with a disease of interest and another expression dataset from matching normal tissues. DISCERN estimates the extent to which each gene is perturbed—having distinct regulator connectivity in the inferred gene-regulator dependencies between the disease and normal conditions. This approach has distinct advantages over existing methods. First, DISCERN infers conditional dependencies between candidate regulators and genes, where conditional dependence relationships discriminate the evidence for direct interactions from indirect interactions more precisely than pairwise correlation. Second, DISCERN uses a new likelihood-based scoring function to alleviate concerns about accuracy of the specific edges inferred in a particular network. DISCERN identifies perturbed genes more accurately in synthetic data than existing methods to identify perturbed genes between distinct states. In expression datasets from patients with acute myeloid leukemia (AML), breast cancer and lung cancer, genes with high DISCERN scores in each cancer are enriched for known tumor drivers, genes associated with the biological processes known to be important in the disease, and genes associated with patient prognosis, in the respective cancer. Finally, we show that DISCERN can uncover potential mechanisms underlying network perturbation by explaining observed epigenomic activity patterns in cancer and normal tissue types more accurately than alternative methods, based on the available epigenomic data from the ENCODE project. PMID:27145341

  16. Identifying crash patterns on roundabouts.

    PubMed

    Polders, Evelien; Daniels, Stijn; Casters, Winfried; Brijs, Tom

    2015-01-01

    Roundabouts are a type of circular intersection control generally associated with a favorable influence on traffic safety. International studies of intersections converted to roundabouts indicate a strong reduction in injury crashes, particularly for crashes with fatal or serious injuries. Nevertheless, some crashes still occur at roundabouts. The present study aims to improve the understanding of roundabout safety by identifying crash types, locations, and factors that are associated with roundabout crashes. An analysis of 399 crashes on 28 roundabouts in Flanders, Belgium, was carried out based on detailed crash descriptions; that is, crash data and collision diagrams. The crashes are sampled from police-reported crashes at roundabouts in the region of Flanders, Belgium. Collision diagrams of the registered crashes were used to distinguish 8 different crash types. The roundabout itself is divided into 11 detailed and different typical segments, according to previously established knowledge on the occurrence of crashes at roundabouts. The 8 roundabout crash types are examined by injury severity, crash location within the roundabout, type of roundabout, type of cycle facility, and type of involved road user. Four dominant crash types are identified: rear-end crashes, collisions with vulnerable road users, entering-circulating crashes, and single-vehicle collisions with the central island. Crashes with vulnerable road users and collisions with the central island are characterized by significantly higher proportions of injury crashes. About 80% of the crashes occurred on the entry lanes and the circulatory road (segments 1-4). Road users who are the most at risk to be involved in serious injury crashes at roundabouts are cyclists and moped riders. The main goal of this study was to identify and analyze dominant crash types at roundabouts by taking into account detailed information on the crash location. Some connections between certain roundabout crash types, their

  17. Identifying the health conscious consumer.

    PubMed

    Kraft, F B; Goodell, P W

    1993-01-01

    Individuals who lead a "wellness-oriented" lifestyle are concerned with nutrition, fitness, stress, and their environment. They accept responsibility for their health and are excellent customers for health-related products and services. Those who lack a wellness orientation are identified as higher health risks and become candidates for health promotion program intervention. The authors report a new scale by which to measure the wellness-oriented lifestyle. Scale development procedures are detailed, followed by information from five studies that support its validity. The authors suggest ways health care marketers may use the Wellness Scale to segment and target potential customers and position their products and services.

  18. Featured Image: Identifying Weird Galaxies

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2017-08-01

    Hoags Object, an example of a ring galaxy. [NASA/Hubble Heritage Team/Ray A. Lucas (STScI/AURA)]The above image (click for the full view) shows PanSTARRSobservationsof some of the 185 galaxies identified in a recent study as ring galaxies bizarre and rare irregular galaxies that exhibit stars and gas in a ring around a central nucleus. Ring galaxies could be formed in a number of ways; one theory is that some might form in a galaxy collision when a smaller galaxy punches through the center of a larger one, triggering star formation around the center. In a recent study, Ian Timmis and Lior Shamir of Lawrence Technological University in Michigan explore ways that we may be able to identify ring galaxies in the overwhelming number of images expected from large upcoming surveys. They develop a computer analysis method that automatically finds ring galaxy candidates based on their visual appearance, and they test their approach on the 3 million galaxy images from the first PanSTARRS data release. To see more of the remarkable galaxies the authors found and to learn more about their identification method, check out the paper below.CitationIan Timmis and Lior Shamir 2017 ApJS 231 2. doi:10.3847/1538-4365/aa78a3

  19. Detecting and identifying offset gaze.

    PubMed

    Balsdon, Tarryn; Clifford, Colin W G

    2017-06-14

    A number of experiments have demonstrated that observers can accurately identify stimuli that they fail to detect (Rollman and Nachmias, 1972; Harris and Fahle, 1995; Allik et al. 1982, 2014). Using a 2x2AFC double judgements procedure, we demonstrated an analogous pattern of performance in making judgements about the direction of eye gaze. Participants were shown two faces in succession: one with direct gaze and one with gaze offset to the left or right. We found that they could identify the direction of gaze offset (left/right) better than they could detect which face contained the offset gaze. A simple Thurstonian model, under which the detection judgement is shown to be more computationally complex, was found to explain the empirical data. A further experiment incorporated metacognitive ratings into the double judgements procedure to measure observers' metacognitive awareness (Meta-d') across the two judgements and to assess whether observers were aware of the evidence for offset gaze when detection performance was at and below threshold. Results suggest that metacognitive awareness is tied to performance, with approximately equal Meta-d' across the two judgements, when sensitivity is taken into account. These results show that both performance and metacognitive awareness rely not only on the strength of sensory evidence but also on the computational complexity of the decision, which determines the relative distance of that evidence from the decision axes.

  20. Identifying teaching in wild animals.

    PubMed

    Thornton, Alex; Raihani, Nichola J

    2010-08-01

    After a long period of neglect, the study of teaching in nonhuman animals is beginning to take a more prominent role in research on social learning. Unlike other forms of social learning, teaching requires knowledgeable individuals to play an active role in facilitating learning by the naive. Casting aside anthropocentric requirements for cognitive mechanisms assumed to underpin teaching in our own species, researchers are now beginning to discover evidence for teaching across a wide range of taxa. Nevertheless, unequivocal evidence for teaching remains scarce, with convincing experimental data limited to meerkats, pied babblers, and tandem-running ants. In this review, our aim is to stimulate further research in different species and contexts by providing conceptual and methodological guidelines for identifying teaching, with a focus on natural populations. We begin by highlighting the fact that teaching is a form of cooperative behavior that functions to promote learning in others and show that consideration of these key characteristics is critical in helping to identify suitable targets for future research. We then go on to discuss potential observational, experimental, and statistical techniques that may assist researchers in providing evidence that the criteria that make up the accepted operational definition of teaching have been met. Supplemental materials for this article may be downloaded from http://lb.psychonomic-journals.org/content/supplemental.

  1. Identifying future zoonotic disease threats

    PubMed Central

    Cooper, Natalie; Nunn, Charles L.

    2013-01-01

    Background and objectives: Emerging infectious diseases often originate in wildlife, making it important to identify infectious agents in wild populations. It is widely acknowledged that wild animals are incompletely sampled for infectious agents, especially in developing countries, but it is unclear how much more sampling is needed, and where that effort should focus in terms of host species and geographic locations. Here, we identify these gaps in primate parasites, many of which have already emerged as threats to human health. Methodology: We obtained primate host–parasite records and other variables from existing databases. We then investigated sampling effort within primates relative to their geographic range size, and within countries relative to their primate species richness. We used generalized linear models, controlling for phylogenetic or spatial autocorrelation, to model variation in sampling effort across primates and countries. Finally, we used species richness estimators to extrapolate parasite species richness. Results: We found uneven sampling effort within all primate groups and continents. Sampling effort among primates was influenced by their geographic range size and substrate use, with terrestrial species receiving more sampling. Our parasite species richness estimates suggested that, among the best sampled primates and countries, almost half of primate parasites remain to be sampled; for most primate hosts, the situation is much worse. Conclusions and implications: Sampling effort for primate parasites is uneven and low. The sobering message is that we know little about even the best studied primates, and even less regarding the spatial and temporal distribution of parasitism within species. PMID:24481184

  2. Identifying infection hotspots early on.

    PubMed

    Shabha, Ghasson

    2012-04-01

    According to many published studies, 'ducting in ventilation and air-conditioning are largely overlooked and ignored, as they are out of sight and out of mind', despite mounting evidence indicating a higher risk in spreading airborne infections'. So says Ghasson Shabha BSc (Arch) MSc, PhD (Arch), MBIFM, Associate CIBSE, PG Cert Ed, of the Faculty of Technology, Engineering and the Environment (TEE), at the Birmingham School of the Built Environment (BSBE) at Birmingham City University, who adds that CIBSE estimates that fewer than 5% of buildings with air-conditioning systems above 12 kW have been inspected so far. Here he argues that incorporating 3D building information modelling software into existing computer-aided facilities management software systems will enable hospitals' 'infection hotspots' to be far more quickly identified, and subsequently monitored, to prevent future problems.

  3. Identifying and mapping community vulnerability.

    PubMed

    Morrow, B H

    1999-03-01

    Disaster vulnerability is socially constructed, i.e., it arises out of the social and economic circumstances of everyday living. Most often discussed from the perspective of developing nations, this article extends the argument using American demographic trends. Examples from recent disasters, Hurricane Andrew in particular, illustrate how certain categories of people, such as the poor, the elderly, women-headed households and recent residents, are at greater risk throughout the disaster response process. Knowledge of where these groups are concentrated within communities and the general nature of their circumstances is an important step towards effective emergency management. Emergency planners, policy-makers and responding organisations are encouraged to identify and locate high-risk sectors on Community Vulnerability Maps, integrating this information into GIS systems where feasible. Effective disaster management calls for aggressively involving these neighbourhoods and groups at all levels of planning and response, as well as mitigation efforts that address the root causes of vulnerability.

  4. Identifying methamphetamine exposure in children

    PubMed Central

    Castaneto, Marisol S.; Barnes, Allan J.; Scheidweiler, Karl B.; Schaffer, Michael; Rogers, Kristen K.; Stewart, Deborah; Huestis, Marilyn A.

    2013-01-01

    Introduction Methamphetamine (MAMP) use, distribution and manufacture remain a serious public health and safety problem in the United States, and children environmentally exposed to MAMP face a myriad of developmental, social and health risks, including severe abuse and neglect necessitating child protection involvement. It is recommended that drug-endangered children receive medical evaluation and care with documentation of overall physical and mental conditions and have urine drug testing.1 The primary aim of this study was to determine the best biological matrix to detect MAMP, amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA) and methylenedioxyethylamphetamine (MDEA) in environmentally exposed children. Method 91 children, environmentally exposed to household MAMP intake, were medically evaluated at the Child and Adolescent Abuse Resource and Evaluation (CAARE) Diagnostic and Treatment Center at the University of California, Davis (UCD) Children's Hospital. MAMP, AMP, MDMA, MDA and MDEA were quantified in urine and oral fluid (OF) by gas chromatography mass spectrometry (GCMS) and in hair by liquid chromatography tandem mass spectrometry (LCMSMS). Results Overall drug detection rates in OF, urine and hair were 6.9%, 22.1% and 77.8%, respectively. Seventy children (79%) tested positive for 1 or more drugs in 1 or more matrices. MAMP was the primary analyte detected in all 3 biological matrices. All positive OF (n=5) and 18 of 19 positive urine specimens also had a positive hair test. Conclusion Hair analysis offered a more sensitive tool for identifying MAMP, AMP and MDMA environmental exposure in children than urine or OF testing. A negative urine, or hair test does not exclude the possibility of drug exposure, but hair testing provided the greatest sensitivity for identifying drug-exposed children. PMID:24263642

  5. Identifying problem and compulsive gamblers.

    PubMed Central

    van Es, R.

    2000-01-01

    OBJECTIVE: To present a meta-analysis of current research on the prevalence, identification, and treatment of problem and compulsive gamblers. QUALITY OF EVIDENCE: Problem and compulsive gambling was not a socio-scientific concern until the last two decades. Hence research on this topic is limited. The summary and analysis for this paper relied on computer searches of journal and news abstracts in addition to direct contact with organizations addressing the identification and treatment of compulsive gamblers. MAIN MESSAGE: An estimated 5% of those who gamble run into problems. About 1% of those who gamble are predicted to experience serious problems. Successful treatment of problem and compulsive gambling continues to be a challenge. Although cognitive therapy has been the favoured approach, a combination of several therapeutic approaches is advocated. CONCLUSIONS: Problem and compulsive gambling can present a real health threat. As with other addictions, treatment strategies continue to be a baffling social problem. Aware and informed physicians can have a pivotal role in the difficult process of identifying, acknowledging, and remediating problem and compulsive gambling. PMID:10907572

  6. Identifying and Bounding Ethnic Neighborhoods

    PubMed Central

    Logan, John R.; Spielman, Seth; Xu, Hongwei; Klein, Philip N.

    2013-01-01

    This study presents three novel approaches to the question of how best to identify ethnic neighborhoods (or more generally, neighborhoods defined any aspect of their population composition) and to define their boundaries. It takes advantage of unusual data on the residential locations of all residents of Newark, NJ, in 1880 to avoid having to accept arbitrary administrative units (like census tracts) as the building blocks of neighborhoods. For theoretical reasons the street segment is chosen as the basic unit of analysis. All three methods use information on the ethnic composition of buildings or street segments and the ethnicity of their neighbors. One approach is a variation of k-functions calculated for each adult resident, which are then subjected to a cluster analysis to detect discrete patterns. The second is an application of an energy minimization algorithm commonly used to enhance digital images. The third is a Bayesian approach previously used to study county-level disability data. Results of all three methods depend on decisions about technical procedures and criteria that are made by the investigator. Resulting maps are roughly similar, but there is no one best solution. We conclude that researchers should continue to seek alternative methods, and that the preferred method depends on how one’s conceptualization of neighborhoods matches the empirical approach. PMID:24039327

  7. RECOVIR Software for Identifying Viruses

    NASA Technical Reports Server (NTRS)

    Chakravarty, Sugoto; Fox, George E.; Zhu, Dianhui

    2013-01-01

    Most single-stranded RNA (ssRNA) viruses mutate rapidly to generate a large number of strains with highly divergent capsid sequences. Determining the capsid residues or nucleotides that uniquely characterize these strains is critical in understanding the strain diversity of these viruses. RECOVIR (an acronym for "recognize viruses") software predicts the strains of some ssRNA viruses from their limited sequence data. Novel phylogenetic-tree-based databases of protein or nucleic acid residues that uniquely characterize these virus strains are created. Strains of input virus sequences (partial or complete) are predicted through residue-wise comparisons with the databases. RECOVIR uses unique characterizing residues to identify automatically strains of partial or complete capsid sequences of picorna and caliciviruses, two of the most highly diverse ssRNA virus families. Partition-wise comparisons of the database residues with the corresponding residues of more than 300 complete and partial sequences of these viruses resulted in correct strain identification for all of these sequences. This study shows the feasibility of creating databases of hitherto unknown residues uniquely characterizing the capsid sequences of two of the most highly divergent ssRNA virus families. These databases enable automated strain identification from partial or complete capsid sequences of these human and animal pathogens.

  8. Identifying Chemical Groups for Biomonitoring

    PubMed Central

    Krowech, Gail; Hoover, Sara; Plummer, Laurel; Sandy, Martha; Zeise, Lauren; Solomon, Gina

    2016-01-01

    Summary: Regulatory agencies face daunting challenges identifying emerging chemical hazards because of the large number of chemicals in commerce and limited data on exposure and toxicology. Evaluating one chemical at a time is inefficient and can lead to replacement with uncharacterized chemicals or chemicals with structural features already linked to toxicity. The Office of Environmental Health Hazard Assessment (OEHHA) has developed a process for constructing and assessing chemical groups for potential biomonitoring in California. We screen for chemicals with significant exposure potential and propose possible chemical groups, based on structure and function. To support formal consideration of these groups by Biomonitoring California’s Scientific Guidance Panel, we conduct a detailed review of exposure and toxicity data and examine the likelihood of detection in biological samples. To date, 12 chemical groups have been constructed and added to the pool of chemicals that can be selected for Biomonitoring California studies, including p,p´-bisphenols, brominated and chlorinated organic compounds used as flame retardants, non-halogenated aromatic phosphates, and synthetic polycyclic musks. Evaluating chemical groups, rather than individual chemicals, is an efficient way to respond to shifts in chemical use and the emergence of new chemicals. This strategy can enable earlier identification of important chemicals for monitoring and intervention. PMID:27905275

  9. Identifying acid salts of magnesium

    SciTech Connect

    Plumb, R.; Thivierge, R.F. Jr.; Xu, W.W.

    1987-11-05

    In preliminary work they found that significant quantities of certain nitrogen oxides and of sulfuric acid were absorbed by lower hydrates of magnesium sulfate. It appeared that acid salts were being formed but the known chemistry of group IIA (group 2) sulfates and acid sulfates which was worked out many years ago did not provide an explanation of their observations. They developed a new technique for delineating the solidus boundary of ternary mixtures using friability tests and applied it to the systems of interest. Magnesium acid salt hydrates with compositions on the solidus boundary could be readily identified. X-ray powder patterns confirmed the existence of two previously unknown ternary compounds, Mg/sub 2/(HSO/sub 4/)/sub 2/SO/sub 4/ x 4H/sub 2/O and Mg(HSO/sub 4/)/sub 2/ x H/sub 2/SO/sub 4/ x 3H/sub 2/O. Mixed acid sulfate-nitrate-hydrates could be detected but fuming at room temperatures interfered with quantitative determinations of the solidus boundary and X-ray measurements.

  10. Identifying and Inactivating Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Newcombe, David; Dekas, Anne; Venkateswaran, Kasthuri

    2009-01-01

    Problems associated with, and new strategies for, inactivating resistant organisms like Bacillus canaveralius (found at Kennedy Space Center during a survey of three NASA cleanrooms) have been defined. Identifying the particular component of the spore that allows its heightened resistance can guide the development of sterilization procedures that are targeted to the specific molecules responsible for resistance, while avoiding using unduly harsh methods that jeopardize equipment. The key element of spore resistance is a multilayered protein shell that encases the spore called the spore coat. The coat of the best-studied spore-forming microbe, B. subtilis, consists of at least 45 proteins, most of which are poorly characterized. Several protective roles for the coat are well characterized including resistance to desiccation, large toxic molecules, ortho-phthalaldehyde, and ultraviolet (UV) radiation. One important long-term specific goal is an improved sterilization procedure that will enable NASA to meet planetary protection requirements without a terminal heat sterilization step. This would support the implementation of planetary protection policies for life-detection missions. Typically, hospitals and government agencies use biological indicators to ensure the quality control of sterilization processes. The spores of B. canaveralius that are more resistant to osmotic stress would serve as a better biological indicator for potential survival than those in use currently.

  11. Priority Planetary Science Missions Identified

    NASA Astrophysics Data System (ADS)

    Showstack, Randy

    2011-03-01

    The U.S. National Research Council's (NRC) planetary science decadal survey report, released on 7 March, lays out a grand vision for priority planetary science missions for 2013-2022 within a tightly constrained fiscal environment. The cost-conscious report, issued by NRC's Committee on the Planetary Science Decadal Survey, identifies high-priority flagship missions, recommends a number of potential midsized missions, and indicates support for some smaller missions. The report states that the highest-priority flagship mission for the decade is the Mars Astrobiology Explorer-Cacher (MAX-C)—the first of three components of a NASA/European Space Agency Mars sample return campaign—provided that the mission scope can be reduced so that MAX-C costs no more than $2.5 billion. The currently estimated mission cost of $3.5 billion “would take up a disproportionate near-term share of the overall budget for NASA's Planetary Science Division,” the report notes.

  12. Identifying ELIXIR Core Data Resources

    PubMed Central

    Durinx, Christine; McEntyre, Jo; Appel, Ron; Apweiler, Rolf; Barlow, Mary; Blomberg, Niklas; Cook, Chuck; Gasteiger, Elisabeth; Kim, Jee-Hyub; Lopez, Rodrigo; Redaschi, Nicole; Stockinger, Heinz; Teixeira, Daniel; Valencia, Alfonso

    2017-01-01

    The core mission of ELIXIR is to build a stable and sustainable infrastructure for biological information across Europe. At the heart of this are the data resources, tools and services that ELIXIR offers to the life-sciences community, providing stable and sustainable access to biological data. ELIXIR aims to ensure that these resources are available long-term and that the life-cycles of these resources are managed such that they support the scientific needs of the life-sciences, including biological research. ELIXIR Core Data Resources are defined as a set of European data resources that are of fundamental importance to the wider life-science community and the long-term preservation of biological data. They are complete collections of generic value to life-science, are considered an authority in their field with respect to one or more characteristics, and show high levels of scientific quality and service. Thus, ELIXIR Core Data Resources are of wide applicability and usage. This paper describes the structures, governance and processes that support the identification and evaluation of ELIXIR Core Data Resources. It identifies key indicators which reflect the essence of the definition of an ELIXIR Core Data Resource and support the promotion of excellence in resource development and operation. It describes the specific indicators in more detail and explains their application within ELIXIR’s sustainability strategy and science policy actions, and in capacity building, life-cycle management and technical actions. The identification process is currently being implemented and tested for the first time. The findings and outcome will be evaluated by the ELIXIR Scientific Advisory Board in March 2017. Establishing the portfolio of ELIXIR Core Data Resources and ELIXIR Services is a key priority for ELIXIR and publicly marks the transition towards a cohesive infrastructure. PMID:27803796

  13. Identifying ELIXIR Core Data Resources.

    PubMed

    Durinx, Christine; McEntyre, Jo; Appel, Ron; Apweiler, Rolf; Barlow, Mary; Blomberg, Niklas; Cook, Chuck; Gasteiger, Elisabeth; Kim, Jee-Hyub; Lopez, Rodrigo; Redaschi, Nicole; Stockinger, Heinz; Teixeira, Daniel; Valencia, Alfonso

    2016-01-01

    The core mission of ELIXIR is to build a stable and sustainable infrastructure for biological information across Europe. At the heart of this are the data resources, tools and services that ELIXIR offers to the life-sciences community, providing stable and sustainable access to biological data. ELIXIR aims to ensure that these resources are available long-term and that the life-cycles of these resources are managed such that they support the scientific needs of the life-sciences, including biological research. ELIXIR Core Data Resources are defined as a set of European data resources that are of fundamental importance to the wider life-science community and the long-term preservation of biological data. They are complete collections of generic value to life-science, are considered an authority in their field with respect to one or more characteristics, and show high levels of scientific quality and service. Thus, ELIXIR Core Data Resources are of wide applicability and usage. This paper describes the structures, governance and processes that support the identification and evaluation of ELIXIR Core Data Resources. It identifies key indicators which reflect the essence of the definition of an ELIXIR Core Data Resource and support the promotion of excellence in resource development and operation. It describes the specific indicators in more detail and explains their application within ELIXIR's sustainability strategy and science policy actions, and in capacity building, life-cycle management and technical actions. The identification process is currently being implemented and tested for the first time. The findings and outcome will be evaluated by the ELIXIR Scientific Advisory Board in March 2017. Establishing the portfolio of ELIXIR Core Data Resources and ELIXIR Services is a key priority for ELIXIR and publicly marks the transition towards a cohesive infrastructure.

  14. A novel single cell method to identify the genetic composition at a single nuclear body

    PubMed Central

    Anchel, David; Ching, Reagan W.; Cotton, Rachel; Li, Ren; Bazett-Jones, David P.

    2016-01-01

    Gene loci make specific associations with compartments of the nucleus (e.g. the nuclear envelope, nucleolus, and transcription factories) and this association may determine or reflect a mechanism of genetic control. With current methods, it is not possible to identify sets of genes that converge to form a “gene hub” as there is a reliance on loci-specific probes, or immunoprecipitation of a particular protein from bulk cells. We introduce a method that will allow for the identification of loci contained within the vicinity of a single nuclear body in a single cell. For the first time, we demonstrate that the DNA sequences originating from a single sub-nuclear structure in a single cell targeted by two-photon irradiation can be determined, and mapped to a particular locus. Its application to single PML nuclear bodies reveals ontologically related loci that frequently associate with each other and with PML bodies in a population of cells, and a possible nuclear body targeting role for specific transcription factor binding sites. PMID:27389808

  15. Drosophila Imp iCLIP identifies an RNA assemblage coordinating F-actin formation.

    PubMed

    Hansen, Heidi Theil; Rasmussen, Simon Horskjær; Adolph, Sidsel Kramshøj; Plass, Mireya; Krogh, Anders; Sanford, Jeremy; Nielsen, Finn Cilius; Christiansen, Jan

    2015-06-09

    Post-transcriptional RNA regulons ensure coordinated expression of monocistronic mRNAs encoding functionally related proteins. In this study, we employ a combination of RIP-seq and short- and long-wave individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) technologies in Drosophila cells to identify transcripts associated with cytoplasmic ribonucleoproteins (RNPs) containing the RNA-binding protein Imp. We find extensive binding of Imp to 3' UTRs of transcripts that are involved in F-actin formation. A common denominator of the RNA-protein interface is the presence of multiple motifs with a central UA-rich element flanked by CA-rich elements. Experiments in single cells and intact flies reveal compromised actin cytoskeletal dynamics associated with low Imp levels. The former shows reduced F-actin formation and the latter exhibits abnormal neuronal patterning. This demonstrates a physiological significance of the defined RNA regulon. Our data imply that Drosophila Imp RNPs may function as cytoplasmic mRNA assemblages that encode proteins which participate in actin cytoskeletal remodeling. Thus, they may facilitate coordinated protein expression in sub-cytoplasmic locations such as growth cones.

  16. Nucleus-localized antisense small RNAs with 5'-polyphosphate termini regulate long term transcriptional gene silencing in Entamoeba histolytica G3 strain.

    PubMed

    Zhang, Hanbang; Alramini, Hussein; Tran, Vy; Singh, Upinder

    2011-12-30

    In the deep-branching eukaryotic parasite Entamoeba histolytica, transcriptional gene silencing (TGS) of the Amoebapore A gene (ap-a) in the G3 strain has been reported with subsequent development of this parasite strain for gene silencing. However, the mechanisms underlying this gene silencing approach are poorly understood. Here we report that antisense small RNAs (sRNAs) specific to the silenced ap-a gene can be identified in G3 parasites. Furthermore, when additional genes are silenced in the G3 strain, antisense sRNAs to the newly silenced genes can also be detected. Characterization of these sRNAs demonstrates that they are ~27 nucleotides in size, have 5'-polyphosphate termini, and persist even after removal of the silencing plasmid. Immunofluorescence analysis (IFA) and fluorescence in situ hybridization (FISH) show that both the Argonaute protein EhAGO2-2 and antisense sRNAs to the silenced genes are localized to the parasite nucleus. Furthermore, α-EhAGO2-2 immunoprecipitation confirmed the direct association of the antisense sRNAs with EhAGO2-2. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that the loci of the silenced genes are enriched for histone H3 and EhAGO2-2, indicating that both chromatin modification and the RNA-induced transcriptional silencing complex are involved in permanent gene silencing in G3 parasites. In conclusion, our data demonstrate that G3-based gene silencing in E. histolytica is mediated by an siRNA pathway, which utilizes antisense 5'-polyphosphate sRNAs. To our knowledge, this is the first study to show that 5'- polyphosphate antisense sRNAs can mediate TGS, and it is the first example of RNAi-mediated TGS in protozoan parasites.

  17. Overview Article: Identifying transcriptional cis-regulatory modules in animal genomes

    PubMed Central

    Suryamohan, Kushal; Halfon, Marc S.

    2014-01-01

    Gene expression is regulated through the activity of transcription factors and chromatin modifying proteins acting on specific DNA sequences, referred to as cis-regulatory elements. These include promoters, located at the transcription initiation sites of genes, and a variety of distal cis-regulatory modules (CRMs), the most common of which are transcriptional enhancers. Because regulated gene expression is fundamental to cell differentiation and acquisition of new cell fates, identifying, characterizing, and understanding the mechanisms of action of CRMs is critical for understanding development. CRM discovery has historically been challenging, as CRMs can be located far from the genes they regulate, have few readily-identifiable sequence characteristics, and for many years were not amenable to high-throughput discovery methods. However, the recent availability of complete genome sequences and the development of next-generation sequencing methods has led to an explosion of both computational and empirical methods for CRM discovery in model and non-model organisms alike. Experimentally, CRMs can be identified through chromatin immunoprecipitation directed against transcription factors or histone post-translational modifications, identification of nucleosome-depleted “open” chromatin regions, or sequencing-based high-throughput functional screening. Computational methods include comparative genomics, clustering of known or predicted transcription factor binding sites, and supervised machine-learning approaches trained on known CRMs. All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications. Experimental confirmation of predictions is essential, although shortcomings in current methods suggest that additional means of validation need to be developed. PMID:25704908

  18. A genome-wide survey of CD4+ lymphocyte regulatory genetic variants identifies novel asthma genes

    PubMed Central

    Sharma, Sunita; Zhou, Xiaobo; Thibault, Derek M.; Himes, Blanca E.; Liu, Andy; Szefler, Stanley J.; Strunk, Robert; Castro, Mario; Hansel, Nadia N.; Diette, Gregory B.; Vonakis, Becky M.; Adkinson, N. Franklin; Avila, Lydiana; Soto-Quiros, Manuel; Barraza-Villareal, Albino; Lemanske, Robert F.; Solway, Julian; Krishnan, Jerry; White, Steven R.; Cheadle, Chris; Berger, Alan E.; Fan, Jinshui; Boorgula, Meher Preethi; Nicolae, Dan; Gilliland, Frank; Barnes, Kathleen; London, Stephanie J.; Martinez, Fernando; Ober, Carole; Celedón, Juan C.; Carey, Vincent J.; Weiss, Scott T.; Raby, Benjamin A.

    2014-01-01

    Background Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. Objective We evaluated 6,706 cis-acting expression-associated variants (eSNP) identified through a genome-wide eQTL survey of CD4+ lymphocytes for association with asthma. Methods eSNP were tested for association with asthma in 359 asthma cases and 846 controls from the Childhood Asthma Management Program, with verification using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE)-qPCR and Chromatin-Immunoprecipitation (ChIP)-PCR in lung derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. Results Cis-acting eSNP demonstrated associations with asthma in both cohorts. We confirmed the previously-reported association of ORMDL3/GSDMB variants with asthma (combined p=2.9 × 108). Reproducible associations were also observed for eSNP in three additional genes: FADS2 (p=0.002), NAGA (p=0.0002), and F13A1 (p=0.0001). We subsequently demonstrated that FADS2 mRNA is increased in CD4+ lymphocytes in asthmatics, and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. Conclusions Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes, and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma. PMID:24934276

  19. Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

    PubMed

    Alajem, Adi; Biran, Alva; Harikumar, Arigela; Sailaja, Badi Sri; Aaronson, Yair; Livyatan, Ilana; Nissim-Rafinia, Malka; Sommer, Andreia Gianotti; Mostoslavsky, Gustavo; Gerbasi, Vincent R; Golden, Daniel E; Datta, Arnab; Sze, Siu Kwan; Meshorer, Eran

    2015-03-31

    Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

  20. An in vivo hypoxia metagene identifies the novel hypoxia inducible factor target gene SLCO1B3.

    PubMed

    Ramachandran, Anassuya; Betts, Guy; Bhana, Sara; Helme, Gemma; Blick, Christopher; Moller-Levet, Carla; Saunders, Emma; Valentine, Helen; Pepper, Stuart; Miller, Crispin J; Buffa, Francesca; Harris, Adrian L; West, Catharine M L

    2013-05-01

    A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes.

  1. Experimental methods for identifying failure mechanisms

    NASA Technical Reports Server (NTRS)

    Daniel, I. M.

    1983-01-01

    Experimental methods for identifying failure mechanisms in fibrous composites are studied. Methods to identify failure in composite materials includes interferometry, holography, fractography and ultrasonics.

  2. Characterization of Human Macrophage Antigens Identified by Monoclonal Antibodies

    DTIC Science & Technology

    1984-01-01

    Total extracts of iodinated glycoproteins or iodinated surface proteins were dis- solved in 50 pLl O’Farrell Buffer A [9,5 M urea. 2% (w,’v) nonidet -P...the c:ll surface labeled by vectorial iodination. p40 skas not immunoprecipitable from an iodinated concanavalin A-glycoprotein fraction of U937...of U937 cells. T cell clone. B cell clone, and peripheral blood granulocytes and monocytes. but not lymphocytes (Table 3). p40 was also detected in

  3. Computational analysis of siRNA recognition by the Ago2 PAZ domain and identification of the determinants of RNA-induced gene silencing.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2013-01-01

    RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.

  4. Application of genomic DNA affinity chromatography identifies multiple interferon-alpha-regulated Stat2 complexes.

    PubMed

    Ghislain, J J; Fish, E N

    1996-05-24

    Interferon-alpha (IFN-alpha)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo-and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA affinity chromatography, for the identification of IFN-alpha-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA affinity chromatography results and identified a Stat2:1 complex that binds with low affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-alpha dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-alpha activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.

  5. Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease

    SciTech Connect

    Kasyapa, Chitta S.; Kunapuli, Padmaja; Cowell, John K. . E-mail: John.Cowell@RoswellPark.org

    2005-09-10

    ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interaction between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.

  6. Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

    PubMed

    Heyder, Tina; Kohler, Maxie; Tarasova, Nataliya K; Haag, Sabrina; Rutishauser, Dorothea; Rivera, Natalia V; Sandin, Charlotta; Mia, Sohel; Malmström, Vivianne; Wheelock, Åsa M; Wahlström, Jan; Holmdahl, Rikard; Eklund, Anders; Zubarev, Roman A; Grunewald, Johan; Ytterberg, A Jimmy

    2016-09-01

    Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients

  7. Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples *

    PubMed Central

    Heyder, Tina; Kohler, Maxie; Tarasova, Nataliya K.; Haag, Sabrina; Rutishauser, Dorothea; Rivera, Natalia V.; Sandin, Charlotta; Mia, Sohel; Malmström, Vivianne; Wheelock, Åsa M.; Wahlström, Jan; Holmdahl, Rikard; Eklund, Anders; Zubarev, Roman A.; Grunewald, Johan; Ytterberg, A. Jimmy

    2016-01-01

    Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides. Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 106 bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10–15 × 106 cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls. In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 106 cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and

  8. International Genome-Wide Association Study Consortium Identifies Novel Loci Associated With Blood Pressure in Children and Adolescents

    PubMed Central

    Parmar, Priyakumari Ganesh; Taal, H. Rob; Timpson, Nicholas J.; Howe, Laura D.; Verwoert, Germaine; Aalto, Ville; Uitterlinden, Andre G.; Briollais, Laurent; Evans, Dave M.; Wright, Margie J.; Newnham, John P.; Whitfield, John B.; Lyytikäinen, Leo-Pekka; Rivadeneira, Fernando; Boomsma, Dorrett I.; Viikari, Jorma; Gillman, Matthew W.; St Pourcain, Beate; Hottenga, Jouke-Jan; Montgomery, Grant W.; Hofman, Albert; Kähönen, Mika; Martin, Nicholas G.; Tobin, Martin D.; Raitakari, Ollie; Vioque, Jesus; Jaddoe, Vincent W.V.; Jarvelin, Marjo-Riita; Beilin, Lawrence J.; Heinrich, Joachim; van Duijn, Cornelia M.; Pennell, Craig E.; Lawlor, Debbie A.; Palmer, Lyle J.

    2017-01-01

    Background Our aim was to identify genetic variants associated with blood pressure (BP) in childhood and adolescence. Methods and Results Genome-wide association study data from participating European ancestry cohorts of the Early Genetics and Lifecourse Epidemiology (EAGLE) Consortium was meta-analyzed across 3 epochs; prepuberty (4–7 years), puberty (8–12 years), and postpuberty (13–20 years). Two novel loci were identified as having genome-wide associations with systolic BP across specific age epochs: rs1563894 (ITGA11, located in active H3K27Ac mark and transcription factor chromatin immunoprecipitation and 5′-C-phosphate-G-3′ methylation site) during prepuberty (P=2.86×10–8) and rs872256 during puberty (P=8.67×10–9). Several single-nucleotide polymorphism clusters were also associated with childhood BP at P<5×10–3. Using a P value threshold of <5×10–3, we found some overlap in variants across the different age epochs within our study and between several single-nucleotide polymorphisms in any of the 3 epochs and adult BP-related single-nucleotide polymorphisms. Conclusions Our results suggest that genetic determinants of BP act from childhood, develop over the lifecourse, and show some evidence of age-specific effects. PMID:26969751

  9. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

    PubMed

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L

    2012-08-01

    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  10. Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State

    PubMed Central

    Pells, Steve; Koutsouraki, Eirini; Morfopoulou, Sofia; Valencia-Cadavid, Sara; Tomlinson, Simon R.; Kalathur, Ravi; Futschik, Matthias E.; De Sousa, Paul A.

    2015-01-01

    Human embryonic stem cells (hESCs) undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency “signature” would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI) methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5) induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells. PMID:26151932

  11. Meta-analysis of IDH-mutant cancers identifies EBF1 as an interaction partner for TET2.

    PubMed

    Guilhamon, Paul; Eskandarpour, Malihe; Halai, Dina; Wilson, Gareth A; Feber, Andrew; Teschendorff, Andrew E; Gomez, Valenti; Hergovich, Alexander; Tirabosco, Roberto; Fernanda Amary, M; Baumhoer, Daniel; Jundt, Gernot; Ross, Mark T; Flanagan, Adrienne M; Beck, Stephan

    2013-01-01

    Isocitrate dehydrogenase (IDH) genes 1 and 2 are frequently mutated in acute myeloid leukaemia (AML), low-grade glioma, cholangiocarcinoma (CC) and chondrosarcoma (CS). For AML, low-grade glioma and CC, mutant IDH status is associated with a DNA hypermethylation phenotype, implicating altered epigenome dynamics in the aetiology of these cancers. Here we show that the IDH variants in CS are also associated with a hypermethylation phenotype and display increased production of the oncometabolite 2-hydroxyglutarate, supporting the role of mutant IDH-produced 2-hydroxyglutarate as an inhibitor of TET-mediated DNA demethylation. Meta-analysis of the acute myeloid leukaemia, low-grade glioma, cholangiocarcinoma and CS methylation data identifies cancer-specific effectors within the retinoic acid receptor activation pathway among the hypermethylated targets. By analysing sequence motifs surrounding hypermethylated sites across the four cancer types, and using chromatin immunoprecipitation and western blotting, we identify the transcription factor EBF1 (early B-cell factor 1) as an interaction partner for TET2, suggesting a sequence-specific mechanism for regulating DNA methylation.

  12. A Synthetic Interaction Screen Identifies Factors Selectively Required for Proliferation and TERT Transcription in p53-Deficient Human Cancer Cells

    PubMed Central

    Park, Sung Mi; Zhu, Lihua J.; Debily, Marie-anne; Kittler, Ellen L. W.; Zapp, Maria L.; Lapointe, David; Gobeil, Stephane; Virbasius, Ching-Man; Green, Michael R.

    2012-01-01

    Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)–based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53−) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53− human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53− cells, RNAi–mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53− but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53− cancer cells. PMID:23284306

  13. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

    PubMed Central

    Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; Whitfield, Michael L.

    2012-01-01

    We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. PMID:22740631

  14. Ugene, a newly identified protein that is commonly over-expressed in cancer, and that binds uracil DNA-glycosylase

    PubMed Central

    Guo, Chunguang; Zhang, Xiaodong; Fink, Stephen P; Platzer, Petra; Wilson, Keith; Willson, James K. V.; Wang, Zhenghe; Markowitz, Sanford D

    2008-01-01

    Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including, breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA-glycosylase 2 (UNG2), an important enzyme in the base excision repair pathway, was identified as a partner protein that binds to Ugene. Co-immunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH2-terminus. We suggest Ugene induction in cancer may contribute to the cancer phenotype by interacting with the base excision repair pathway. PMID:18676834

  15. 10 Ways to Identify Hearing Loss

    MedlinePlus

    Skip Navigation Bar Home Current Issue Past Issues 10 Ways to Identify Hearing Loss Past Issues / Spring ... due to excessive noise exposure and other causes. 10 Ways to Identify Hearing Loss Take the following ...

  16. 10 Ways to Identify Hearing Loss

    MedlinePlus

    ... page please turn JavaScript on. Feature: Hearing Loss 10 Ways to Identify Hearing Loss Past Issues / Spring ... Research / Screening Newborns / How Loud Is Too Loud? / 10 Ways to Identify Hearing Loss Spring 2015 Issue: ...

  17. Structural Identifiability of Viscoelastic Mechanical Systems

    PubMed Central

    2014-01-01

    We solve the local and global structural identifiability problems for viscoelastic mechanical models represented by networks of springs and dashpots. We propose a very simple characterization of both local and global structural identifiability based on identifiability tables, with the purpose of providing a guideline for constructing arbitrarily complex, identifiable spring-dashpot networks. We illustrate how to use our results in a number of examples and point to some applications in cardiovascular modeling. PMID:24523860

  18. Method of identifying plant pathogen tolerance

    DOEpatents

    Ecker, Joseph R.; Staskawicz, Brian J.; Bent, Andrew F.; Innes, Roger W.

    1997-10-07

    A process for identifying a plant having disease tolerance comprising administering to a plant an inhibitory amount of ethylene and screening for ethylene insensitivity, thereby identifying a disease tolerant plant, is described. Plants identified by the foregoing process are also described.

  19. 29 CFR 4010.7 - Identifying information.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Identifying information. 4010.7 Section 4010.7 Labor... DISCLOSURE REQUIREMENTS ANNUAL FINANCIAL AND ACTUARIAL INFORMATION REPORTING § 4010.7 Identifying information..., http://www.pbgc.gov, the following identifying information with respect to each member of the filer's...

  20. 29 CFR 4010.7 - Identifying information.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 9 2012-07-01 2012-07-01 false Identifying information. 4010.7 Section 4010.7 Labor... DISCLOSURE REQUIREMENTS ANNUAL FINANCIAL AND ACTUARIAL INFORMATION REPORTING § 4010.7 Identifying information..., http://www.pbgc.gov, the following identifying information with respect to each member of the filer's...

  1. 29 CFR 4010.7 - Identifying information.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 9 2013-07-01 2013-07-01 false Identifying information. 4010.7 Section 4010.7 Labor... DISCLOSURE REQUIREMENTS ANNUAL FINANCIAL AND ACTUARIAL INFORMATION REPORTING § 4010.7 Identifying information..., http://www.pbgc.gov, the following identifying information with respect to each member of the filer's...

  2. Ability of Slovakian Pupils to Identify Birds

    ERIC Educational Resources Information Center

    Prokop, Pavol; Rodak, Rastislav

    2009-01-01

    A pupil's ability to identify common organisms is necessary for acquiring further knowledge of biology. We investigated how pupils were able to identify 25 bird species following their song, growth habits, or both features presented simultaneously. Just about 19% of birds were successfully identified by song, about 39% by growth habit, and 45% of…

  3. Method of identifying plant pathogen tolerance

    DOEpatents

    Ecker, J.R.; Staskawicz, B.J.; Bent, A.F.; Innes, R.W.

    1997-10-07

    A process for identifying a plant having disease tolerance comprising administering to a plant an inhibitory amount of ethylene and screening for ethylene insensitivity, thereby identifying a disease tolerant plant, is described. Plants identified by the foregoing process are also described. 7 figs.

  4. Ability of Slovakian Pupils to Identify Birds

    ERIC Educational Resources Information Center

    Prokop, Pavol; Rodak, Rastislav

    2009-01-01

    A pupil's ability to identify common organisms is necessary for acquiring further knowledge of biology. We investigated how pupils were able to identify 25 bird species following their song, growth habits, or both features presented simultaneously. Just about 19% of birds were successfully identified by song, about 39% by growth habit, and 45% of…

  5. Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

    PubMed Central

    Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

    2013-01-01

    Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

  6. Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

    PubMed

    Sazonova, Olga; Zhao, Yuqi; Nürnberg, Sylvia; Miller, Clint; Pjanic, Milos; Castano, Victor G; Kim, Juyong B; Salfati, Elias L; Kundaje, Anshul B; Bejerano, Gill; Assimes, Themistocles; Yang, Xia; Quertermous, Thomas

    2015-05-01

    To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including "growth factor binding," "matrix interaction," and "smooth muscle contraction." We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

  7. Identifying Candidate Chemical-Disease Linkages ...

    EPA Pesticide Factsheets

    Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment. Presentation at meeting on Environmental and Epigenetic Determinants of IBD in New York, NY on identifying candidate chemical-disease linkages by using AOPs to identify molecular initiating events and using relevant high throughput assays to screen for candidate chemicals. This hazard information is combined with exposure models to inform risk assessment.

  8. On the identifiability of metabolic network models.

    PubMed

    Berthoumieux, Sara; Brilli, Matteo; Kahn, Daniel; de Jong, Hidde; Cinquemani, Eugenio

    2013-12-01

    A major problem for the identification of metabolic network models is parameter identifiability, that is, the possibility to unambiguously infer the parameter values from the data. Identifiability problems may be due to the structure of the model, in particular implicit dependencies between the parameters, or to limitations in the quantity and quality of the available data. We address the detection and resolution of identifiability problems for a class of pseudo-linear models of metabolism, so-called linlog models. Linlog models have the advantage that parameter estimation reduces to linear or orthogonal regression, which facilitates the analysis of identifiability. We develop precise definitions of structural and practical identifiability, and clarify the fundamental relations between these concepts. In addition, we use singular value decomposition to detect identifiability problems and reduce the model to an identifiable approximation by a principal component analysis approach. The criterion is adapted to real data, which are frequently scarce, incomplete, and noisy. The test of the criterion on a model with simulated data shows that it is capable of correctly identifying the principal components of the data vector. The application to a state-of-the-art dataset on central carbon metabolism in Escherichia coli yields the surprising result that only 4 out of 31 reactions, and 37 out of 100 parameters, are identifiable. This underlines the practical importance of identifiability analysis and model reduction in the modeling of large-scale metabolic networks. Although our approach has been developed in the context of linlog models, it carries over to other pseudo-linear models, such as generalized mass-action (power-law) models. Moreover, it provides useful hints for the identifiability analysis of more general classes of nonlinear models of metabolism.

  9. Directed Proteomics Identifies a Plant-Specific Protein Rapidly Phosphorylated in Response to Bacterial and Fungal Elicitors

    PubMed Central

    Peck, Scott C.; Nühse, Thomas S.; Hess, Daniel; Iglesias, Alejandro; Meins, Fred; Boller, Thomas

    2001-01-01

    The perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used radioactive orthophosphate to pulse-label suspension-cultured cells of Arabidopsis in conjunction with two-dimensional gel electrophoresis and mass spectrometry to identify proteins that are phosphorylated rapidly in response to bacterial and fungal elicitors. One of these proteins, AtPhos43, and related proteins in tomato and rice, are phosphorylated within minutes after treatment with flagellin or chitin fragments. By measuring 32P incorporation into AtPhos43 immunoprecipitated from extracts of elicitor-treated hormone and defense-response mutants, we found that phosphorylation of AtPhos43 after flagellin treatment but not chitin treatment is dependent on FLS2, a receptor-like kinase involved in flagellin perception. Induction by both elicitors is not dependent on salicylic acid or EDS1, a putative lipase involved in defense signaling. PMID:11402173

  10. Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb.

    PubMed

    Zhao, Liang; Glazov, Evgeny A; Pattabiraman, Diwakar R; Al-Owaidi, Faisal; Zhang, Ping; Brown, Matthew A; Leo, Paul J; Gonda, Thomas J

    2011-06-01

    To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation.

  11. Portable Radiometer Identifies Minerals in the Field

    NASA Technical Reports Server (NTRS)

    Goetz, A. F. H.; Machida, R. A.

    1982-01-01

    Hand-held optical instrument aids in identifying minerals in field. Can be used in exploration for minerals on foot or by aircraft. The radiometer is especially suitable for identifying clay and carbonate minerals. Radiometer measures reflectances of mineral at two wavelengths, computes ratio of reflectances, and displays ratio to user.

  12. Identifying Bilingual Semantic Neural Representations across Languages

    ERIC Educational Resources Information Center

    Buchweitz, Augusto; Shinkareva, Svetlana V.; Mason, Robert A.; Mitchell, Tom M.; Just, Marcel Adam

    2012-01-01

    The goal of the study was to identify the neural representation of a noun's meaning in one language based on the neural representation of that same noun in another language. Machine learning methods were used to train classifiers to identify which individual noun bilingual participants were thinking about in one language based solely on their…

  13. Healthcare Identifiers legislation: a whiff of fourberie.

    PubMed

    Mendelson, Danuta

    2010-05-01

    The Healthcare Identifiers Bill 2010 (Cth), which will establish "the national e-health Healthcare Identifiers Service to provide that patients, healthcare providers and provider organisations can be consistently identified", is in the process of being enacted by the Australian Federal Parliament. The legislation will enable the government to assign to each "healthcare recipient" a 26-digit electronic "Healthcare Identifier", which will be accessible, with or without the recipient's consent, to a broad range of health care service providers as well as other entities. The individual Healthcare Identifier file will initially contain such identifying information as, where applicable, the Medicare number and/or the Veterans' Affairs number; name; address; gender; date of birth; and "the date of birth accuracy indicator" presumably birth certificate. However, since each "service" provided by a health care provider to a health care recipient will be automatically recorded on each individual's Healthcare Identifier file, in time these electronic files should contain a full record of such services or contacts. Moreover, the Healthcare Identifiers are considered a "key" to, or a "foundation stone" for, the implementation of the shared electronic health records scheme, because they will enable linkage with and retrieval of each patient's clinical records throughout the health care service system. However, there has been virtually no discussion about the legal, ethical and social implications of this legislation.

  14. Self-Identifying Emergency Radio Beacons

    NASA Technical Reports Server (NTRS)

    Friedman, Morton L.

    1987-01-01

    Rescue teams aided by knowledge of vehicle in distress. Similar to conventional emergency transmitters except contains additional timing and modulating circuits. Additions to standard emergency transmitter enable transmitter to send rescuers identifying signal in addition to conventional distress signal created by sweep generator. Data generator contains identifying code.

  15. Identifying Bilingual Semantic Neural Representations across Languages

    ERIC Educational Resources Information Center

    Buchweitz, Augusto; Shinkareva, Svetlana V.; Mason, Robert A.; Mitchell, Tom M.; Just, Marcel Adam

    2012-01-01

    The goal of the study was to identify the neural representation of a noun's meaning in one language based on the neural representation of that same noun in another language. Machine learning methods were used to train classifiers to identify which individual noun bilingual participants were thinking about in one language based solely on their…

  16. Identifying Opinion Leaders to Promote Behavior Change

    ERIC Educational Resources Information Center

    Valente, Thomas W.; Pumpuang, Patchareeya

    2007-01-01

    This article reviews 10 techniques used to identify opinion leaders to promote behavior change. Opinion leaders can act as gatekeepers for interventions, help change social norms, and accelerate behavior change. Few studies document the manner in which opinion leaders are identified, recruited, and trained to promote health. The authors categorize…

  17. Marking Identifiable Scripts: Following up Student Concerns

    ERIC Educational Resources Information Center

    Owen, Cathy; Stefaniak, John; Corrigan, Gerry

    2010-01-01

    Introduction: Medical student concern that the submission of named examination scripts to examiners could cause bias initiated a study on the effect of identified and de-identified scripts on assessment outcome. Methods: Data were collected from a convenience examination sample of Year 1 (n = 88 students; n = 29 questions) and Year 2 scripts (n =…

  18. Parameter Identifiability of Fundamental Pharmacodynamic Models

    PubMed Central

    Janzén, David L. I.; Bergenholm, Linnéa; Jirstrand, Mats; Parkinson, Joanna; Yates, James; Evans, Neil D.; Chappell, Michael J.

    2016-01-01

    Issues of parameter identifiability of routinely used pharmacodynamics models are considered in this paper. The structural identifiability of 16 commonly applied pharmacodynamic model structures was analyzed analytically, using the input-output approach. Both fixed-effects versions (non-population, no between-subject variability) and mixed-effects versions (population, including between-subject variability) of each model structure were analyzed. All models were found to be structurally globally identifiable under conditions of fixing either one of two particular parameters. Furthermore, an example was constructed to illustrate the importance of sufficient data quality and show that structural identifiability is a prerequisite, but not a guarantee, for successful parameter estimation and practical parameter identifiability. This analysis was performed by generating artificial data of varying quality to a structurally identifiable model with known true parameter values, followed by re-estimation of the parameter values. In addition, to show the benefit of including structural identifiability as part of model development, a case study was performed applying an unidentifiable model to real experimental data. This case study shows how performing such an analysis prior to parameter estimation can improve the parameter estimation process and model performance. Finally, an unidentifiable model was fitted to simulated data using multiple initial parameter values, resulting in highly different estimated uncertainties. This example shows that although the standard errors of the parameter estimates often indicate a structural identifiability issue, reasonably “good” standard errors may sometimes mask unidentifiability issues. PMID:27994553

  19. Marking Identifiable Scripts: Following up Student Concerns

    ERIC Educational Resources Information Center

    Owen, Cathy; Stefaniak, John; Corrigan, Gerry

    2010-01-01

    Introduction: Medical student concern that the submission of named examination scripts to examiners could cause bias initiated a study on the effect of identified and de-identified scripts on assessment outcome. Methods: Data were collected from a convenience examination sample of Year 1 (n = 88 students; n = 29 questions) and Year 2 scripts (n =…

  20. Identifying Information Focuses in Listening Comprehension

    ERIC Educational Resources Information Center

    Zhang, Hong-yan

    2011-01-01

    The study explains the process of learners' listening comprehension within Halliday's information theory in functional grammar, including the skills of identifying focuses while listening in college English teaching. Identifying information focuses in listening is proved to improve the students' communicative listening ability by the means of a…

  1. EZID: Long term identifiers made easy (Invited)

    NASA Astrophysics Data System (ADS)

    Starr, J.

    2013-12-01

    Scholarly research is producing ever increasing amounts of digital research data, and this data should be managed throughout the research life cycle both as part of good scientific practice, but also to comply with funder mandates, such as the 2013 OSTP Public Access Memo (http://www.whitehouse.gov/sites/default/files/microsites/ostp/ostp_public_access_memo_2013.pdf). By assigning unique and persistent identifiers to data objects, data managers can gain control and flexibility over what can be a daunting task. This is due to the fact that the objects can be moved to new locations without disruption to links, as long as the identifier target is maintained. EZID is a tool that makes assigning and maintaining unique, persistent identifiers easy. It was designed and built by California Digital Library (CDL) and has both a user interface and a RESTful API. EZID currently offers services for two globally unique, persistent identifier schemes: Digital Object Identifiers (DOIs) and Archival Resource Keys (ARKs). DOIs are identifiers originating from the publishing world and are in widespread use for journal articles. CDL is able to offer DOIs because of being a founding member of DataCite (http://www.datacite.org/), an international consortium established to provide easier access to scientific research data on the Internet. ARKs are identifiers originating from the library, archive and museum community. Like DOIs, they become persistent when the objects and identifier forwarding information is maintained. DOIs and ARKs have a key role in data management and, therefore, in data management plans. DOIs are the recommended identifier for use in data citation, and ARKs provide the maximum flexibility needed for data documentation and management throughout the early phases of a project. The two identifier schemes are able to be used together, and EZID is made to work with both. EZID clients, coming from education, research, government, and the private sector, are utilizing the

  2. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner.

    PubMed

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé; Emiliani, Stéphane; Gallois-Montbrun, Sarah

    2016-12-20

    Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.

  3. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner

    PubMed Central

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé

    2017-01-01

    Abstract Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway. PMID:28003477

  4. A Screening of UNF Targets Identifies Rnb, a Novel Regulator of Drosophila Circadian Rhythms.

    PubMed

    Kozlov, Anatoly; Jaumouillé, Edouard; Machado Almeida, Pedro; Koch, Rafael; Rodriguez, Joseph; Abruzzi, Katharine C; Nagoshi, Emi

    2017-07-12

    Behavioral circadian rhythms are controlled by multioscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly Drosophila melanogaster are regulated differently in clock neuron subclasses to support their specific functions (Lee et al., 2016; Top et al., 2016). The nuclear receptor unfulfilled (unf) represents a regulatory node that provides the small ventral lateral neurons (s-LNvs) unique characteristics as the master pacemaker (Beuchle et al., 2012). We previously showed that UNF interacts with the s-LNv molecular clocks by regulating transcription of the core clock gene period (per) (Jaumouillé et al., 2015). To gain more insight into the mechanisms by which UNF contributes to the functioning of the circadian master pacemaker, we identified UNF target genes using chromatin immunoprecipitation. Our data demonstrate that a previously uncharacterized gene CG7837, which we termed R and B (Rnb), acts downstream of UNF to regulate the function of the s-LNvs as the master circadian pacemaker. Mutations and LNv-targeted adult-restricted knockdown of Rnb impair locomotor rhythms. RNB localizes to the nucleus, and its loss-of-function blunts the molecular rhythms and output rhythms of the s-LNvs, particularly the circadian rhythms in PDF accumulation and axonal arbor remodeling. These results establish a second pathway by which UNF interacts with the molecular clocks in the s-LNvs and highlight the mechanistic differences in the molecular clockwork within the pacemaker circuit.SIGNIFICANCE STATEMENT Circadian behavior is generated by a pacemaker circuit comprising diverse classes of pacemaker neurons, each of which contains a molecular clock. In addition to the anatomical and functional diversity, recent studies have shown the mechanistic differences in the molecular clockwork among the pacemaker neurons in Drosophila Here, we identified the molecular characteristics distinguishing

  5. Structural Identifiability of Dynamic Systems Biology Models

    PubMed Central

    Villaverde, Alejandro F.

    2016-01-01

    A powerful way of gaining insight into biological systems is by creating a nonlinear differential equation model, which usually contains many unknown parameters. Such a model is called structurally identifiable if it is possible to determine the values of its parameters from measurements of the model outputs. Structural identifiability is a prerequisite for parameter estimation, and should be assessed before exploiting a model. However, this analysis is seldom performed due to the high computational cost involved in the necessary symbolic calculations, which quickly becomes prohibitive as the problem size increases. In this paper we show how to analyse the structural identifiability of a very general class of nonlinear models by extending methods originally developed for studying observability. We present results about models whose identifiability had not been previously determined, report unidentifiabilities that had not been found before, and show how to modify those unidentifiable models to make them identifiable. This method helps prevent problems caused by lack of identifiability analysis, which can compromise the success of tasks such as experiment design, parameter estimation, and model-based optimization. The procedure is called STRIKE-GOLDD (STRuctural Identifiability taKen as Extended-Generalized Observability with Lie Derivatives and Decomposition), and it is implemented in a MATLAB toolbox which is available as open source software. The broad applicability of this approach facilitates the analysis of the increasingly complex models used in systems biology and other areas. PMID:27792726

  6. Identifiability of PBPK Models with Applications to ...

    EPA Pesticide Factsheets

    Any statistical model should be identifiable in order for estimates and tests using it to be meaningful. We consider statistical analysis of physiologically-based pharmacokinetic (PBPK) models in which parameters cannot be estimated precisely from available data, and discuss different types of identifiability that occur in PBPK models and give reasons why they occur. We particularly focus on how the mathematical structure of a PBPK model and lack of appropriate data can lead to statistical models in which it is impossible to estimate at least some parameters precisely. Methods are reviewed which can determine whether a purely linear PBPK model is globally identifiable. We propose a theorem which determines when identifiability at a set of finite and specific values of the mathematical PBPK model (global discrete identifiability) implies identifiability of the statistical model. However, we are unable to establish conditions that imply global discrete identifiability, and conclude that the only safe approach to analysis of PBPK models involves Bayesian analysis with truncated priors. Finally, computational issues regarding posterior simulations of PBPK models are discussed. The methodology is very general and can be applied to numerous PBPK models which can be expressed as linear time-invariant systems. A real data set of a PBPK model for exposure to dimethyl arsinic acid (DMA(V)) is presented to illustrate the proposed methodology. We consider statistical analy

  7. Structural Identifiability of Dynamic Systems Biology Models.

    PubMed

    Villaverde, Alejandro F; Barreiro, Antonio; Papachristodoulou, Antonis

    2016-10-01

    A powerful way of gaining insight into biological systems is by creating a nonlinear differential equation model, which usually contains many unknown parameters. Such a model is called structurally identifiable if it is possible to determine the values of its parameters from measurements of the model outputs. Structural identifiability is a prerequisite for parameter estimation, and should be assessed before exploiting a model. However, this analysis is seldom performed due to the high computational cost involved in the necessary symbolic calculations, which quickly becomes prohibitive as the problem size increases. In this paper we show how to analyse the structural identifiability of a very general class of nonlinear models by extending methods originally developed for studying observability. We present results about models whose identifiability had not been previously determined, report unidentifiabilities that had not been found before, and show how to modify those unidentifiable models to make them identifiable. This method helps prevent problems caused by lack of identifiability analysis, which can compromise the success of tasks such as experiment design, parameter estimation, and model-based optimization. The procedure is called STRIKE-GOLDD (STRuctural Identifiability taKen as Extended-Generalized Observability with Lie Derivatives and Decomposition), and it is implemented in a MATLAB toolbox which is available as open source software. The broad applicability of this approach facilitates the analysis of the increasingly complex models used in systems biology and other areas.

  8. Identifying Codes on Directed De Bruijn Graphs

    DTIC Science & Technology

    2014-12-19

    ar X iv :1 41 2. 58 42 v1 [ m at h. C O ] 1 8 D ec 2 01 4 Identifying Codes on Directed De Bruijn Graphs Debra Boutin ∗ Department of...Mathematics Hamilton College Victoria Horan † Air Force Research Laboratory Information Directorate December 19, 2014 Abstract For a directed graph G, a t...length at most t is both non-empty and unique. A graph is called t-identifiable if there exists a t-identifying code. This paper shows that the de

  9. Anti-MDA5 autoantibodies in juvenile dermatomyositis identify a distinct clinical phenotype: a prospective cohort study

    PubMed Central

    2014-01-01

    Introduction The aim of this study was to define the frequency and associated clinical phenotype of anti-MDA5 autoantibodies in a large UK based, predominantly Caucasian, cohort of patients with juvenile dermatomyositis (JDM). Methods Serum samples and clinical data were obtained from 285 patients with JDM recruited to the UK Juvenile Dermatomyositis Cohort and Biomarker Study. The presence of anti-MDA5 antibodies was determined by immunoprecipitation and confirmed by ELISA using recombinant MDA5 protein. Results were compared with matched clinical data, muscle biopsies (scored by an experienced paediatric neuropathologist) and chest imaging (reviewed by an experienced paediatric radiologist). Results Anti-MDA5 antibodies were identified in 7.4% of JDM patients and were associated with a distinct clinical phenotype including skin ulceration (P = 0.03) oral ulceration (P = 0.01), arthritis (P <0.01) and milder muscle disease both clinically (as determined by Childhood Myositis Assessment Score (P = 0.03)) and histologically (as determined by a lower JDM muscle biopsy score (P <0.01)) than patients who did not have anti-MDA5 antibodies. A greater proportion of children with anti-MDA5 autoantibodies achieved disease inactivity at two years post-diagnosis according to PRINTO criteria (P = 0.02). A total of 4 out of 21 children with anti-MDA5 had interstitial lung disease; none had rapidly progressive interstitial lung disease. Conclusions Anti-MDA5 antibodies can be identified in a small but significant proportion of patients with JDM and identify a distinctive clinical sub-group. Screening for anti-MDA5 autoantibodies at diagnosis would be useful to guide further investigation for lung disease, inform on prognosis and potentially confirm the diagnosis, as subtle biopsy changes could otherwise be missed. PMID:24989778

  10. Combined Antibody/Lectin Enrichment Identifies Extensive Changes in the O-GlcNAc Sub-proteome upon Oxidative Stress.

    PubMed

    Lee, Albert; Miller, Devin; Henry, Roger; Paruchuri, Venkata D P; O'Meally, Robert N; Boronina, Tatiana; Cole, Robert N; Zachara, Natasha E

    2016-12-02

    O-Linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies and regulates over 3000 nuclear, cytoplasmic, and mitochondrial proteins. Upon exposure to stress and injury, cells and tissues increase the O-GlcNAc modification, or O-GlcNAcylation, of numerous proteins promoting the cellular stress response and thus survival. The aim of this study was to identify proteins that are differentially O-GlcNAcylated upon acute oxidative stress (H2O2) to provide insight into the mechanisms by which O-GlcNAc promotes survival. We achieved this goal by employing Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and a novel "G5-lectibody" immunoprecipitation strategy that combines four O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39, and HGAC85) and the lectin WGA. Using the G5-lectibody column in combination with basic reversed phase chromatography and C18 RPLC-MS/MS, 990 proteins were identified and quantified. Hundreds of proteins that were identified demonstrated increased (>250) or decreased (>110) association with the G5-lectibody column upon oxidative stress, of which we validated the O-GlcNAcylation status of 24 proteins. Analysis of proteins with altered glycosylation suggests that stress-induced changes in O-GlcNAcylation cluster into pathways known to regulate the cell's response to injury and include protein folding, transcriptional regulation, epigenetics, and proteins involved in RNA biogenesis. Together, these data suggest that stress-induced O-GlcNAcylation regulates numerous and diverse cellular pathways to promote cell and tissue survival.

  11. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.

    PubMed

    Kuo, Taiyi; Lew, Michelle J; Mayba, Oleg; Harris, Charles A; Speed, Terence P; Wang, Jen-Chywan

    2012-07-10

    Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes.

  12. An integrated genomic approach identifies persistent tumor suppressive effects of transforming growth factor-β in human breast cancer

    PubMed Central

    2014-01-01

    Introduction Transforming growth factor-βs (TGF-βs) play a dual role in breast cancer, with context-dependent tumor-suppressive or pro-oncogenic effects. TGF-β antagonists are showing promise in early-phase clinical oncology trials to neutralize the pro-oncogenic effects. However, there is currently no way to determine whether the tumor-suppressive effects of TGF-β are still active in human breast tumors at the time of surgery and treatment, a situation that could lead to adverse therapeutic responses. Methods Using a breast cancer progression model that exemplifies the dual role of TGF-β, promoter-wide chromatin immunoprecipitation and transcriptomic approaches were applied to identify a core set of TGF-β-regulated genes that specifically reflect only the tumor-suppressor arm of the pathway. The clinical significance of this signature and the underlying biology were investigated using bioinformatic analyses in clinical breast cancer datasets, and knockdown validation approaches in tumor xenografts. Results TGF-β-driven tumor suppression was highly dependent on Smad3, and Smad3 target genes that were specifically enriched for involvement in tumor suppression were identified. Patterns of Smad3 binding reflected the preexisting active chromatin landscape, and target genes were frequently regulated in opposite directions in vitro and in vivo, highlighting the strong contextuality of TGF-β action. An in vivo-weighted TGF-β/Smad3 tumor-suppressor signature was associated with good outcome in estrogen receptor-positive breast cancer cohorts. TGF-β/Smad3 effects on cell proliferation, differentiation and ephrin signaling contributed to the observed tumor suppression. Conclusions Tumor-suppressive effects of TGF-β persist in some breast cancer patients at the time of surgery and affect clinical outcome. Carefully tailored in vitro/in vivo genomic approaches can identify such patients for exclusion from treatment with TGF-β antagonists. PMID:24890385

  13. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling

    PubMed Central

    Kuo, Taiyi; Lew, Michelle J.; Mayba, Oleg; Harris, Charles A.; Speed, Terence P.; Wang, Jen-Chywan

    2012-01-01

    Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes. PMID:22733784

  14. Helping You Identify Quality Laboratory Services

    MedlinePlus

    Helping You Identify Quality Laboratory Services Selecting quality health care services for yourself, a relative or friend requires special thought and attention. The Joint Commission has prepared this information ...

  15. Study Identifies New Lymphoma Treatment Target

    Cancer.gov

    NCI researchers have identified new therapeutic targets for diffuse large B-cell lymphoma. Drugs that hit these targets are under clinical development and the researchers hope to begin testing them in clinical trials of patients with DLBCL.

  16. Identifying associations between genomic alterations in tumors.

    PubMed

    George, Joshy; Gorringe, Kylie L; Smyth, Gordon K; Bowtell, David D L

    2013-01-01

    Single-nucleotide polymorphism (SNP) mapping arrays are a reliable method for identifying somatic copy number alterations in cancer samples. Though this is immensely useful to identify potential driver genes, it is not sufficient to identify genes acting in a concerted manner. In cancer cells, co-amplified genes have been shown to provide synergistic effects, and genomic alterations targeting a pathway have been shown to occur in a mutually exclusive manner. We therefore developed a bioinformatic method for detecting such gene pairs using an integrated analysis of genomic copy number and gene expression data. This approach allowed us to identify a gene pair that is co-amplified and co-expressed in high-grade serous ovarian cancer. This finding provided information about the interaction of specific genetic events that contribute to the development and progression of this disease.

  17. Determining the identifiability of DNA database entries.

    PubMed Central

    Malin, B.; Sweeney, L.

    2000-01-01

    CleanGene is a software program that helps determine the identifiability of sequenced DNA, independent of any explicit demographics or identifiers maintained with the DNA. The program computes the likelihood that the release of DNA database entries could be related to specific individuals that are the subjects of the data. The engine within CleanGene relies on publicly available health care data and on knowledge of particular diseases to help relate identified individuals to DNA entries. Over 20 diseases, ranging over ataxias, blood diseases, and sex-linked mutations are accounted for, with 98-100% of individuals found identifiable. We assume the genetic material is released in a linear sequencing format from an individual's genome. CleanGene and its related experiments are useful tools for any institution seeking to provide anonymous genetic material for research purposes. PMID:11079941

  18. Identifying Consumer Education Content for Industrial Arts.

    ERIC Educational Resources Information Center

    Sharpe, Dennis B.; Baker, Glenn E.

    1982-01-01

    The survey described here identified suitable consumer topics which can be infused into industrial courses at all grade levels. The survey reinforced the development of sound consumer skills as an important objective of industrial arts education. (SK)

  19. Asteroid Redirect Mission: Identify, Redirect, Explore

    NASA Image and Video Library

    NASA is developing a first-ever mission to identify, capture and redirect a near-Earth asteroid to a stable orbit around the moon, where astronauts will explore it in the 2020s, returning with samp...

  20. Identifying regional opportunities for accelerated timber managemnet

    Treesearch

    David A. Gansner; Joseph E. Barnard; Samuel F. Gingrich; Samuel F. Gingrich

    1973-01-01

    Describes a procedure for identifying regional opportunities for accelerated timber management and demonstrates its application. Results provide a basis for rational choices among alternative management strategies and permit meaningful micro- and macro-evaluations of treatment response.

  1. Identifying signs of intimate partner violence.

    PubMed

    Ali, Parveen; McGarry, Julie; Dhingra, Katie

    2016-02-01

    Intimate partner violence is a major public health and social problem that affects people everywhere. Nurses can play an important role in identifying victims who present to healthcare settings with domestic abuse-related health issues. Evidence suggests that most women who present to emergency departments have experienced domestic abuse at some point in their lives, but that only 5% are identified by healthcare professionals. To identify and respond to victims effectively, emergency nurses must understand domestic abuse and its associated complexities. This article provides an overview of these issues, including the different types of abuse, and their prevalence, causes and effects on health. The article also explores how emergency nurses can identify and manage the effects of violence at work.

  2. IDENTIFYING KEY CONTRIBUTIONS TO INFORMATION SCIENCE,

    DTIC Science & Technology

    Several alternative approaches were examined to determine how one might identify some of the key (written) contributions to ’ information science ’. The...references. The unclear selective patterns in current bibliographies in the information science field also present problems. It is suggested that in...identifying key contributions we are far from common agreement on the conceptual, methodological or practical contributions to the information science field

  3. Identifying ECM Mediators of Tumor Cell Dormancy

    DTIC Science & Technology

    2008-05-01

    proliferating cells. f) At time of euthanasia, stage of estrous will be determined by vaginal smear so that inter- animal variation due to estrous...ratio of the number of distinct peptides identified for each protein divided by the total number of peptides identified in the two hour run are...displayed as the peptide count ratio. Using this peptide counting label-free quantification approach, surprisingly, suggested that the three fibrillar

  4. Identifying ECM Mediators of Tumor Cell Dormancy

    DTIC Science & Technology

    2009-05-01

    determined by vaginal smear so that inter- animal variation due to estrous can be controlled for across groups. Further, cervical samples will be... peptides identified for each protein divided by the total number of peptides identified in the two hour run are displayed as the peptide count ratio...Using this peptide counting label-free quantification approach, surprisingly, suggested that the three fibrillar collagen chains were at lower levels in

  5. Newly identified YSO candidates towards LDN 1188

    NASA Astrophysics Data System (ADS)

    Marton , G.; Verebélyi, E.; Kiss, Cs.; Smidla, J.

    2013-11-01

    We present an analysis of young stellar object (YSO) candidates towards the LDN 1188 molecular cloud. The YSO candidates were selected from the WISE all-sky catalogue, based on a statistical method. We found 601 candidates in the region, and classified them as Class I, Flat, and Class II YSOs. Groups were identified and described with the Minimal Spanning Tree (MST) method. Previously identified molecular cores show evidence of ongoing star formation at different stages throughout the cloud complex.

  6. ORCID Author Identifiers: A Primer for Librarians.

    PubMed

    Akers, Katherine G; Sarkozy, Alexandra; Wu, Wendy; Slyman, Alison

    2016-01-01

    The ORCID (Open Researcher and Contributor ID) registry helps disambiguate authors and streamline research workflows by assigning unique 16-digit author identifiers that enable automatic linkages between researchers and their scholarly activities. This article describes how ORCID works, the benefits of using ORCID, and how librarians can promote ORCID at their institutions by raising awareness of ORCID, helping researchers create and populate ORCID profiles, and integrating ORCID identifiers into institutional repositories and other university research information systems.

  7. Identifying Codes on Directed De Bruijn Graphs

    DTIC Science & Technology

    2015-08-27

    2. 15. SUBJECT TERMS Identifying Code; De Bruijn Network; Graph Theory 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT UU 18. NUMBER...in graphs . IEEE Trans. Inform. Theory , 44(2):599–611, 1998. 1 [10] Samir Khuller, Balaji Raghavachari, and Azriel Rosenfeld. Landmarks in graphs ...JOURNAL ARTICLE (POST PRINT) 3. DATES COVERED (From - To) JUN 2013 – AUG 2015 4. TITLE AND SUBTITLE IDENTIFYING CODES ON DIRECTED DE BRUIJN GRAPHS 5a

  8. Exploiting intrinsic fluctuations to identify model parameters.

    PubMed

    Zimmer, Christoph; Sahle, Sven; Pahle, Jürgen

    2015-04-01

    Parameterisation of kinetic models plays a central role in computational systems biology. Besides the lack of experimental data of high enough quality, some of the biggest challenges here are identification issues. Model parameters can be structurally non-identifiable because of functional relationships. Noise in measured data is usually considered to be a nuisance for parameter estimation. However, it turns out that intrinsic fluctuations in particle numbers can make parameters identifiable that were previously non-identifiable. The authors present a method to identify model parameters that are structurally non-identifiable in a deterministic framework. The method takes time course recordings of biochemical systems in steady state or transient state as input. Often a functional relationship between parameters presents itself by a one-dimensional manifold in parameter space containing parameter sets of optimal goodness. Although the system's behaviour cannot be distinguished on this manifold in a deterministic framework it might be distinguishable in a stochastic modelling framework. Their method exploits this by using an objective function that includes a measure for fluctuations in particle numbers. They show on three example models, immigration-death, gene expression and Epo-EpoReceptor interaction, that this resolves the non-identifiability even in the case of measurement noise with known amplitude. The method is applied to partially observed recordings of biochemical systems with measurement noise. It is simple to implement and it is usually very fast to compute. This optimisation can be realised in a classical or Bayesian fashion.

  9. IDENTIFYING COLLISIONAL FAMILIES IN THE KUIPER BELT

    SciTech Connect

    Marcus, Robert A.; Ragozzine, Darin; Murray-Clay, Ruth A.; Holman, Matthew J.

    2011-05-20

    The identification and characterization of numerous collisional families-clusters of bodies with a common collisional origin-in the asteroid belt has added greatly to the understanding of asteroid belt formation and evolution. More recent study has also led to an appreciation of physical processes that had previously been neglected (e.g., the Yarkovsky effect). Collisions have certainly played an important role in the evolution of the Kuiper Belt as well, though only one collisional family has been identified in that region to date, around the dwarf planet Haumea. In this paper, we combine insights into collisional families from numerical simulations with the current observational constraints on the dynamical structure of the Kuiper Belt to investigate the ideal sizes and locations for identifying collisional families. We find that larger progenitors (r {approx} 500 km) result in more easily identifiable families, given the difficulty in identifying fragments of smaller progenitors in magnitude-limited surveys, despite their larger spread and less frequent occurrence. However, even these families do not stand out well from the background. Identifying families as statistical overdensities is much easier than characterizing families by distinguishing individual members from interlopers. Such identification seems promising, provided the background population is well known. In either case, families will also be much easier to study where the background population is small, i.e., at high inclinations. Overall, our results indicate that entirely different techniques for identifying families will be needed for the Kuiper Belt, and we provide some suggestions.

  10. Identifying nineteenth century genealogical links from genotypes.

    PubMed

    Stankovich, Jim; Bahlo, Melanie; Rubio, Justin P; Wilkinson, Christopher R; Thomson, Russell; Banks, Annette; Ring, Maree; Foote, Simon J; Speed, Terence P

    2005-07-01

    We have developed a likelihood method to identify moderately distant genealogical relationships from genomewide scan data. The aim is to compare the genotypes of many pairs of people and identify those pairs most likely to be related to one another. We have tested the algorithm using the genotypes of 170 Tasmanians with multiple sclerosis recruited into a haplotype association study. It is estimated from genealogical records that approximately 65% of Tasmania's current population of 470,000 are direct descendants of the 13,000 female founders living in this island state of Australia in the mid-nineteenth century. All cases and four to five relatives of each case have been genotyped with microsatellite markers at a genomewide average density of 4 cM. Previous genealogical research has identified 51 pairwise relationships linking 56 of the 170 cases. Testing the likelihood calculation on these known relative pairs, we have good power to identify relationships up to degree eight (e.g. third cousins once removed). Applying the algorithm to all other pairs of cases, we have identified a further 61 putative relative pairs, with an estimated false discovery rate of 10%. The power to identify genealogical links should increase when the new, denser sets of SNP markers are used. Except in populations where there is a searchable electronic database containing virtually all genealogical links in the past six generations, the algorithm should be a useful aid for genealogists working on gene-mapping projects, both linkage studies and association studies.

  11. A novel ex vivo immunoproteomic approach characterising Fasciola hepatica tegumental antigens identified using immune antibody from resistant sheep.

    PubMed

    Cameron, Timothy C; Cooke, Ira; Faou, Pierre; Toet, Hayley; Piedrafita, David; Young, Neil; Rathinasamy, Vignesh; Beddoe, Travis; Anderson, Glenn; Dempster, Robert; Spithill, Terry W

    2017-08-01

    A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica, we hypothesised that antibody from F. gigantica-infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica. Purified IgG from the sera of F. gigantica-infected Indonesian Thin Tail sheep collected pre-infection and 4weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica, suggesting that the Indonesian Thin Tail week

  12. Distributed design approach in persistent identifiers systems

    NASA Astrophysics Data System (ADS)

    Golodoniuc, Pavel; Car, Nicholas; Klump, Jens

    2017-04-01

    The need to identify both digital and physical objects is ubiquitous in our society. Past and present persistent identifier (PID) systems, of which there is a great variety in terms of technical and social implementations, have evolved with the advent of the Internet, which has allowed for globally unique and globally resolvable identifiers. PID systems have catered for identifier uniqueness, integrity, persistence, and trustworthiness, regardless of the identifier's application domain, the scope of which has expanded significantly in the past two decades. Since many PID systems have been largely conceived and developed by small communities, or even a single organisation, they have faced challenges in gaining widespread adoption and, most importantly, the ability to survive change of technology. This has left a legacy of identifiers that still exist and are being used but which have lost their resolution service. We believe that one of the causes of once successful PID systems fading is their reliance on a centralised technical infrastructure or a governing authority. Golodoniuc et al. (2016) proposed an approach to the development of PID systems that combines the use of (a) the Handle system, as a distributed system for the registration and first-degree resolution of persistent identifiers, and (b) the PID Service (Golodoniuc et al., 2015), to enable fine-grained resolution to different information object representations. The proposed approach solved the problem of guaranteed first-degree resolution of identifiers, but left fine-grained resolution and information delivery under the control of a single authoritative source, posing risk to the long-term availability of information resources. Herein, we develop these approaches further and explore the potential of large-scale decentralisation at all levels: (i) persistent identifiers and information resources registration; (ii) identifier resolution; and (iii) data delivery. To achieve large-scale decentralisation

  13. Scalable persistent identifier systems for dynamic datasets

    NASA Astrophysics Data System (ADS)

    Golodoniuc, P.; Cox, S. J. D.; Klump, J. F.

    2016-12-01

    Reliable and persistent identification of objects, whether tangible or not, is essential in information management. Many Internet-based systems have been developed to identify digital data objects, e.g., PURL, LSID, Handle, ARK. These were largely designed for identification of static digital objects. The amount of data made available online has grown exponentially over the last two decades and fine-grained identification of dynamically generated data objects within large datasets using conventional systems (e.g., PURL) has become impractical. We have compared capabilities of various technological solutions to enable resolvability of data objects in dynamic datasets, and developed a dataset-centric approach to resolution of identifiers. This is particularly important in Semantic Linked Data environments where dynamic frequently changing data is delivered live via web services, so registration of individual data objects to obtain identifiers is impractical. We use identifier patterns and pattern hierarchies for identification of data objects, which allows relationships between identifiers to be expressed, and also provides means for resolving a single identifier into multiple forms (i.e. views or representations of an object). The latter can be implemented through (a) HTTP content negotiation, or (b) use of URI querystring parameters. The pattern and hierarchy approach has been implemented in the Linked Data API supporting the United Nations Spatial Data Infrastructure (UNSDI) initiative and later in the implementation of geoscientific data delivery for the Capricorn Distal Footprints project using International Geo Sample Numbers (IGSN). This enables flexible resolution of multi-view persistent identifiers and provides a scalable solution for large heterogeneous datasets.

  14. Identifying Cell Type-Specific Transcription Factors by Integrating ChIP-seq and eQTL Data-Application to Monocyte Gene Regulation.

    PubMed

    Choudhury, Mudra; Ramsey, Stephen A

    2016-01-01

    We describe a novel computational approach to identify transcription factors (TFs) that are candidate regulators in a human cell type of interest. Our approach involves integrating cell type-specific expression quantitative trait locus (eQTL) data and TF data from chromatin immunoprecipitation-to-tag-sequencing (ChIP-seq) experiments in cell lines. To test the method, we used eQTL data from human monocytes in order to screen for TFs. Using a list of known monocyte-regulating TFs, we tested the hypothesis that the binding sites of cell type-specific TF regulators would be concentrated in the vicinity of monocyte eQTLs. For each of 397 ChIP-seq data sets, we obtained an enrichment ratio for the number of ChIP-seq peaks that are located within monocyte eQTLs. We ranked ChIP-seq data sets according to their statistical significances for eQTL overlap, and from this ranking, we observed that monocyte-regulating TFs are more highly ranked than would be expected by chance. We identified 27 TFs that had significant monocyte enrichment scores and mapped them into a protein interaction network. Our analysis uncovered two novel candidate monocyte-regulating TFs, BCLAF1 and SIN3A. Our approach is an efficient method to identify candidate TFs that can be used for any cell/tissue type for which eQTL data are available.

  15. Identifying Cell Type-Specific Transcription Factors by Integrating ChIP-seq and eQTL Data–Application to Monocyte Gene Regulation

    PubMed Central

    Choudhury, Mudra; Ramsey, Stephen A.

    2016-01-01

    We describe a novel computational approach to identify transcription factors (TFs) that are candidate regulators in a human cell type of interest. Our approach involves integrating cell type-specific expression quantitative trait locus (eQTL) data and TF data from chromatin immunoprecipitation-to-tag-sequencing (ChIP-seq) experiments in cell lines. To test the method, we used eQTL data from human monocytes in order to screen for TFs. Using a list of known monocyte-regulating TFs, we tested the hypothesis that the binding sites of cell type-specific TF regulators would be concentrated in the vicinity of monocyte eQTLs. For each of 397 ChIP-seq data sets, we obtained an enrichment ratio for the number of ChIP-seq peaks that are located within monocyte eQTLs. We ranked ChIP-seq data sets according to their statistical significances for eQTL overlap, and from this ranking, we observed that monocyte-regulating TFs are more highly ranked than would be expected by chance. We identified 27 TFs that had significant monocyte enrichment scores and mapped them into a protein interaction network. Our analysis uncovered two novel candidate monocyte-regulating TFs, BCLAF1 and SIN3A. Our approach is an efficient method to identify candidate TFs that can be used for any cell/tissue type for which eQTL data are available. PMID:28008225

  16. On the relationship between sloppiness and identifiability.

    PubMed

    Chis, Oana-Teodora; Villaverde, Alejandro F; Banga, Julio R; Balsa-Canto, Eva

    2016-12-01

    Dynamic models of biochemical networks are often formulated as sets of non-linear ordinary differential equations, whose states are the concentrations or abundances of the network components. They typically have a large number of kinetic parameters, which must be determined by calibrating the model with experimental data. In recent years it has been suggested that dynamic systems biology models are universally sloppy, meaning that the values of some parameters can be perturbed by several orders of magnitude without causing significant changes in the model output. This observation has prompted calls for focusing on model predictions rather than on parameters. In this work we examine the concept of sloppiness, investigating its links with the long-established notions of structural and practical identifiability. By analysing a set of case studies we show that sloppiness is not equivalent to lack of identifiability, and that sloppy models can be identifiable. Thus, using sloppiness to draw conclusions about the possibility of estimating parameter values can be misleading. Instead, structural and practical identifiability analyses are better tools for assessing the confidence in parameter estimates. Furthermore, we show that, when designing new experiments to decrease parametric uncertainty, designs that optimize practical identifiability criteria are more informative than those that minimize sloppiness. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Identifying Surfaces on the Pale Blue Dot

    NASA Astrophysics Data System (ADS)

    Cowan, Nicolas B.; Strait, T. E.

    2013-01-01

    The changing color of an unresolved planet hint at its cloud structure and surface features. Principal component analysis has previously been shown to robustly identify the number of surfaces contributing to disk-integrated observations. We have taken an important next step by showing that one can identify the reflectance spectra of unknown surface types, based solely on time-resolved, disk-integrated multi-band photometry. We test our method on disk-integrated observations of Earth taken by the Deep Impact spacecraft as part of the EPOXI mission. Critically, we borrow analysis tools from the remote sensing community, where spatially-resolved spectral data are routinely analyzed in an automated way to identify known (eg. boreal forest) as well as unknown surfaces (eg. military bunkers). Our study indicates that planned missions like the Terrestrial Planet Finder (TPF) will be able to identify new surfaces on exoplanets, and also offers a relatively model-independent avenue to identifying surface liquid water on other worlds.

  18. Identifying Turbulent Structures through Topological Segmentation

    SciTech Connect

    Bremer, Peer-Timo; Gruber, Andrea; Bennett, Janine C.; Gyulassy, Attila; Kolla, Hemanth; Chen, Jacqueline H.; Grout, Ray W.

    2016-01-01

    A new method of extracting vortical structures from a turbulent flow is proposed whereby topological segmentation of an indicator function scalar field is used to identify the regions of influence of the individual vortices. This addresses a long-standing challenge in vector field topological analysis: indicator functions commonly used produce a scalar field based on the local velocity vector field; reconstructing regions of influence for a particular structure requires selecting a threshold to define vortex extent. In practice, the same threshold is rarely meaningful throughout a given flow. By also considering the topology of the indicator field function, the characteristics of vortex strength and extent can be separated and the ambiguity in the choice of the threshold reduced. The proposed approach is able to identify several types of vortices observed in a jet in cross-flow configuration simultaneously where no single threshold value for a selection of common indicator functions appears able to identify all of these vortex types.

  19. Scientometric methods for identifying emerging technologies

    DOEpatents

    Abercrombie, Robert K; Schlicher, Bob G; Sheldon, Frederick T

    2015-11-03

    Provided is a method of generating a scientometric model that tracks the emergence of an identified technology from initial discovery (via original scientific and conference literature), through critical discoveries (via original scientific, conference literature and patents), transitioning through Technology Readiness Levels (TRLs) and ultimately on to commercial application. During the period of innovation and technology transfer, the impact of scholarly works, patents and on-line web news sources are identified. As trends develop, currency of citations, collaboration indicators, and on-line news patterns are identified. The combinations of four distinct and separate searchable on-line networked sources (i.e., scholarly publications and citation, worldwide patents, news archives, and on-line mapping networks) are assembled to become one collective network (a dataset for analysis of relations). This established network becomes the basis from which to quickly analyze the temporal flow of activity (searchable events) for the example subject domain.

  20. Identifying non-resonant Kepler planetary systems

    NASA Astrophysics Data System (ADS)

    Veras, Dimitri; Ford, Eric B.

    2012-02-01

    The Kepler mission has discovered a plethora of multiple transiting planet candidate exosystems, many of which feature putative pairs of planets near mean motion resonance commensurabilities. Identifying potentially resonant systems could help guide future observations and enhance our understanding of planetary formation scenarios. We develop and apply an algebraic method to determine which Kepler two-planet systems cannot be in a first-fourth order resonance, given the current, publicly available data. This method identifies when any potentially resonant angle of a system must circulate. We identify and list 70 near-resonant systems which cannot actually reside in resonance, assuming a widely used formulation for deriving planetary masses from their observed radii and that these systems do not contain unseen bodies that affect the interactions of the observed planets. This work strengthens the argument that a high fraction of exoplanetary systems may be near resonance but not actually in resonance.

  1. Bayesian inference to identify parameters in viscoelasticity

    NASA Astrophysics Data System (ADS)

    Rappel, Hussein; Beex, Lars A. A.; Bordas, Stéphane P. A.

    2017-08-01

    This contribution discusses Bayesian inference (BI) as an approach to identify parameters in viscoelasticity. The aims are: (i) to show that the prior has a substantial influence for viscoelasticity, (ii) to show that this influence decreases for an increasing number of measurements and (iii) to show how different types of experiments influence the identified parameters and their uncertainties. The standard linear solid model is the material description of interest and a relaxation test, a constant strain-rate test and a creep test are the tensile experiments focused on. The experimental data are artificially created, allowing us to make a one-to-one comparison between the input parameters and the identified parameter values. Besides dealing with the aforementioned issues, we believe that this contribution forms a comprehensible start for those interested in applying BI in viscoelasticity.

  2. Photoacoustic tomography to identify inflammatory arthritis

    NASA Astrophysics Data System (ADS)

    Rajian, Justin Rajesh; Girish, Gandikota; Wang, Xueding

    2012-09-01

    Identifying neovascularity (angiogenesis) as an early feature of inflammatory arthritis can help in early accurate diagnosis and treatment monitoring of this disease. Photoacoustic tomography (PAT) is a hybrid imaging modality which relies on intrinsic differences in the optical absorption among the tissues being imaged. Since blood has highly absorbing chromophores including both oxygenated and deoxygenated hemoglobin, PAT holds potential in identifying early angiogenesis associated with inflammatory joint diseases. PAT is used to identify changes in the development of inflammatory arthritis in a rat model. Imaging at two different wavelengths, 1064 nm and 532 nm, on rats revealed that there is a significant signal enhancement in the ankle joints of the arthritis affected rats when compared to the normal control group. Histology images obtained from both the normal and the arthritis affected rats correlated well with the PAT findings. Results support the fact that the emerging PAT could become a new tool for clinical management of inflammatory arthritis.

  3. Computational methods to identify new antibacterial targets.

    PubMed

    McPhillie, Martin J; Cain, Ricky M; Narramore, Sarah; Fishwick, Colin W G; Simmons, Katie J

    2015-01-01

    The development of resistance to all current antibiotics in the clinic means there is an urgent unmet need for novel antibacterial agents with new modes of action. One of the best ways of finding these is to identify new essential bacterial enzymes to target. The advent of a number of in silico tools has aided classical methods of discovering new antibacterial targets, and these programs are the subject of this review. Many of these tools apply a cheminformatic approach, utilizing the structural information of either ligand or protein, chemogenomic databases, and docking algorithms to identify putative antibacterial targets. Considering the wealth of potential drug targets identified from genomic research, these approaches are perfectly placed to mine this rich resource and complement drug discovery programs.

  4. Psychosocial counselling of identifiable sperm donors.

    PubMed

    Visser, M; Mochtar, M H; de Melker, A A; van der Veen, F; Repping, S; Gerrits, T

    2016-05-01

    What do identifiable sperm donors feel about psychosocial counselling? Identifiable sperm donors found it important that psychosocial counselling focused on emotional consequences and on rules and regulations and they expected to have access to psychosocial counselling at the time that donor-offspring actually sought contact. Most studies on sperm donors are on anonymous donors and focus on recruitment, financial compensation, anonymity and motivations. There is limited knowledge on the value that identifiable sperm donors place on psychosocial counselling and what their needs are in this respect. We performed a qualitative study from March until June 2014 with 25 identifiable sperm donors, who were or had been a donor at the Centre for Reproductive Medicine of the Academic Medical Centre in Amsterdam any time between 1989 and 2014. We held semi-structured in-depth interviews with identifiable sperm donors with an average age of 44 years. The interviews were fully transcribed and analysed using the constant comparative method of grounded theory. Twelve out of 15 donors (former donors ITALIC! n = 8, active donors ITALIC! n = 7) who had received a counselling session during their intake procedure found it important that they had been able to talk about issues such as the emotional consequences of donation, disclosure to their own children, family and friends, future contact with donor-offspring and rules and regulations. Of the 10 former donors who had received no counselling session, 8 had regretted the lack of intensive counselling. In the years following their donation, most donors simply wanted to know how many offspring had been born using their sperm and had no need for further counselling. Nevertheless, they frequently mentioned that they were concerned about the well-being of 'their' offspring. In addition, they would value the availability of psychosocial counselling in the event that donor-offspring actually sought contact. A limitation of our study is its

  5. On identifiability of flexible structure parameters

    NASA Technical Reports Server (NTRS)

    Joshi, S. M.; Goglia, G. L.

    1983-01-01

    This report investigates the identifiability of modal parameters of flexible structures. Expressions are derived for Cramer-Rao lower bounds for the modal parameters, that is, frequencies, damping ratios and mode shapes or slopes. The optimal initial state, which maximizes the trace of the Fisher information matrix in the absence of persistent input, is obtained. The concepts discussed are applied to a finite-element model of the 122 meter hoop/column antenna. The numerical results show that the identifiability of the structural frequencies is excellent, followed by that of the damping ratios and the mode-slopes.

  6. Identifying gaps in international food safety regulation.

    PubMed

    McGrady, Benn; Ho, Christina S

    2011-01-01

    The rise in food importation in countries such as the United States, coupled with food safety incidents, has led to increased concern with the safety of imported food. This concern has prompted discussion of how international law and governance mechanisms might enhance food safety. This paper identifies the objectives underlying multilateral approaches to food safety such as raising food safety standards abroad, information sharing and ensuring market access. The paper then explores how these objectives are integrated into the international system and identifies how the current state of the law creates imbalances in the pursuit of these objectives.

  7. Molecular Fingerprints to Identify Candida Species

    PubMed Central

    Spampinato, Claudia

    2013-01-01

    A wide range of molecular techniques have been developed for genotyping Candida species. Among them, multilocus sequence typing (MLST) and microsatellite length polymorphisms (MLP) analysis have recently emerged. MLST relies on DNA sequences of internal regions of various independent housekeeping genes, while MLP identifies microsatellite instability. Both methods generate unambiguous and highly reproducible data. Here, we review the results achieved by using these two techniques and also provide a brief overview of a new method based on high-resolution DNA melting (HRM). This method identifies sequence differences by subtle deviations in sample melting profiles in the presence of saturating fluorescent DNA binding dyes. PMID:23844370

  8. Identifying the substance abuser in clinical practice.

    PubMed

    Bell, K

    1992-01-01

    Illicit drugs are used regularly by 14.5 million Americans. By identifying patients who abuse substances, the nurse will be better able to provide for the treatment interventions needed and omit ineffective treatment interventions. The patient will benefit by receiving timely and appropriate care. To identify substance abusers, the nurse must know effects of commonly abused drugs, their routes of administration, withdrawal signs, and the physical assessments that should be performed. The most common drugs abused are narcotics, depressants, antidepressants, anti-anxiety drugs, stimulants, hallucinogens, and marijuana.

  9. Minimal covariant observables identifying all pure states

    NASA Astrophysics Data System (ADS)

    Carmeli, Claudio; Heinosaari, Teiko; Toigo, Alessandro

    2013-09-01

    It has been recently shown by Heinosaari, Mazzarella and Wolf (2013) [1] that an observable that identifies all pure states of a d-dimensional quantum system has minimally 4d-4 outcomes or slightly less (the exact number depending on d). However, no simple construction of this type of minimal observable is known. We investigate covariant observables that identify all pure states and have minimal number of outcomes. It is shown that the existence of this kind of observables depends on the dimension of the Hilbert space.

  10. Identifying node importance in complex networks

    NASA Astrophysics Data System (ADS)

    Hu, Ping; Fan, Wenli; Mei, Shengwei

    2015-07-01

    In this paper, we propose a novel node importance evaluation method from the perspective of the existence of mutual dependence among nodes. The node importance comprises its initial importance and the importance contributions from both the adjacent and non-adjacent nodes according to the dependence strength between them. From the simulation analyses on an example network and the ARPA network, we observe that our method can well identify the node importance. Then, the cascading failures on the Netscience and E-mail networks demonstrate that the networks are more vulnerable when continuously removing the important nodes identified by our method, which further proves the accuracy of our method.

  11. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    PubMed

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  12. Field guide for identifying fuel loading models

    Treesearch

    Pamela G. Sikkink; Duncan C. Lutes; Robert E. Keane

    2009-01-01

    This report details a procedure for identifying fuel loading models (FLMs) in the field. FLMs are a new classification system for predicting fire effects from on-site fuels. Each FLM class represents fuel beds that have similar fuel loadings and produce similar emissions and soil surface heating when burned using computer simulations. We describe how to estimate fuel...

  13. Problems Identifying Independent and Dependent Variables

    ERIC Educational Resources Information Center

    Leatham, Keith R.

    2012-01-01

    This paper discusses one step from the scientific method--that of identifying independent and dependent variables--from both scientific and mathematical perspectives. It begins by analyzing an episode from a middle school mathematics classroom that illustrates the need for students and teachers alike to develop a robust understanding of…

  14. [Identifying Gifted American Indian Students.] What Works.

    ERIC Educational Resources Information Center

    Davidson, Karen

    1997-01-01

    In Hardin (Montana) schools, where 55% of students are American Indians, the same identification methods are used to identify gifted students among all cultural groups. These methods include nonverbal standardized tests and subjective recommendations based on the Frasier Talent Assessment Profile. Other equitable practices include equal…

  15. Identifying Concrete and Formal Operational Children.

    ERIC Educational Resources Information Center

    Docherty, Edward M.

    This paper presents a study designed to determine if groups of concrete and formal operational children can be identified through the technique of cluster analysis, using a battery of Piagetian tasks. A Total of 64 subjects, 8 boys and 8 girls from each of the second, fourth, sixth, and eighth grade levels, were selected from a public elementary…

  16. International Team Identifies Biomarker for Scleroderma

    MedlinePlus

    ... that are under development for this disease," said study author Robert Lafyatis, M.D., of the Boston University School of Medicine. "This would enable us to more rapidly identify new therapies in early clinical trials." With this goal in mind, Dr. Lafyatis helped form an international network of ...

  17. Professional Development: Identifying Effective Instructional Coaching Activities

    ERIC Educational Resources Information Center

    Mannino, Gina

    2014-01-01

    The purpose of this study was to identify the instructional coaching activities most used by instructional coaches in southeast Texas school districts and to test if there was a relationship between the use of instructional coaching and perceived improvement in the instructional practices of teachers and student achievement. The participants for…

  18. Methods for Identifying Versioned and Plagiarized Documents.

    ERIC Educational Resources Information Center

    Hoad, Timothy C.; Zobel, Justin

    2003-01-01

    Describes research that was conducted to develop and evaluate techniques for identifying plagiarism, revisions, and different versions of online documents. Highlights include ranking; parsing; similarity measures; identity measures; fingerprinting documents; measuring effectiveness via recall and precision; and experiments on two document…

  19. Identifying Depression in Students with Mental Retardation.

    ERIC Educational Resources Information Center

    Stough, Laura M.; Baker, Lynn

    1999-01-01

    Offers guidelines to teachers for identifying depression in students with mental retardation. Discusses prevalence and symptoms of depression, causes of depression, difficulty of diagnosis in students with mental retardation, detecting symptoms in the classroom, treatment of depression, and psychological services. Inserts list ideas for helping…

  20. Identify, Organize, and Retrieve Items Using Zotero

    ERIC Educational Resources Information Center

    Clark, Brian; Stierman, John

    2009-01-01

    Librarians build collections. To do this they use tools that help them identify, organize, and retrieve items for the collection. Zotero (zoh-TAIR-oh) is such a tool that helps the user build a library of useful books, articles, web sites, blogs, etc., discovered while surfing online. A visit to Zotero's homepage, www.zotero.org, shows a number of…

  1. National Board Certification Identifies Strong Teachers

    ERIC Educational Resources Information Center

    Education Digest: Essential Readings Condensed for Quick Review, 2009

    2009-01-01

    Advanced certification through the National Board for Professional Teaching Standards (NBPTS) is an effective way to identify highly skilled teachers, according to a congressionally mandated report from the National Research Council. Students taught by NBPTS-certified teachers make greater gains on achievement tests than students taught by…

  2. Identifying Ethical Hypernorms for Accounting Educators

    ERIC Educational Resources Information Center

    Siegel, Philip H.; Mintz, Steven; Naser-Tavakolian, Mohsen; O'Shaughnessy, John

    2012-01-01

    Accounting educators have a unique role in academe because students learn about codes of ethics that will guide their actions as professionals. We identify hypernorms related to internal auditing educators that reflect unethical behaviors believed to be universally unacceptable by that community. We then compare the results to a prior survey of…

  3. Identifying the Context of Postsecondary Instructional Programs.

    ERIC Educational Resources Information Center

    Levinson-Rose, Judith; Menges, Robert J.

    A framework for articulating postsecondary instructional programs is described. The framework identifies two important sources of influence on programs: the type of institution within which the program operates and the program objectives. These factors influence both program components and how these components are linked and processes that are…

  4. How to Identify High-Growth Schools

    ERIC Educational Resources Information Center

    Pfeiffer, Linda E.

    2015-01-01

    When researching school options, parents may want to look for schools with high-growth scores which, according to research, may be indicators of other characteristics such as programming, leadership, culture, and size. This quick guide offers parents tips on how to identify high-growth schools and what to ask when evaluating school options. An…

  5. Ideas Identified and Distributed through Project IDEA.

    ERIC Educational Resources Information Center

    American Alliance for Health, Physical Education, and Recreation, Washington, DC.

    This document contains ideas on a variety of subjects directed at the physical educator. The work was compiled by Project IDEA (Identify, Distribute, Exchange for Action). Topics include the following: (a) scheduling, (b) curriculum, (c) games, (d) specific courses, (e) life sports, (f) fitness, (g) adaptive Physical education, (h) course methods,…

  6. Sputum induction problems identified through genetic fingerprinting.

    PubMed

    Nivin, Beth; O'Flaherty, Tholief; Leibert, Eric; Zhao, Ben Yang; Driscoll, Jeffrey

    2002-10-01

    To identify the contamination source of a cluster of eight positive Mycobacterium tuberculosis isolates from one laboratory session. Spoligotyping was performed on M. tuberculosis isolates processed during one laboratory session. Laboratory and sputum induction protocols and records were reviewed. Sputum induction staff were interviewed. An environmental assessment of the sputum induction booth was performed. Spoligotyping identified a unique strain of susceptible M. tuberculosis from five induced sputa collected at Clinic A on the same day. Three specimens processed concurrently from other clinics had spoligotypes different from each other and from the cluster strain. A laboratory investigation revealed no procedural lapses. Sputum induction records from Clinic A indicated that patient 1 in the sputum induction booth had prior culture-confirmed tuberculosis. Patient 2 had a history of a drug-resistant strain. Patient 3 had completed tuberculosis treatment, with positive cultures 7 months earlier. Patients 4 and 5 were new to the clinic and had no subsequent positive M. tuberculosis specimens. The sputum induction booth was working within normal parameters. Sputum induction that day was overseen by a new employee with limited training and no supervision. A review of the sputum induction protocol identified ambiguity regarding care of the ultrasonic nebulizer between patients, which may have led to reuse of the discarded nebulizer solution from patient 1. A break in the sputum induction protocol may have contributed to contamination of patient specimens. Sputum induction is complicated, mandating adequate staff training and supervision and patient preparation. Spoligotyping identified a potential source of M. tuberculosis contamination.

  7. Techniques for identifying predators of goose nests

    USGS Publications Warehouse

    Anthony, R. Michael; Grand, J.B.; Fondell, T.F.; Miller, David A.

    2006-01-01

    We used cameras and artificial eggs to identify nest predators of dusky Canada goose Branta canadensis occidentalis nests during 1997-2000. Cameras were set up at 195 occupied goose nests and 60 artificial nests. We placed wooden eggs and domestic goose eggs that were emptied and then filled with wax or foam in an additional 263 natural goose nests to identify predators from marks in the artificial eggs. All techniques had limitations, but each correctly identified predators and estimated their relative importance. Nests with cameras had higher rates of abandonment than natural nests, especially during laying. Abandonment rates were reduced by deploying artificial eggs late in laying and reducing time at nests. Predation rates for nests with cameras were slightly lower than for nests without cameras. Wax-filled artificial eggs caused mortality of embryos in natural nests, but were better for identifying predator marks at artificial nests. Use of foam-filled artificial eggs in natural nests was the most cost effective means of monitoring nest predation. ?? Wildlife Biology (2006).

  8. Identifying the Multiple Intelligences of Your Students

    ERIC Educational Resources Information Center

    McClellan, Joyce A.; Conti, Gary J.

    2008-01-01

    One way of addressing individual differences among adult learners is to identify the Multiple Intelligences of the learner. Multiple Intelligences refers to the concept developed by Howard Gardner that challenges the traditional view of intelligence and explains the presence of nine different Multiple Intelligences. The purpose of this study was…

  9. Identifying Fallacies of Reference in Argumentation

    ERIC Educational Resources Information Center

    Gough, Jim

    2009-01-01

    The experience of teaching informal logic (sometimes called practical logic) at the introductory level over the last fifteen years has allowed the author the opportunity to identify some interesting problems. These problems have been encountered by students attempting to understand some of the ideas presented in the informal logic course and by…

  10. Student Success Factors: Identifying Key Predictors

    ERIC Educational Resources Information Center

    Sulaiman, Ainin; Mohezar, Suhana

    2006-01-01

    The authors' main aim in this study was to identify key predictors of Master of Business Administration (MBA) students' academic performance. The authors measured performance by the students' cumulative grade point average achieved, using data from the Students Information Systems and Application database. The authors found that a student's…

  11. Identifying the Rhetoric of Uncertainty Reduction.

    ERIC Educational Resources Information Center

    Williams, David E.

    Offering a rhetorical perspective of uncertainty reduction, this paper (1) discusses uncertainty reduction theory and dramatism; (2) identifies rhetorical strategies inherent in C. W. Berger and R. J. Calabrese's theory; (3) extends predicted outcome value to influenced outcome value; and (4) argues that the goal of uncertainty reduction and…

  12. National Board Certification Identifies Strong Teachers

    ERIC Educational Resources Information Center

    Education Digest: Essential Readings Condensed for Quick Review, 2009

    2009-01-01

    Advanced certification through the National Board for Professional Teaching Standards (NBPTS) is an effective way to identify highly skilled teachers, according to a congressionally mandated report from the National Research Council. Students taught by NBPTS-certified teachers make greater gains on achievement tests than students taught by…

  13. Identifiability and Equivalence of GLLIRM Models

    ERIC Educational Resources Information Center

    Revuelta, Javier

    2009-01-01

    The generalized logit-linear item response model (GLLIRM) is a linearly constrained nominal categories model (NCM) that computes the scale and intercept parameters for categories as a weighted sum of basic parameters. This paper addresses the problems of the identifiability of the basic parameters and the equivalence between different GLLIRM…

  14. Biogeographic methods identify gymnosperm biodiversity hotspots

    NASA Astrophysics Data System (ADS)

    Contreras-Medina, Raúl; Morrone, Juan J.; Luna Vega, Isolda

    2001-10-01

    A remarkable congruence among areas of endemism, panbiogeographic nodes, and refugia in western North America, Japan, south-western China, Tasmania, and New Caledonia indicates that these areas deserve special status for conservation. Here we propose that areas identified by different biogeographic methods are significant candidates for designation as hotspots.

  15. Problems Identifying Independent and Dependent Variables

    ERIC Educational Resources Information Center

    Leatham, Keith R.

    2012-01-01

    This paper discusses one step from the scientific method--that of identifying independent and dependent variables--from both scientific and mathematical perspectives. It begins by analyzing an episode from a middle school mathematics classroom that illustrates the need for students and teachers alike to develop a robust understanding of…

  16. Identifying Advanced Technologies for Education's Future.

    ERIC Educational Resources Information Center

    Moore, Gwendolyn B.; Yin, Robert K.

    A study to determine how three advanced technologies might be applied to the needs of special education students helped inspire the development of a new method for identifying such applications. This new method, named the "Hybrid Approach," combines features of the two traditional methods: technology-push and demand-pull. Technology-push involves…

  17. Identifying Mentors for Student Employees on Campus

    ERIC Educational Resources Information Center

    Frock, David

    2015-01-01

    Purpose: This exploratory research project aims to seek an effective process for identifying supervisors of part-time student employees who also serve in a mentoring capacity. Design/methodology/approach: This paper is based on a review of literature and an evaluation process focused on established traits and functions of mentoring as applied to…

  18. Using lice to identify cowbird hosts

    USGS Publications Warehouse

    Hahn, D.C.; Osenton, P.C.; Price, R.W.

    1995-01-01

    Avian lice may link fledgling Brown-headed Cowbirds to the host species that raised them. Lice, if host-specific and transferred to nestling cowbirds, could serve to identify the principal host species raising cowbirds in a local area. This approach of trapping cowbird fledglings in a feeding flock, then collecting and identifying the lice they carry is economical. The alternative requires a team of people to locate large numbers of parasitized host nests. We trapped 250 cowbird fledglings during June-August 1994 on Patuxent Research Center, and from them we collected 426 lice identified as representing 6 genera and 12 species. We. also collected and identified 347 lice from 30 known host species that were mist-netted on our Center. The lice found on cowbird fledglings in this population can be linked to Wood Thrush, Red-eyed Vireo, Common Yellowthroat, Rufous-sided Towhee, Red-winged Blackbird, Common Grackle, Song Sparrow, Field Sparrow, and Tree sparrow, based on this study and also on published reports.

  19. Avoiding medication mixups. Identifiable imprint codes.

    PubMed Central

    Vasudevan, P; Del Gianni, T; Robertson, W O

    1996-01-01

    This study was done to determine if current imprinting of solid medication forms permits health care professionals to identify the manufacturers involved so as to be able to activate the hierarchic identification system mandated by the Food and Drug Administration. We tested 15 representatives of 6 groups of health professionals for their ability to identify the manufacturer after having examined 30 solid-dosage forms drawn from a pseudo-random sample of stock hospital formulary products. The correct identification of the manufacturer was the sole criterion. Of the 2,700 opportunities, the manufacturer was able to be identified for only 43%. Nurses and medical students had a 35% success rate, pharmacists and poison center specialists a 55% success rate, and residents and attending physicians a 40% rate. None approached 95% accuracy. Currently employed imprints fail in their objective to permit health care professionals--or the general public--to rapidly identify prescription drugs. The manufacturers' logotypes need to be modified if this identification system is to be implemented. We propose a simple voluntary collaborative effort by the pharmaceutical industry to solve the problem. PMID:9000855

  20. Identifying Benefit Segments among College Students.

    ERIC Educational Resources Information Center

    Brown, Joseph D.

    1991-01-01

    Using concept of market segmentation (dividing market into distinct groups requiring different product benefits), surveyed 398 college students to determine benefit segments among students selecting a college to attend and factors describing each benefit segment. Identified one major segment of students (classroomers) plus three minor segments…

  1. Identifying Teaching Methods that Engage Entrepreneurship Students

    ERIC Educational Resources Information Center

    Balan, Peter; Metcalfe, Mike

    2012-01-01

    Purpose: Entrepreneurship education particularly requires student engagement because of the complexity of the entrepreneurship process. The purpose of this paper is to describe how an established measure of engagement can be used to identify relevant teaching methods that could be used to engage any group of entrepreneurship students.…

  2. A screening cascade to identify ERβ ligands

    PubMed Central

    Filgueira, Carly S.; Benod, Cindy; Lou, Xiaohua; Gunamalai, Prem S.; Villagomez, Rosa A.; Strom, Anders; Gustafsson, Jan-Åke; Berkenstam, Anders L.; Webb, Paul

    2014-01-01

    The establishment of effective high throughput screening cascades to identify nuclear receptor (NR) ligands that will trigger defined, therapeutically useful sets of NR activities is of considerable importance. Repositioning of existing approved drugs with known side effect profiles can provide advantages because de novo drug design suffers from high developmental failure rates and undesirable side effects which have dramatically increased costs. Ligands that target estrogen receptor β (ERβ) could be useful in a variety of diseases ranging from cancer to neurological to cardiovascular disorders. In this context, it is important to minimize cross-reactivity with ERα, which has been shown to trigger increased rates of several types of cancer. Because of high sequence similarities between the ligand binding domains of ERα and ERβ, preferentially targeting one subtype can prove challenging. Here, we describe a sequential ligand screening approach comprised of complementary in-house assays to identify small molecules that are selective for ERβ. Methods include differential scanning fluorimetry, fluorescence polarization and a GAL4 transactivation assay. We used this strategy to screen several commercially-available chemical libraries, identifying thirty ERβ binders that were examined for their selectivity for ERβ versus ERα, and tested the effects of selected ligands in a prostate cancer cell proliferation assay. We suggest that this approach could be used to rapidly identify candidates for drug repurposing. PMID:25422593

  3. 34 CFR 300.32 - Personally identifiable.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 2 2010-07-01 2010-07-01 false Personally identifiable. 300.32 Section 300.32 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION...

  4. Identifying Specific Comprehension Deficits in Children

    ERIC Educational Resources Information Center

    Gifford, Diane Baty

    2013-01-01

    Research has shown that educators may be missing an under-identified population of approximately 10 percent of typically developing children, who have fluent, age-appropriate decoding and word recognition skills, yet have specific difficulties with other higher-level text processing factors. These children are said to have specific comprehension…

  5. Diagnostics Tools Identify Faults Prior to Failure

    NASA Technical Reports Server (NTRS)

    2013-01-01

    Through the SBIR program, Rochester, New York-based Impact Technologies LLC collaborated with Ames Research Center to commercialize the Center s Hybrid Diagnostic Engine, or HyDE, software. The fault detecting program is now incorporated into a software suite that identifies potential faults early in the design phase of systems ranging from printers to vehicles and robots, saving time and money.

  6. Interchange. Program Improvement Products Identified through Networking.

    ERIC Educational Resources Information Center

    Ohio State Univ., Columbus. National Center for Research in Vocational Education.

    This catalog lists exemplary field-based program improvement products identified by the Dissemination and Utilization Products and Services Program (D&U) at the National Center for Research in Vocational Education. It is designed to increase awareness of these products among vocational educators and to provide information about them that…

  7. Diffraction gratings used as identifying markers

    DOEpatents

    Deason, Vance A.; Ward, Michael B.

    1991-01-01

    A finely detailed defraction grating is applied to an object as an identifier or tag which is unambiguous, difficult to duplicate, or remove and transfer to another item, and can be read and compared with prior readings with relative ease. The exact pattern of the defraction grating is mapped by diffraction moire techniques and recorded for comparison with future readings of the same grating.

  8. Identifying personal microbiomes using metagenomic codes.

    PubMed

    Franzosa, Eric A; Huang, Katherine; Meadow, James F; Gevers, Dirk; Lemon, Katherine P; Bohannan, Brendan J M; Huttenhower, Curtis

    2015-06-02

    Community composition within the human microbiome varies across individuals, but it remains unknown if this variation is sufficient to uniquely identify individuals within large populations or stable enough to identify them over time. We investigated this by developing a hitting set-based coding algorithm and applying it to the Human Microbiome Project population. Our approach defined body site-specific metagenomic codes: sets of microbial taxa or genes prioritized to uniquely and stably identify individuals. Codes capturing strain variation in clade-specific marker genes were able to distinguish among 100s of individuals at an initial sampling time point. In comparisons with follow-up samples collected 30-300 d later, ∼30% of individuals could still be uniquely pinpointed using metagenomic codes from a typical body site; coincidental (false positive) matches were rare. Codes based on the gut microbiome were exceptionally stable and pinpointed >80% of individuals. The failure of a code to match its owner at a later time point was largely explained by the loss of specific microbial strains (at current limits of detection) and was only weakly associated with the length of the sampling interval. In addition to highlighting patterns of temporal variation in the ecology of the human microbiome, this work demonstrates the feasibility of microbiome-based identifiability-a result with important ethical implications for microbiome study design. The datasets and code used in this work are available for download from huttenhower.sph.harvard.edu/idability.

  9. 34 CFR 300.32 - Personally identifiable.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 2 2013-07-01 2013-07-01 false Personally identifiable. 300.32 Section 300.32 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION...

  10. 34 CFR 300.32 - Personally identifiable.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 2 2014-07-01 2013-07-01 true Personally identifiable. 300.32 Section 300.32 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION ASSISTANCE TO STATES FOR THE EDUCATION OF CHILDREN...

  11. Identifying and Prioritizing Critical Hardwood Resources

    Treesearch

    Sam C. Doak; Sharon Johnson; Marlyce Myers

    1991-01-01

    A logical framework is required to provide a focus for the implementation of a variety of landowner incentive techniques in accordance with existing goals to protect and enhance hardwood resources. A system is presented for identifying and prioritizing critical hardwood resources for site specific conservation purposes. Flexibility is built into this system so that...

  12. Methods for Identifying Versioned and Plagiarized Documents.

    ERIC Educational Resources Information Center

    Hoad, Timothy C.; Zobel, Justin

    2003-01-01

    Describes research that was conducted to develop and evaluate techniques for identifying plagiarism, revisions, and different versions of online documents. Highlights include ranking; parsing; similarity measures; identity measures; fingerprinting documents; measuring effectiveness via recall and precision; and experiments on two document…

  13. Identifying Protein-Calorie Malnutrition Workshop.

    ERIC Educational Resources Information Center

    Walker, Susan S.; Barker, Ellen M.

    Instructional materials are provided for a workshop to enable participants to assist in identifying patients at risk with protein-calorie malnutrition and in corrrecting this nutritional deficiency. Representative topics are nutrients; protein, mineral, and vitamin sources, functions, and deficiency symptoms; malnutrition; nutritional deficiency…

  14. Identifying Benefit Segments among College Students.

    ERIC Educational Resources Information Center

    Brown, Joseph D.

    1991-01-01

    Using concept of market segmentation (dividing market into distinct groups requiring different product benefits), surveyed 398 college students to determine benefit segments among students selecting a college to attend and factors describing each benefit segment. Identified one major segment of students (classroomers) plus three minor segments…

  15. Identify Your Brand, Before You Market.

    ERIC Educational Resources Information Center

    Claggett, Laura

    2002-01-01

    Discusses marketing in special libraries and suggests that librarians need to identify library services that set them apart from others. Highlights include the competitive environment and alternatives for the consumer; value that the library offers; targeting consumers; return on investment; and determining why consumers choose your services. (LRW)

  16. Neoplasms identified in free-flying birds

    USGS Publications Warehouse

    Siegfried, L.M.

    1983-01-01

    Nine neoplasms were identified in carcasses of free-flying wild birds received at the National Wildlife Health Laboratory; gross and microscopic descriptions are reported herein. The prevalence of neoplasia in captive and free-flying birds is discussed, and lesions in the present cases are compared with those previously described in mammals and birds.

  17. How to Identify High-Growth Schools

    ERIC Educational Resources Information Center

    Pfeiffer, Linda E.

    2015-01-01

    When researching school options, parents may want to look for schools with high-growth scores which, according to research, may be indicators of other characteristics such as programming, leadership, culture, and size. This quick guide offers parents tips on how to identify high-growth schools and what to ask when evaluating school options. An…

  18. Identifying Teaching Methods that Engage Entrepreneurship Students

    ERIC Educational Resources Information Center

    Balan, Peter; Metcalfe, Mike

    2012-01-01

    Purpose: Entrepreneurship education particularly requires student engagement because of the complexity of the entrepreneurship process. The purpose of this paper is to describe how an established measure of engagement can be used to identify relevant teaching methods that could be used to engage any group of entrepreneurship students.…

  19. Identifying Mentors for Student Employees on Campus

    ERIC Educational Resources Information Center

    Frock, David

    2015-01-01

    Purpose: This exploratory research project aims to seek an effective process for identifying supervisors of part-time student employees who also serve in a mentoring capacity. Design/methodology/approach: This paper is based on a review of literature and an evaluation process focused on established traits and functions of mentoring as applied to…

  20. Problems identified with home infusion pumps.

    PubMed

    Koeppen, M A; Caspers, S M

    1994-01-01

    A variety of infusion pumps and devices are available on the market today. In this article, the authors examine these products based on questionnaires sent out to typical consumers, including hospitals and caregivers. Using the results of this questionnaire, the authors identify whether or not users of home infusion pumps and devices find them difficult to operate.

  1. Serial Item Contribution Identifier: New SISAC Code.

    ERIC Educational Resources Information Center

    Computers in Libraries, 1993

    1993-01-01

    Discusses the benefits of the Serial Item Contribution Identifier (SICI) standard for serials handling. Developed by the Serials Industry Systems Advisory Committee (SISAC), SICI applications through the use of a SISAC barcode are expected to benefit shipping, ordering, serials processing and claiming, document delivery, and information exchange.…

  2. Do Colleges Identify or Develop Intelligence?

    ERIC Educational Resources Information Center

    Moore, Randy

    2004-01-01

    Most colleges and universities emphasize identifying smartness much more than developing smartness. This value is made explicit in the many influential rankings of colleges and universities, in which elitist schools who recruit students with high SAT scores, grade point averages, and class rankings are declared "better" than other…

  3. Zebrafish phenotypic screen identifies novel Notch antagonists.

    PubMed

    Velaithan, Vithya; Okuda, Kazuhide Shaun; Ng, Mei Fong; Samat, Norazwana; Leong, Sze Wei; Faudzi, Siti Munirah Mohd; Abas, Faridah; Shaari, Khozirah; Cheong, Sok Ching; Tan, Pei Jean; Patel, Vyomesh

    2017-04-01

    Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27(KIP1). Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

  4. Identifying Depression in Students with Mental Retardation.

    ERIC Educational Resources Information Center

    Stough, Laura M.; Baker, Lynn

    1999-01-01

    Offers guidelines to teachers for identifying depression in students with mental retardation. Discusses prevalence and symptoms of depression, causes of depression, difficulty of diagnosis in students with mental retardation, detecting symptoms in the classroom, treatment of depression, and psychological services. Inserts list ideas for helping…

  5. Identifying Effectiveness Criteria for Internet Payment Systems.

    ERIC Educational Resources Information Center

    Shon, Tae-Hwan; Swatman, Paula M. C.

    1998-01-01

    Examines Internet payment systems (IPS): third-party, card, secure Web server, electronic token, financial electronic data interchange (EDI), and micropayment based. Reports the results of a Delphi survey of experts identifying and classifying IPS effectiveness criteria and classifying types of IPS providers. Includes the survey invitation letter…

  6. Identifying the Cognitive Decrements Caused By HIV

    DTIC Science & Technology

    1994-06-10

    regions with specific tests. Thus, probable sites of lesions or other forms of damage can be identified. Information processing tests generally cannot be...Relationship to severity of disease and brain atrophy. Acta Neurologica Scandinavica, 87, 88-94o Ratcliff, R. (1993). Methods for dealing with reaction time

  7. An Empirical Approach to Identifying Effective Schools.

    ERIC Educational Resources Information Center

    Webster, William J.; Olson, George H.

    One approach to identifying effective schools defines effectiveness in terms of student achievement in reading, mathematics, and language usage. Exceptional school achievement is indicated by performance above or below the level expected if students were merely to maintain their previous rate of growth. Regression analysis is used to compute…

  8. Identifying Protein-Calorie Malnutrition Workshop.

    ERIC Educational Resources Information Center

    Walker, Susan S.; Barker, Ellen M.

    Instructional materials are provided for a workshop to enable participants to assist in identifying patients at risk with protein-calorie malnutrition and in corrrecting this nutritional deficiency. Representative topics are nutrients; protein, mineral, and vitamin sources, functions, and deficiency symptoms; malnutrition; nutritional deficiency…

  9. A test to identify persistent picky eaters.

    PubMed

    Toyama, Hannah; Agras, W Stewart

    2016-12-01

    Picky eating is common and usually relatively brief as new foods are accepted. Persistent picky eating, however, is often associated with comorbid psychopathology. The aim of this study was to use parent-reported child feeding behaviors to identify which picky eaters persist. Participants were a subsample from the Stanford Infant Growth Study a prospective study of child development. Out of the 216 infants, 86 were identified as picky eaters. Picky eaters were separated into two groups using a median split: short-term (n=40) and persistent picky eaters (n=46). Recursive Partitioning detected three significant parent-reported feeding questions that may identify persistent picky eaters at an early age: Is your child a picky eater? (yes), does s/he have strong likes with regard to food (yes), does your child accept new foods readily? (no). These results provide a first step allowing providers to identify persistent picky eaters and possibly enable intervention at an early age. Further studies are needed to replicate and extend these findings in another sample of picky eaters. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Identifying the Knowledge Base for School Improvement

    ERIC Educational Resources Information Center

    Beach, Robert H.; Lindahl, Ron

    2004-01-01

    In 1983, a landmark event occurred in public education in the US: the National Commission on Excellence in Education published its now famous report, A Nation at Risk. The report was generally regarded as the catalyst for decades of public and political pressure for large-scale school improvement or reform. Here, Beach and Lindahl identify a…

  11. Embedded sensor having an identifiable orientation

    DOEpatents

    Bennett, Thomas E.; Nelson, Drew V.

    2002-01-01

    An apparatus and method is described wherein a sensor, such as a mechanical strain sensor, embedded in a fiber core, is "flagged" to identify a preferred orientation of the sensor. The identifying "flag" is a composite material, comprising a plurality of non-woven filaments distributed in a resin matrix, forming a small planar tab. The fiber is first subjected to a stimulus to identify the orientation providing the desired signal response, and then sandwiched between first and second layers of the composite material. The fiber, and therefore, the sensor orientation is thereby captured and fixed in place. The process for achieving the oriented fiber includes, after identifying the fiber orientation, carefully laying the oriented fiber onto the first layer of composite, moderately heating the assembled layer for a short period in order to bring the composite resin to a "tacky" state, heating the second composite layer as the first, and assembling the two layers together such that they merge to form a single consolidated block. The consolidated block achieving a roughly uniform distribution of composite filaments near the embedded fiber such that excess resin is prevented from "pooling" around the periphery of the fiber.

  12. Identifying Barriers to Study Abroad Program Participation

    ERIC Educational Resources Information Center

    McKinley, Karen E.

    2014-01-01

    University administrators, industry professionals, and government leaders encourage college students to participate in study abroad programs. Despite an increase in the number of students going abroad, the percentage of students participating in global programs remain low. This study identified barriers to study abroad program participation at a…

  13. Identifying marker typing incompatibilities in linkage analysis

    SciTech Connect

    Stringham, H.M.; Boehnke, M.

    1996-10-01

    A common problem encountered in linkage analyses is that execution of the computer program is halted because of genotypes in the data that are inconsistent with Mendelian inheritance. Such inconsistencies may arise because of pedigree errors or errors in typing. In some cases, the source of the inconsistencies is easily identified by examining the pedigree. In others, the error is not obvious, and substantial time and effort are required to identify the responsible genotypes. We have developed two methods for automatically identifying those individuals whose genotypes are most likely the cause of the inconsistencies. First, we calculate the posterior probability of genotyping error for each member of the pedigree, given the marker data on all pedigree members and allowing anyone in the pedigree to have an error. Second, we identify those individuals whose genotypes could be solely responsible for the inconsistency in the pedigree. We illustrate these methods with two examples: one a pedigree error, the second a genotyping error. These methods have been implemented as a module of the pedigree analysis program package MENDEL. 9 refs., 2 figs., 2 tabs.

  14. Professional Development: Identifying Effective Instructional Coaching Activities

    ERIC Educational Resources Information Center

    Mannino, Gina

    2014-01-01

    The purpose of this study was to identify the instructional coaching activities most used by instructional coaches in southeast Texas school districts and to test if there was a relationship between the use of instructional coaching and perceived improvement in the instructional practices of teachers and student achievement. The participants for…

  15. Data Identifiers and Citations Enable Reproducible Science

    NASA Astrophysics Data System (ADS)

    Tilmes, C.

    2011-12-01

    Modern science often involves data processing with tremendous volumes of data. Keeping track of that data has been a growing challenge for data center. Researchers who access and use that data don't always reference and cite their data sources adequately for consumers of their research to follow their methodology or reproduce their analyses or experiments. Recent research has led to recommendations for good identifiers and citations that can help address this problem. This paper will describe some of the best practices in data identifiers, reference and citation. Using a simplified example scenario based on a long term remote sensing satellite mission, it will explore issues in identifying dynamic data sets and the importance of good data citations for reproducibility. It will describe the difference between granule and collection level identifiers, using UUIDs and DOIs to illustrate some recommendations for developing identifiers and assigning them during data processing. As data processors create data products, the provenance of the input products and precise steps that led to their creation are recorded and published for users of the data to see. As researchers access the data from an archive, they can use the provenance to help understand the genesis of the data, which could have effects on their usage of the data. By citing the data on publishing their research, others can retrieve the precise data used in their research and reproduce the analyses and experiments to confirm the results. Describing the experiment to a sufficient extent to reproduce the research enforces a formal approach that lends credibility to the results, and ultimately, to the policies of decision makers depending on that research.

  16. Identifying data sharing in biomedical literature.

    PubMed

    Piwowar, Heather A; Chapman, Wendy W; Chapman, Wendy

    2008-11-06

    Many policies and projects now encourage investigators to share their raw research data with other scientists. Unfortunately, it is difficult to measure the effectiveness of these initiatives because data can be shared in such a variety of mechanisms and locations. We propose a novel approach to finding shared datasets: using NLP techniques to identify declarations of dataset sharing within the full text of primary research articles. Using regular expression patterns and machine learning algorithms on open access biomedical literature, our system was able to identify 61% of articles with shared datasets with 80% precision. A simpler version of our classifier achieved higher recall (86%), though lower precision (49%). We believe our results demonstrate the feasibility of this approach and hope to inspire further study of dataset retrieval techniques and policy evaluation.

  17. Trustworthy persistent identifier systems of the future

    NASA Astrophysics Data System (ADS)

    Golodoniuc, Pavel; Klump, Jens; Car, Nicholas

    2016-04-01

    Over the last two decades, persistent identifier (PID) systems have seen some significant changes in their governance policies, system capabilities, and technology. The development of most systems was driven by two main application areas, namely archives and libraries. Guidelines and criteria for trustworthy PID systems have been clearly devised (Bütikofer, 2009) and many PID system implementations for the identification of static digital objects have been built (e.g., PURL). However systems delivering persistent identifiers for dynamic datasets are not yet mature. There has been a rapid proliferation of different PID systems caused by the specific technical or organisational requirements of various communities that could not be met by existing systems such as DOI, ISBN, and EAN. Many of these different systems were limited by their inability to provide native means of persistent identifier resolution. This has prompted a decoupling of PID-associated data from the resolution service and this is where the Handle system has played a significant role. The Handle allowed to build a distributed system of independently managed resolver services. A trustworthy PID system must be designed to outlive the objects it provides persistent identifiers for, which may cease to exist or otherwise be deprecated, and the technology used to implement it, which will certainly need to change with time. We propose that such a system should rest on four pillars of agreements - (i) definitions, (ii) policies, (iii) services, and (iv) data services, to ensure longevity. While we believe all four pillars are equally important, we intentionally leave regulating aspects of issuing of identifiers and their registration out of the scope of this paper and focus on the agreements that have to be established between PID resolver services and the data sources indicated by the persistent identifiers. We propose an approach to development of PID systems that combines the use of (a) the Handle system

  18. Identifying essential genes in Arabidopsis thaliana.

    PubMed

    Meinke, David; Muralla, Rosanna; Sweeney, Colleen; Dickerman, Allan

    2008-09-01

    Eight years after publication of the Arabidopsis genome sequence and two years before completing the first phase of an international effort to characterize the function of every Arabidopsis gene, plant biologists remain unable to provide a definitive answer to the following basic question: what is the minimal gene set required for normal growth and development? The purpose of this review is to summarize different strategies employed to identify essential genes in Arabidopsis, an important component of the minimal gene set in plants, to present an overview of the datasets and specific genes identified to date, and to discuss the prospects for future saturation of this important class of genes. The long-term goal of this collaborative effort is to facilitate basic research in plant biology and complement ongoing research with other model organisms.

  19. Framework for Identifying Cybersecurity Risks in Manufacturing

    DOE PAGES

    Hutchins, Margot J.; Bhinge, Raunak; Micali, Maxwell K.; ...

    2015-10-21

    Increasing connectivity, use of digital computation, and off-site data storage provide potential for dramatic improvements in manufacturing productivity, quality, and cost. However, there are also risks associated with the increased volume and pervasiveness of data that are generated and potentially accessible to competitors or adversaries. Enterprises have experienced cyber attacks that exfiltrate confidential and/or proprietary data, alter information to cause an unexpected or unwanted effect, and destroy capital assets. Manufacturers need tools to incorporate these risks into their existing risk management processes. This article establishes a framework that considers the data flows within a manufacturing enterprise and throughout its supplymore » chain. The framework provides several mechanisms for identifying generic and manufacturing-specific vulnerabilities and is illustrated with details pertinent to an automotive manufacturer. Finally, in addition to providing manufacturers with insights into their potential data risks, this framework addresses an outcome identified by the NIST Cybersecurity Framework.« less

  20. Guidelines for identifying suspect/counterfeit material

    SciTech Connect

    1995-09-01

    These guidelines are intended to assist users of products in identifying: substandard, misrepresented, or fraudulently marked items. The guidelines provide information about such topics as: precautions, inspection and testing, dispositioning identified items, installed inspection and reporting suspect/counterfeit materials. These guidelines apply to users who are developing procurement documents, product acceptance/verification methods, company procedures, work instructions, etc. The intent of these SM guidelines in relation to the Quality Assurance Program Description (QAPD) and implementing company Management Control Procedures is not to substitute or replace existing requirements, as defined in either the QAPD or company implementing instructions (Management Control Procedures). Instead, the guidelines are intended to provide a consolidated source of information addressing the issue of Suspect/Counterfeit materials. These guidelines provide an extensive suspect component listing and suspect indications listing. Users can quickly check their suspect items against the list of manufacturers products (i.e., type, LD. number, and nameplate information) by consulting either of these listings.

  1. Framework for Identifying Cybersecurity Risks in Manufacturing

    SciTech Connect

    Hutchins, Margot J.; Bhinge, Raunak; Micali, Maxwell K.; Robinson, Stefanie L.; Sutherland, John W.; Dornfeld, David

    2015-10-21

    Increasing connectivity, use of digital computation, and off-site data storage provide potential for dramatic improvements in manufacturing productivity, quality, and cost. However, there are also risks associated with the increased volume and pervasiveness of data that are generated and potentially accessible to competitors or adversaries. Enterprises have experienced cyber attacks that exfiltrate confidential and/or proprietary data, alter information to cause an unexpected or unwanted effect, and destroy capital assets. Manufacturers need tools to incorporate these risks into their existing risk management processes. This article establishes a framework that considers the data flows within a manufacturing enterprise and throughout its supply chain. The framework provides several mechanisms for identifying generic and manufacturing-specific vulnerabilities and is illustrated with details pertinent to an automotive manufacturer. Finally, in addition to providing manufacturers with insights into their potential data risks, this framework addresses an outcome identified by the NIST Cybersecurity Framework.

  2. Persistent Identifiers for Dutch cultural heritage institutions

    NASA Astrophysics Data System (ADS)

    Ras, Marcel; Kruithof, Gijsbert

    2016-04-01

    Over the past years, more and more collections belonging to archives, libraries, media, museums, and knowledge institutes are being digitised and made available online. These are exciting times for ALM institutions. They are realising that, in the information society, their collections are goldmines. Unfortunately most heritage institutions in the Netherlands do not yet meet the basic preconditions for long-term availability of their collections. The digital objects often have no long lasting fixed reference yet. URL's and web addresses change. Some digital objects that were referenced in Europeana and other portals can no longer be found. References in scientific articles have a very short life span, which is damaging for scholarly research. In 2015, the Dutch Digital Heritage Network (NDE) has started a two-year work program to co-ordinate existing initiatives in order to improve the (long-term) accessibility of the Dutch digital heritage for a wide range of users, anytime, anyplace. The Digital Heritage Network is a partnership established on the initiative of the Ministry of Education, Culture and Science. The members of the NDE are large, national institutions that strive to professionally preserve and manage digital data, e.g. the National Library, The Netherlands Institute for Sound and Vision, the Netherlands Cultural Heritage Agency, the Royal Netherlands Academy of Arts and Sciences, the National Archive of the Netherlands and the DEN Foundation, and a growing number of associations and individuals both within and outside the heritage sector. By means of three work programmes the goals of the Network should be accomplished and improve the visibility, the usability and the sustainability of digital heritage. Each programme contains of a set of projects. Within the sustainability program a project on creating a model for persistent identifiers is taking place. The main goals of the project are (1) raise awareness among cultural heritage institutions on the

  3. Wham: Identifying Structural Variants of Biological Consequence

    PubMed Central

    Kronenberg, Zev N.; Osborne, Edward J.; Cone, Kelsey R.; Kennedy, Brett J.; Domyan, Eric T.; Shapiro, Michael D.; Elde, Nels C.; Yandell, Mark

    2015-01-01

    Existing methods for identifying structural variants (SVs) from short read datasets are inaccurate. This complicates disease-gene identification and efforts to understand the consequences of genetic variation. In response, we have created Wham (Whole-genome Alignment Metrics) to provide a single, integrated framework for both structural variant calling and association testing, thereby bypassing many of the difficulties that currently frustrate attempts to employ SVs in association testing. Here we describe Wham, benchmark it against three other widely used SV identification tools–Lumpy, Delly and SoftSearch–and demonstrate Wham’s ability to identify and associate SVs with phenotypes using data from humans, domestic pigeons, and vaccinia virus. Wham and all associated software are covered under the MIT License and can be freely downloaded from github (https://github.com/zeeev/wham), with documentation on a wiki (http://zeeev.github.io/wham/). For community support please post questions to https://www.biostars.org/. PMID:26625158

  4. Identifying Groundwater Recharge in Arid Regions

    NASA Astrophysics Data System (ADS)

    Thomas, B. F.; Famiglietti, J. S.

    2015-12-01

    Recharge epodicity in arid regions provides a method to estimate annual groundwater recharge given a relationship expressed as the recharge to precipitation ratio. Traditionally, in-situ observations are required to identify aquifer recharge events, while more advanced approaches such as the water-table fluctuation method or the episodic master recession method are necessary to delineate the recharge event. Our study uses the Gravity Recovery and Climate Experiment (GRACE) observations to estimate monthly changes in groundwater storage which are attributed to the combination of groundwater abstraction and episodic recharge in the arid southwestern United States. Our results illustrate the ability of remote sensing technologies to identify episodic groundwater recharge in arid regions which can be used within sustainable groundwater management frameworks to effectively manage groundwater resources.

  5. Wham: Identifying Structural Variants of Biological Consequence.

    PubMed

    Kronenberg, Zev N; Osborne, Edward J; Cone, Kelsey R; Kennedy, Brett J; Domyan, Eric T; Shapiro, Michael D; Elde, Nels C; Yandell, Mark

    2015-12-01

    Existing methods for identifying structural variants (SVs) from short read datasets are inaccurate. This complicates disease-gene identification and efforts to understand the consequences of genetic variation. In response, we have created Wham (Whole-genome Alignment Metrics) to provide a single, integrated framework for both structural variant calling and association testing, thereby bypassing many of the difficulties that currently frustrate attempts to employ SVs in association testing. Here we describe Wham, benchmark it against three other widely used SV identification tools-Lumpy, Delly and SoftSearch-and demonstrate Wham's ability to identify and associate SVs with phenotypes using data from humans, domestic pigeons, and vaccinia virus. Wham and all associated software are covered under the MIT License and can be freely downloaded from github (https://github.com/zeeev/wham), with documentation on a wiki (http://zeeev.github.io/wham/). For community support please post questions to https://www.biostars.org/.

  6. Identifying Potential Noise Sources within Acoustic Signals

    NASA Astrophysics Data System (ADS)

    Holcomb, Victoria; Lewalle, Jacques

    2013-11-01

    We test a new algorithm for its ability to detect sources of noise within random background. The goal of these tests is to better understand how to identify sources within acoustic signals while simultaneously determining the strengths and weaknesses of the algorithm in question. Unlike previously published algorithms, the antenna method does not pinpoint events by looking for the most energetic portions of a signal. The algorithm searches for the ideal lag combinations between three signals by taking excerpts of possible events. The excerpt with the lowest calculated minimum distance between possible events is how the algorithm identifies sources. At the minimum distance, the events are close in time and frequency. This method can be compared to the cross correlation and denoising methods to better understand its effectiveness. This work is supported in part by Spectral Energies LLC, under an SBIR grant from AFRL, as well as the Syracuse University MAE department.

  7. Identifying sex and age of akiapolaau

    USGS Publications Warehouse

    Pratt, T.K.; Fancy, S.G.; Harada, C.K.; Lindsey, G.D.; Jacobi, J.D.

    1994-01-01

    Methods for identifying the sex and age of the Akiapolaau (Hemignathus munroi), an endangered honeycreeper found only on the island of Hawaii, were developed by examination and measurement of 73 museum specimens and 24 live birds captured in mist nests. Akiapolaau probably undergo a single annual molt, with most birds molting between February and July. The mottled juvenal plumage is replaced by a first basic plumage characterized by yellowish-gray or yellowish-green underparts and often by retained wingbars. Male Akiapolaau may not attain adult plumage until their third molt. In adult females, only the throat and upper breast become yellow, whereas in adult males the superciliaries, cheeks, and entire underparts are yellow. Adult males have greater exposed culmen, gonys, wing chord, tail, and tarsus lengths than do females. Akiapolaau in first prebasic molt or older can be identified as to sex by culmen length, that of males being >23.4 mm.

  8. Chemical Proteomic Platform To Identify Citrullinated Proteins

    PubMed Central

    2015-01-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases. PMID:26360112

  9. Using filtering effects to identify objects

    NASA Astrophysics Data System (ADS)

    Carroll, T. L.; Rachford, Frederic J.

    2012-06-01

    Reflecting signals off of targets is a method widely used to locate objects, but the reflected signal also contains information that can be used to identify the object. In radar or sonar, the signal amplitudes used are small enough that only linear effects are present, so we can consider the effect of the target on the signal as a linear filter. Using the known effects of linear filters on chaotic signals, we can create a reference that allows us to match a particular target to a particular reflected signal. Furthermore, if some parts of this "filter" vary only slowly as the aspect angle of the object changes, we can produce a reference that averages out the parts that are highly angle dependent so that one reference can be used to identify the target over a range of angles.

  10. Identifying Cellular and Molecular Mechanisms for Magnetosensation

    PubMed Central

    Clites, Benjamin L.; Pierce, Jonathan T.

    2017-01-01

    Diverse animals ranging from worms and insects to birds and turtles perf orm impressive journeys using the magnetic field of the earth as a cue. Although major cellular and molecular mechanisms for sensing mechanical and chemical cues have been elucidated over the past three decades, the mechanisms that animals use to sense magnetic fields remain largely mysterious. Here we survey progress on the search for magnetosensory neurons and magnetosensitive molecules important for animal behaviors. Emphasis is placed on magnetosensation in insects and birds, as well as on the magnetosensitive neuron pair AFD in the nematode Caenorhabditis elegans. We also review conventional criteria used to define animal magnetoreceptors and suggest how approaches used to identify receptors for other sensory modalities may be adapted for magnetoreceptors. Finally, we discuss prospects for under-utilized and novel approaches to identify the elusive magnetoreceptors in animals. PMID:28772099

  11. Identifying bilingual semantic neural representations across languages

    PubMed Central

    Buchweitz, Augusto; Shinkareva, Svetlana V.; Mason, Robert A.; Mitchell, Tom M.; Just, Marcel Adam

    2015-01-01

    The goal of the study was to identify the neural representation of a noun's meaning in one language based on the neural representation of that same noun in another language. Machine learning methods were used to train classifiers to identify which individual noun bilingual participants were thinking about in one language based solely on their brain activation in the other language. The study shows reliable (p < .05) pattern-based classification accuracies for the classification of brain activity for nouns across languages. It also shows that the stable voxels used to classify the brain activation were located in areas associated with encoding information about semantic dimensions of the words in the study. The identification of the semantic trace of individual nouns from the pattern of cortical activity demonstrates the existence of a multi-voxel pattern of activation across the cortex for a single noun common to both languages in bilinguals. PMID:21978845

  12. Identifying a novel locus for psoriatic arthritis

    PubMed Central

    Budu-Aggrey, Ashley; Bowes, John

    2016-01-01

    A number of studies have identified genetic risk loci for PsA, the majority of which also confer risk for psoriasis. The stronger heritability of PsA in comparison with psoriasis suggests that there should be risk loci that are specific for PsA. Identifying such loci could potentially inform therapy development to provide more effective treatments for PsA patients, especially with a considerable proportion being non-responsive to current therapies. Evidence of a PsA-specific locus has been previously found at HLA-B27 within the MHC region. A recent study has provided evidence of non-HLA risk loci that are specific for PsA at IL23R, PTPN22 and on chromosome 5q31. Functional characterization of these loci will provide further understanding of the pathways underlying PsA, and enable us to apply genetic findings for patient benefit. PMID:26255310

  13. Identifiability of linear systems in physical coordinates

    NASA Technical Reports Server (NTRS)

    Su, Tzu-Jeng; Juang, Jer-Nan

    1992-01-01

    Identifiability of linear, time-invariant systems in physical coordinates is discussed. It is shown that identification of the system matrix in physical coordinates can be accomplished by determining a transformation matrix that relates the physical locations of actuators and sensors to the test-data-derived input and output matrices. For systems with symmetric matrices, the solution of a constrained optimization problem is used to characterize all the possible solutions of the transformation matrix. Conditions for the existence of a unique transformation matrix are established easily from the explicit form of the solutions. For systems with limited inputs and outputs, the question about which part of the system can be uniquely identified is also answered. A simple mass-spring system is used to verify the conclusions of this study.

  14. Identifying Proper Names in Parallel Medical Terminologies

    PubMed Central

    Bodenreider, Olivier; Zweigenbaum, Pierre

    2006-01-01

    We propose several criteria to identify proper names in biomedical terminologies. Traditional, pattern-based methods that rely on the immediate context of a proper name are not applicable. However, the availability of translations of some terminologies supports methods based on invariant words instead. A combination of five criteria achieved 86% precision and 88% recall on the 16,401 word forms of the International Classification of Diseases. PMID:11187591

  15. Which functional unit to identify sustainable foods?

    PubMed

    Masset, Gabriel; Vieux, Florent; Darmon, Nicole

    2015-09-01

    In life-cycle assessment, the functional unit defines the unit for calculation of environmental indicators. The objective of the present study was to assess the influence of two functional units, 100 g and 100 kcal (420 kJ), on the associations between three dimensions for identifying sustainable foods, namely environmental impact (via greenhouse gas emissions (GHGE)), nutritional quality (using two distinct nutrient profiling systems) and price. GHGE and price data were collected for individual foods, and were each expressed per 100 g and per 100 kcal. Two nutrient profiling models, SAIN,LIM and UK Ofcom, were used to assess foods' nutritional quality. Spearman correlations were used to assess associations between variables. Sustainable foods were identified as those having more favourable values for all three dimensions. The French Individual and National Dietary Survey (INCA2), 2006-2007. Three hundred and seventy-three foods highly consumed in INCA2, covering 65 % of total energy intake of adult participants. When GHGE and price were expressed per 100 g, low-GHGE foods had a lower price and higher SAIN,LIM and Ofcom scores (r=0·59, -0·34 and -0·43, respectively), suggesting a compatibility between the three dimensions; 101 and 100 sustainable foods were identified with SAIN,LIM and Ofcom, respectively. When GHGE and price were expressed per 100 kcal, low-GHGE foods had a lower price but also lower SAIN,LIM and Ofcom scores (r=0·67, 0·51 and 0·47, respectively), suggesting that more environment-friendly foods were less expensive but also less healthy; thirty-four sustainable foods were identified with both SAIN,LIM and Ofcom. The choice of functional unit strongly influenced the compatibility between the sustainability dimensions and the identification of sustainable foods.

  16. Identifying Topics in Microblogs Using Wikipedia

    PubMed Central

    Yıldırım, Ahmet; Üsküdarlı, Suzan; Özgür, Arzucan

    2016-01-01

    Twitter is an extremely high volume platform for user generated contributions regarding any topic. The wealth of content created at real-time in massive quantities calls for automated approaches to identify the topics of the contributions. Such topics can be utilized in numerous ways, such as public opinion mining, marketing, entertainment, and disaster management. Towards this end, approaches to relate single or partial posts to knowledge base items have been proposed. However, in microblogging systems like Twitter, topics emerge from the culmination of a large number of contributions. Therefore, identifying topics based on collections of posts, where individual posts contribute to some aspect of the greater topic is necessary. Models, such as Latent Dirichlet Allocation (LDA), propose algorithms for relating collections of posts to sets of keywords that represent underlying topics. In these approaches, figuring out what the specific topic(s) the keyword sets represent remains as a separate task. Another issue in topic detection is the scope, which is often limited to specific domain, such as health. This work proposes an approach for identifying domain-independent specific topics related to sets of posts. In this approach, individual posts are processed and then aggregated to identify key tokens, which are then mapped to specific topics. Wikipedia article titles are selected to represent topics, since they are up to date, user-generated, sophisticated articles that span topics of human interest. This paper describes the proposed approach, a prototype implementation, and a case study based on data gathered during the heavily contributed periods corresponding to the four US election debates in 2012. The manually evaluated results (0.96 precision) and other observations from the study are discussed in detail. PMID:26991442

  17. Data Identifiers, Versioning, and Micro-citation

    NASA Astrophysics Data System (ADS)

    Parsons, M. A.; Duerr, R. E.

    2012-12-01

    Data citation, especially using Digital Object Identifiers (DOIs), is an increasingly accepted scientific practice. For example, the AGU Council asserts that data "publications" should "be credited and cited like the products of any other scientific activity," and Thomson Reuters has recently announced a data citation index built from DOIs assigned to data sets. Correspondingly, formal guidelines for how to cite a data set (using DOIs or similar identifiers/locators) have recently emerged, notably those from the international DataCite consortium, the UK Digital Curation Centre, and the US Federation of Earth Science Information Partners. These different data citation guidelines are largely congruent. They agree on the basic practice and elements of data citation, especially for relatively static, whole data collections. There is less agreement on some of the more subtle nuances of data citation. They define different methods for handling different data set versions, especially for the very dynamic, growing data sets that are common in Earth Sciences. They also differ in how people should cite specific, arbitrarily large elements, "passages," or subsets of a larger data collection, i.e., the precise data records actually used in a study. This detailed "micro-citation", and careful reference to exact versions of data are essential to ensure scientific reproducibility. Identifiers such as DOIs are necessary but not sufficient for the precise, detailed, references necessary. Careful practice must be coupled with the use of curated identifiers. In this paper we review the pros and cons of different approaches to versioning and micro-citation. We suggest a workable solution for most existing Earth science data and suggest a more rigorous path forward for the future.

  18. Diffraction gratings used as identifying markers

    DOEpatents

    Deason, V.A.; Ward, M.B.

    1991-03-26

    A finely detailed diffraction grating is applied to an object as an identifier or tag which is unambiguous, difficult to duplicate, or remove and transfer to another item, and can be read and compared with prior readings with relative ease. The exact pattern of the diffraction grating is mapped by diffraction moire techniques and recorded for comparison with future readings of the same grating. 7 figures.

  19. Diffraction gratings used as identifying markers

    SciTech Connect

    Deason, V.A.; Ward, M.B.

    1989-08-03

    A finely detailed defraction grating is applied to an object as an identifier or tag which is unambiguous, difficult to duplicate, or remove and transfer to another item, and can be read and compared with prior readings with relative ease. The exact pattern of the defraction grating is mapped by diffraction moire techniques and recorded for comparison with future readings of the same grating. 7 figs.

  20. Identifying spatial priorities for protecting ecosystem services

    PubMed Central

    Luck, Gary W

    2012-01-01

    Priorities for protecting ecosystem services must be identified to ensure future human well-being. Approaches to broad-scale spatial prioritization of ecosystem services are becoming increasingly popular and are a vital precursor to identifying locations where further detailed analyses of the management of ecosystem services is required (e.g., examining trade-offs among management actions). Prioritization approaches often examine the spatial congruence between priorities for protecting ecosystem services and priorities for protecting biodiversity; therefore, the spatial prioritization method used is crucial because it will influence the alignment of service protection and conservation goals. While spatial prioritization of ecosystem services and prioritization for conservation share similarities, such as the need to document threats and costs, the former differs substantially from the latter owing to the requirement to measure the following components: supply of services; availability of human-derived alternatives to service provision; capacity to meet beneficiary demand; and site dependency in and scale of service delivery. We review studies that identify broad-scale spatial priorities for managing ecosystem services and demonstrate that researchers have used different approaches and included various measures for identifying priorities, and most studies do not consider all of the components listed above. We describe a conceptual framework for integrating each of these components into spatial prioritization of ecosystem services and illustrate our approach using a worked example for water provision. A fuller characterization of the biophysical and social context for ecosystem services that we call for should improve future prioritization and the identification of locations where ecosystem-service management is especially important or cost effective. PMID:24555017

  1. Laser Weather Identifier: Present and Future.

    NASA Astrophysics Data System (ADS)

    Wang, Ting-I.; Lataitis, R.; Lawrence, R. S.; Ochs, G. R.

    1982-11-01

    Prototype Laser Weather Identifier (LWI) systems designed to detect fog, rain and snow were tested for several months at Stapleton International Airport in Denver, and at the AFGL Weather Test Facility at Otis Air Force Base, Massachusetts. We present a detailed analysis of the performance of these systems, compared with human weather observations and tipping-bucket raingages, and suggest modifications for future operational instruments.

  2. Identifying Topics in Microblogs Using Wikipedia.

    PubMed

    Yıldırım, Ahmet; Üsküdarlı, Suzan; Özgür, Arzucan

    2016-01-01

    Twitter is an extremely high volume platform for user generated contributions regarding any topic. The wealth of content created at real-time in massive quantities calls for automated approaches to identify the topics of the contributions. Such topics can be utilized in numerous ways, such as public opinion mining, marketing, entertainment, and disaster management. Towards this end, approaches to relate single or partial posts to knowledge base items have been proposed. However, in microblogging systems like Twitter, topics emerge from the culmination of a large number of contributions. Therefore, identifying topics based on collections of posts, where individual posts contribute to some aspect of the greater topic is necessary. Models, such as Latent Dirichlet Allocation (LDA), propose algorithms for relating collections of posts to sets of keywords that represent underlying topics. In these approaches, figuring out what the specific topic(s) the keyword sets represent remains as a separate task. Another issue in topic detection is the scope, which is often limited to specific domain, such as health. This work proposes an approach for identifying domain-independent specific topics related to sets of posts. In this approach, individual posts are processed and then aggregated to identify key tokens, which are then mapped to specific topics. Wikipedia article titles are selected to represent topics, since they are up to date, user-generated, sophisticated articles that span topics of human interest. This paper describes the proposed approach, a prototype implementation, and a case study based on data gathered during the heavily contributed periods corresponding to the four US election debates in 2012. The manually evaluated results (0.96 precision) and other observations from the study are discussed in detail.

  3. Identify needs of women living with HIV.

    PubMed

    Jelliman, Pauline

    This article reports on the development of the Between the Sheets project for women living with HIV. It began with an event that provided a forum to address their unmet needs concerning sexual activity and related issues. Development was informed by a focus group of women with HIV, some of whom also presented their personal stories. Attendees' evaluations have identified further unmet needs and ways HIV services might adapt to better meet the needs of these individuals.

  4. Identifying Fossil Bacteria in Martian Materials

    NASA Technical Reports Server (NTRS)

    Westall, F.; McKay, D. S.; Gibson, E. K., Jr.

    1999-01-01

    Within the next decade, robotic missions are going to Mars with the search for evidence for extant and extinct life as at least one of the mission objectives. Moreover, the first Martian samples will be returned to Earth in 2008. It is therefore imperative that we can be certain that we can identify life in Martian rocks. In this paper we will not be discussing extant life but will concentrate on fossil life.

  5. Identifying Fossil Bacteria in Martian Materials

    NASA Technical Reports Server (NTRS)

    Westall, F.; McKay, D. S.; Gibson, E. K., Jr.

    1999-01-01

    Within the next decade, robotic missions are going to Mars with the search for evidence for extant and extinct life as at least one of the mission objectives. Moreover, the first Martian samples will be returned to Earth in 2008. It is therefore imperative that we can be certain that we can identify life in Martian rocks. In this paper we will not be discussing extant life but will concentrate on fossil life.

  6. Interrogator system for identifying electrical circuits

    DOEpatents

    Jatko, William B.; McNeilly, David R.

    1988-01-01

    A system for interrogating electrical leads to correctly ascertain the identity of equipment attached to remote ends of the leads. The system includes a source of a carrier signal generated in a controller/receiver to be sent over the leads and an identifier unit at the equipment. The identifier is activated by command of the carrier and uses a portion of the carrier to produce a supply voltage. Each identifier is uniquely programmed for a specific piece of equipment, and causes the impedance of the circuit to be modified whereby the carrier signal is modulated according to that program. The modulation can be amplitude, frequency or phase modulation. A demodulator in the controller/receiver analyzes the modulated carrier signal, and if a verified signal is recognized displays and/or records the information. This information can be utilized in a computer system to prepare a wiring diagram of the electrical equipment attached to specific leads. Specific circuit values are given for amplitude modulation, and the system is particularly described for use with thermocouples.

  7. Identifying duplicate content using statistically improbable phrases

    PubMed Central

    Errami, Mounir; Sun, Zhaohui; George, Angela C.; Long, Tara C.; Skinner, Michael A.; Wren, Jonathan D.; Garner, Harold R.

    2010-01-01

    Motivation: Document similarity metrics such as PubMed's ‘Find related articles’ feature, which have been primarily used to identify studies with similar topics, can now also be used to detect duplicated or potentially plagiarized papers within literature reference databases. However, the CPU-intensive nature of document comparison has limited MEDLINE text similarity studies to the comparison of abstracts, which constitute only a small fraction of a publication's total text. Extending searches to include text archived by online search engines would drastically increase comparison ability. For large-scale studies, submitting short phrases encased in direct quotes to search engines for exact matches would be optimal for both individual queries and programmatic interfaces. We have derived a method of analyzing statistically improbable phrases (SIPs) for assistance in identifying duplicate content. Results: When applied to MEDLINE citations, this method substantially improves upon previous algorithms in the detection of duplication citations, yielding a precision and recall of 78.9% (versus 50.3% for eTBLAST) and 99.6% (versus 99.8% for eTBLAST), respectively. Availability: Similar citations identified by this work are freely accessible in the Déjà vu database, under the SIP discovery method category at http://dejavu.vbi.vt.edu/dejavu/ Contact: merrami@collin.edu PMID:20472545

  8. Identifying chemicals that are planetary boundary threats.

    PubMed

    MacLeod, Matthew; Breitholtz, Magnus; Cousins, Ian T; de Wit, Cynthia A; Persson, Linn M; Rudén, Christina; McLachlan, Michael S

    2014-10-07

    Rockström et al. proposed a set of planetary boundaries that delimit a "safe operating space for humanity". Many of the planetary boundaries that have so far been identified are determined by chemical agents. Other chemical pollution-related planetary boundaries likely exist, but are currently unknown. A chemical poses an unknown planetary boundary threat if it simultaneously fulfills three conditions: (1) it has an unknown disruptive effect on a vital Earth system process; (2) the disruptive effect is not discovered until it is a problem at the global scale, and (3) the effect is not readily reversible. In this paper, we outline scenarios in which chemicals could fulfill each of the three conditions, then use the scenarios as the basis to define chemical profiles that fit each scenario. The chemical profiles are defined in terms of the nature of the effect of the chemical and the nature of exposure of the environment to the chemical. Prioritization of chemicals in commerce against some of the profiles appears feasible, but there are considerable uncertainties and scientific challenges that must be addressed. Most challenging is prioritizing chemicals for their potential to have a currently unknown effect on a vital Earth system process. We conclude that the most effective strategy currently available to identify chemicals that are planetary boundary threats is prioritization against profiles defined in terms of environmental exposure combined with monitoring and study of the biogeochemical processes that underlie vital Earth system processes to identify currently unknown disruptive effects.

  9. Anesthesiology leadership rounding: identifying opportunities for improvement.

    PubMed

    Gravenstein, Dietrich; Ford, Susan; Enneking, F Kayser

    2012-01-01

    Rounding that includes participation of individuals with authority to implement changes has been advocated as important to the transformation of an institution into a high-quality and safe organization. We describe a Department of Anesthesiology's experience with leadership rounding. The Department Chair or other senior faculty designate, a quality coordinator, up to four residents, the ward charge nurse, and patient nurses participated in rounds at bedsides. During a 23-month period, 14 significant opportunities to improve care were identified. Nurses identified 5 of these opportunities, primary team physicians 2, the rounding team 4, and patients or their family members another 3. The anesthesiology service had sole or shared responsibility for 10 improvements. A variety of organizations track specific measures across all phases of the patient experience to gauge quality of care. Chart auditing tools for detecting threats to safety are often used. These measures and tools missed opportunities for improvement that were discovered only through rounding. We conclude that the introduction of leadership rounding by an anesthesiology service can identify opportunities for improving quality that are not captured by conventional efforts.

  10. Dynamic Method for Identifying Collected Sample Mass

    NASA Technical Reports Server (NTRS)

    Carson, John

    2008-01-01

    G-Sample is designed for sample collection missions to identify the presence and quantity of sample material gathered by spacecraft equipped with end effectors. The software method uses a maximum-likelihood estimator to identify the collected sample's mass based on onboard force-sensor measurements, thruster firings, and a dynamics model of the spacecraft. This makes sample mass identification a computation rather than a process requiring additional hardware. Simulation examples of G-Sample are provided for spacecraft model configurations with a sample collection device mounted on the end of an extended boom. In the absence of thrust knowledge errors, the results indicate that G-Sample can identify the amount of collected sample mass to within 10 grams (with 95-percent confidence) by using a force sensor with a noise and quantization floor of 50 micrometers. These results hold even in the presence of realistic parametric uncertainty in actual spacecraft inertia, center-of-mass offset, and first flexibility modes. Thrust profile knowledge is shown to be a dominant sensitivity for G-Sample, entering in a nearly one-to-one relationship with the final mass estimation error. This means thrust profiles should be well characterized with onboard accelerometers prior to sample collection. An overall sample-mass estimation error budget has been developed to approximate the effect of model uncertainty, sensor noise, data rate, and thrust profile error on the expected estimate of collected sample mass.

  11. Identifying differentially regulated subnetworks from phosphoproteomic data

    PubMed Central

    2010-01-01

    Background Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. Results Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. Conclusions SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data. PMID:20584295

  12. DBSI: DNA-binding site identifier

    PubMed Central

    Zhu, Xiaolei; Ericksen, Spencer S.; Mitchell, Julie C.

    2013-01-01

    In this study, we present the DNA-Binding Site Identifier (DBSI), a new structure-based method for predicting protein interaction sites for DNA binding. DBSI was trained and validated on a data set of 263 proteins (TRAIN-263), tested on an independent set of protein-DNA complexes (TEST-206) and data sets of 29 unbound (APO-29) and 30 bound (HOLO-30) protein structures distinct from the training data. We computed 480 candidate features for identifying protein residues that bind DNA, including new features that capture the electrostatic microenvironment within shells near the protein surface. Our iterative feature selection process identified features important in other models, as well as features unique to the DBSI model, such as a banded electrostatic feature with spatial separation comparable with the canonical width of the DNA minor groove. Validations and comparisons with established methods using a range of performance metrics clearly demonstrate the predictive advantage of DBSI, and its comparable performance on unbound (APO-29) and bound (HOLO-30) conformations demonstrates robustness to binding-induced protein conformational changes. Finally, we offer our feature data table to others for integration into their own models or for testing improved feature selection and model training strategies based on DBSI. PMID:23873960

  13. DBSI: DNA-binding site identifier.

    PubMed

    Zhu, Xiaolei; Ericksen, Spencer S; Mitchell, Julie C

    2013-09-01

    In this study, we present the DNA-Binding Site Identifier (DBSI), a new structure-based method for predicting protein interaction sites for DNA binding. DBSI was trained and validated on a data set of 263 proteins (TRAIN-263), tested on an independent set of protein-DNA complexes (TEST-206) and data sets of 29 unbound (APO-29) and 30 bound (HOLO-30) protein structures distinct from the training data. We computed 480 candidate features for identifying protein residues that bind DNA, including new features that capture the electrostatic microenvironment within shells near the protein surface. Our iterative feature selection process identified features important in other models, as well as features unique to the DBSI model, such as a banded electrostatic feature with spatial separation comparable with the canonical width of the DNA minor groove. Validations and comparisons with established methods using a range of performance metrics clearly demonstrate the predictive advantage of DBSI, and its comparable performance on unbound (APO-29) and bound (HOLO-30) conformations demonstrates robustness to binding-induced protein conformational changes. Finally, we offer our feature data table to others for integration into their own models or for testing improved feature selection and model training strategies based on DBSI.

  14. DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ...

    EPA Pesticide Factsheets

    Hepatitis E virus (HEV) is an emerging pathogen that causes significant illness in the developing world. Like the hepatitis A virus, it is transmitted via the fecal-oral route and can cause short-term, acute hepatitis. In addition, hepatitis E has been found to cause a significant rate of mortality in pregnant women. Thus far, a hepatitis E outbreak has not been reported in the U. S. although a swine variant of the virus is common in Midwestern hogs. Since it will be important to identify the presence of this virus in the water supply, we have developed and are testing a reverse transcription-polymerase chain reaction (RT-PCR) method that should be able to identify all of the known HEV strains. Develop sensitive techniques to detect and identify emerging human waterborne pathogenic viruses and viruses on the CCL.Determine effectiveness of viral indicators to measure microbial quality in water matrices.Support activities: (a) culture and distribution of mammalian cells for Agency and scientific community research needs, (b) provide operator expertise for research requiring confocal and electron microscopy, (c) glassware cleaning, sterilization and biological waste disposal for the Cincinnati EPA facility, (d) operation of infectious pathogenic suite, (e) maintenance of walk-in constant temperature rooms and (f) provide Giardia cysts.

  15. Identifying duplicate content using statistically improbable phrases.

    PubMed

    Errami, Mounir; Sun, Zhaohui; George, Angela C; Long, Tara C; Skinner, Michael A; Wren, Jonathan D; Garner, Harold R

    2010-06-01

    Document similarity metrics such as PubMed's 'Find related articles' feature, which have been primarily used to identify studies with similar topics, can now also be used to detect duplicated or potentially plagiarized papers within literature reference databases. However, the CPU-intensive nature of document comparison has limited MEDLINE text similarity studies to the comparison of abstracts, which constitute only a small fraction of a publication's total text. Extending searches to include text archived by online search engines would drastically increase comparison ability. For large-scale studies, submitting short phrases encased in direct quotes to search engines for exact matches would be optimal for both individual queries and programmatic interfaces. We have derived a method of analyzing statistically improbable phrases (SIPs) for assistance in identifying duplicate content. When applied to MEDLINE citations, this method substantially improves upon previous algorithms in the detection of duplication citations, yielding a precision and recall of 78.9% (versus 50.3% for eTBLAST) and 99.6% (versus 99.8% for eTBLAST), respectively. Similar citations identified by this work are freely accessible in the Déjà vu database, under the SIP discovery method category at http://dejavu.vbi.vt.edu/dejavu/.

  16. DEVELOPMENT OF A MOLECULAR METHOD TO IDENTIFY ...

    EPA Pesticide Factsheets

    Hepatitis E virus (HEV) is an emerging pathogen that causes significant illness in the developing world. Like the hepatitis A virus, it is transmitted via the fecal-oral route and can cause short-term, acute hepatitis. In addition, hepatitis E has been found to cause a significant rate of mortality in pregnant women. Thus far, a hepatitis E outbreak has not been reported in the U. S. although a swine variant of the virus is common in Midwestern hogs. Since it will be important to identify the presence of this virus in the water supply, we have developed and are testing a reverse transcription-polymerase chain reaction (RT-PCR) method that should be able to identify all of the known HEV strains. Develop sensitive techniques to detect and identify emerging human waterborne pathogenic viruses and viruses on the CCL.Determine effectiveness of viral indicators to measure microbial quality in water matrices.Support activities: (a) culture and distribution of mammalian cells for Agency and scientific community research needs, (b) provide operator expertise for research requiring confocal and electron microscopy, (c) glassware cleaning, sterilization and biological waste disposal for the Cincinnati EPA facility, (d) operation of infectious pathogenic suite, (e) maintenance of walk-in constant temperature rooms and (f) provide Giardia cysts.

  17. Identifying Broadband Rotational Spectra with Neural Networks

    NASA Astrophysics Data System (ADS)

    Zaleski, Daniel P.; Prozument, Kirill

    2017-06-01

    A typical broadband rotational spectrum may contain several thousand observable transitions, spanning many species. Identifying the individual spectra, particularly when the dynamic range reaches 1,000:1 or even 10,000:1, can be challenging. One approach is to apply automated fitting routines. In this approach, combinations of 3 transitions can be created to form a "triple", which allows fitting of the A, B, and C rotational constants in a Watson-type Hamiltonian. On a standard desktop computer, with a target molecule of interest, a typical AUTOFIT routine takes 2-12 hours depending on the spectral density. A new approach is to utilize machine learning to train a computer to recognize the patterns (frequency spacing and relative intensities) inherit in rotational spectra and to identify the individual spectra in a raw broadband rotational spectrum. Here, recurrent neural networks have been trained to identify different types of rotational spectra and classify them accordingly. Furthermore, early results in applying convolutional neural networks for spectral object recognition in broadband rotational spectra appear promising. Perez et al. "Broadband Fourier transform rotational spectroscopy for structure determination: The water heptamer." Chem. Phys. Lett., 2013, 571, 1-15. Seifert et al. "AUTOFIT, an Automated Fitting Tool for Broadband Rotational Spectra, and Applications to 1-Hexanal." J. Mol. Spectrosc., 2015, 312, 13-21. Bishop. "Neural networks for pattern recognition." Oxford university press, 1995.

  18. Identifying familiar strangers in human encounter networks

    NASA Astrophysics Data System (ADS)

    Liang, Di; Li, Xiang; Zhang, Yi-Qing

    2016-10-01

    Familiar strangers, pairs of individuals who encounter repeatedly but never know each other, have been discovered for four decades yet lack an effective method to identify. Here we propose a novel method called familiar stranger classifier (FSC) to identify familiar strangers from three empirical datasets, and classify human relationships into four types, i.e., familiar stranger (FS), in-role (IR), friend (F) and stranger (S). The analyses of the human encounter networks show that the average number of FS one may encounter is finite but larger than the Dunbar Number, and their encounters are structurally more stable and denser than those of S, indicating the encounters of FS are not limited by the social capacity, and more robust than the random scenario. Moreover, the temporal statistics of encounters between FS over the whole time span show strong periodicity, which are diverse from the bursts of encounters within one day, suggesting the significance of longitudinal patterns of human encounters. The proposed method to identify FS in this paper provides a valid framework to understand human encounter patterns and analyse complex human social behaviors.

  19. Identifying web usage behavior of bank customers

    NASA Astrophysics Data System (ADS)

    Araya, Sandro; Silva, Mariano; Weber, Richard

    2002-03-01

    The bank Banco Credito e Inversiones (BCI) started its virtual bank in 1996 and its registered customers perform currently more than 10,000 Internet transactions daily, which typically cause les than 10% of traditional transaction costs. Since most of the customers are still not registered for online banking, one of the goals of the virtual bank is to increase then umber of registered customers. Objective of the presented work was to identify customers who are likely to perform online banking but still do not use this medium for their transactions. This objective has been reached by determining profiles of registered customers who perform many transactions online. Based on these profiles the bank's Data Warehouse is explored for twins of these heavy users that are still not registered for online banking. We applied clustering in order to group the registered customers into five classes. One of these classes contained almost 30% of all registered customers and could clearly be identified as class of heavy users. Next a neural network assigned online customers to the previously found five classes. Applying the network trained on online customers to all the bank customers identified twins of heavy users that, however had not performed online transactions so far. A mailing to these candidates informing about the advantages of online banking doubled the number of registrations compared to previous campaigns.

  20. Interrogator system for identifying electrical circuits

    DOEpatents

    Jatko, W.B.; McNeilly, D.R.

    1988-04-12

    A system for interrogating electrical leads to correctly ascertain the identity of equipment attached to remote ends of the leads is disclosed. The system includes a source of a carrier signal generated in a controller/receiver to be sent over the leads and an identifier unit at the equipment. The identifier is activated by command of the carrier and uses a portion of the carrier to produce a supply voltage. Each identifier is uniquely programmed for a specific piece of equipment, and causes the impedance of the circuit to be modified whereby the carrier signal is modulated according to that program. The modulation can be amplitude, frequency or phase modulation. A demodulator in the controller/receiver analyzes the modulated carrier signal, and if a verified signal is recognized displays and/or records the information. This information can be utilized in a computer system to prepare a wiring diagram of the electrical equipment attached to specific leads. Specific circuit values are given for amplitude modulation, and the system is particularly described for use with thermocouples. 6 figs.

  1. Persistent Identifiers, Discoverability and Open Science (Communication)

    NASA Astrophysics Data System (ADS)

    Murphy, Fiona; Lehnert, Kerstin; Hanson, Brooks

    2016-04-01

    Early in 2016, the American Geophysical Union announced it was incorporating ORCIDs into its submission workflows. This was accompanied by a strong statement supporting the use of other persistent identifiers - such as IGSNs, and the CrossRef open registry 'funding data'. This was partly in response to funders' desire to track and manage their outputs. However the more compelling argument, and the reason why the AGU has also signed up to the Center for Open Science's Transparency and Openness Promotion (TOP) Guidelines (http://cos.io/top), is that ultimately science and scientists will be the richer for these initiatives due to increased opportunities for interoperability, reproduceability and accreditation. The AGU has appealed to the wider community to engage with these initiatives, recognising that - unlike the introduction of Digital Object Identifiers (DOIs) for articles by CrossRef - full, enriched use of persistent identifiers throughout the scientific process requires buy-in from a range of scholarly communications stakeholders. At the same time, across the general research landscape, initiatives such as Project CRediT (contributor roles taxonomy), Publons (reviewer acknowledgements) and the forthcoming CrossRef DOI Event Tracker are contributing to our understanding and accreditation of contributions and impact. More specifically for earth science and scientists, the cross-functional Coalition for Publishing Data in the Earth and Space Sciences (COPDESS) was formed in October 2014 and is working to 'provide an organizational framework for Earth and space science publishers and data facilities to jointly implement and promote common policies and procedures for the publication and citation of data across Earth Science journals'. Clearly, the judicious integration of standards, registries and persistent identifiers such as ORCIDs and International Geo Sample Numbers (IGSNs) to the research and research output processes is key to the success of this venture

  2. CORE_TF: a user-friendly interface to identify evolutionary conserved transcription factor binding sites in sets of co-regulated genes

    PubMed Central

    Hestand, Matthew S; van Galen, Michiel; Villerius, Michel P; van Ommen, Gert-Jan B; den Dunnen, Johan T; 't Hoen, Peter AC

    2008-01-01

    Background The identification of transcription factor binding sites is difficult since they are only a small number of nucleotides in size, resulting in large numbers of false positives and false negatives in current approaches. Computational methods to reduce false positives are to look for over-representation of transcription factor binding sites in a set of similarly regulated promoters or to look for conservation in orthologous promoter alignments. Results We have developed a novel tool, "CORE_TF" (Conserved and Over-REpresented Transcription Factor binding sites) that identifies common transcription factor binding sites in promoters of co-regulated genes. To improve upon existing binding site predictions, the tool searches for position weight matrices from the TRANSFACR database that are over-represented in an experimental set compared to a random set of promoters and identifies cross-species conservation of the predicted transcription factor binding sites. The algorithm has been evaluated with expression and chromatin-immunoprecipitation on microarray data. We also implement and demonstrate the importance of matching the random set of promoters to the experimental promoters by GC content, which is a unique feature of our tool. Conclusion The program CORE_TF is accessible in a user friendly web interface at . It provides a table of over-represented transcription factor binding sites in the users input genes' promoters and a graphical view of evolutionary conserved transcription factor binding sites. In our test data sets it successfully predicts target transcription factors and their binding sites. PMID:19036135

  3. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation

    PubMed Central

    Wahba, Lamia; Costantino, Lorenzo; Tan, Frederick J.; Zimmer, Anjali; Koshland, Douglas

    2016-01-01

    R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae. Using this methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast. PMID:27298336

  4. Utility of next-generation RNA-sequencing in identifying chimeric transcription involving human endogenous retroviruses.

    PubMed

    Sokol, Martin; Jessen, Karen Margrethe; Pedersen, Finn Skou

    2016-01-01

    Several studies have shown that human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively HERVs) impose direct regulation on human genes through enhancer and promoter motifs present in their long terminal repeats (LTRs). Although chimeric transcription in which novel gene isoforms containing retroviral and human sequence are transcribed from viral promoters are commonly associated with disease, regulation by HERVs is beneficial in other settings; for example, in human testis chimeric isoforms of TP63 induced by an ERV9 LTR protect the male germ line upon DNA damage by inducing apoptosis, whereas in the human globin locus the γ- and β-globin switch during normal hematopoiesis is mediated by complex interactions of an ERV9 LTR and surrounding human sequence. The advent of deep sequencing or next-generation sequencing (NGS) has revolutionized the way researchers solve important scientific questions and develop novel hypotheses in relation to human genome regulation. We recently applied next-generation paired-end RNA-sequencing (RNA-seq) together with chromatin immunoprecipitation with sequencing (ChIP-seq) to examine ERV9 chimeric transcription in human reference cell lines from Encyclopedia of DNA Elements (ENCODE). This led to the discovery of advanced regulation mechanisms by ERV9s and other HERVs across numerous human loci including transcription of large gene-unannotated genomic regions, as well as cooperative regulation by multiple HERVs and non-LTR repeats such as Alu elements. In this article, well-established examples of human gene regulation by HERVs are reviewed followed by a description of paired-end RNA-seq, and its application in identifying chimeric transcription genome-widely. Based on integrative analyses of RNA-seq and ChIP-seq, data we then present novel examples of regulation by ERV9s of tumor suppressor genes CADM2 and SEMA3A, as well as transcription of an unannotated region. Taken together, this article highlights

  5. Identifying suitable sites for Florida panther reintroduction

    USGS Publications Warehouse

    Thatcher, Cindy; van Manen, Frank T.; Clark, Joseph D.

    2006-01-01

    A major objective of the 1995 Florida Panther (Puma concolor cory) Recovery Plan is the establishment of 2 additional panther populations within the historic range. Our goal was to identify prospective sites for Florida panther reintroduction within the historic range based on quantitative landscape assessments. First, we delineated 86 panther home ranges using telemetry data collected from 1981 to 2001 in south Florida to develop a Mahalanobis distance (D2) habitat model, using 4 anthropogenic variables and 3 landscape variables mapped at a 500-m resolution. From that analysis, we identified 9 potential reintroduction sites of sufficient size to support a panther population. We then developed a similar D2 model at a higher spatial resolution to quantify the area of favorable panther habitat at each site. To address potential for the population to expand, we calculated the amount of favorable habitat adjacent to each prospective reintroduction site within a range of dispersal distances of female panthers. We then added those totals to the contiguous patches to estimate the total amount of effective panther habitat at each site. Finally, we developed an expert-assisted model to rank and incorporate potentially important habitat variables that were not appropriate for our empirical analysis (e.g., area of public lands, livestock density). Anthropogenic factors heavily influenced both the landscape and the expert-assisted models. Of the 9 areas we identified, the Okefenokee National Wildlife Refuge, Ozark National Forest, and Felsenthal National Wildlife Refuge regions had the highest combination of effective habitat area and expert opinion scores. Sensitivity analyses indicated that variability among key model parameters did not affect the high ranking of those sites. Those sites should be considered as starting points for the field evaluation of potential reintroduction sites.

  6. Identifying Airborne Pathogens in Time to Respond

    SciTech Connect

    Hazi, A

    2006-01-25

    Among the possible terrorist activities that might threaten national security is the release of an airborne pathogen such as anthrax. Because the potential damage to human health could be severe, experts consider 1 minute to be an operationally useful time limit for identifying the pathogen and taking action. Many commercial systems can identify airborne pathogenic microbes, but they take days or, at best, hours to produce results. The Department of Homeland Security (DHS) and other U.S. government agencies are interested in finding a faster approach. To answer this national need, a Livermore team, led by scientist Eric Gard, has developed the bioaerosol mass spectrometry (BAMS) system--the only instrument that can detect and identify spores at low concentrations in less than 1 minute. BAMS can successfully distinguish between two related but different spore species. It can also sort out a single spore from thousands of other particles--biological and nonbiological--with no false positives. The BAMS team won a 2005 R&D 100 Award for developing the system. Livermore's Laboratory Directed Research and Development (LDRD) Program funded the biomedical aspects of the BAMS project, and the Department of Defense's Technical Support Working Group and Defense Advanced Research Project Agency funded the biodefense efforts. Developing a detection system that can analyze small samples so quickly has been challenging. Livermore engineer Vincent Riot, who worked on the BAMS project, explains, ''A typical spore weighs approximately one-trillionth of a gram and is dispersed in the atmosphere, which contains naturally occurring particles that could be present at concentrations thousands of times higher. Previous systems also had difficulty separating benign organisms from those that are pathogenic but very similar, which has resulted in false alarms''.

  7. [Identifying children at risk for cardiorespiratory arrest].

    PubMed

    Carrillo Alvarez, A; Martínez Gutiérrez, A; Salvat Germán, F

    2004-08-01

    Cardiorespiratory arrest in children with severe disease does not usually present suddenly or unexpectedly but is often the result of a progressive deterioration of respiratory and/or circulatory function. Before failure of these functions occurs, there is a series of clinical signs that serve as a warning. Health professionals should not only evaluate clinical signs of respiratory and/or circulatory insufficiency but should also be able to identify these warning signs as early as possible, preferably in the compensation phase, given that the possibility that this process can be reversed by therapeutic measures decreases as the process progresses.

  8. Identifying related journals through log analysis

    PubMed Central

    Lu, Zhiyong; Xie, Natalie; Wilbur, W. John

    2009-01-01

    Motivation: With the explosion of biomedical literature and the evolution of online and open access, scientists are reading more articles from a wider variety of journals. Thus, the list of core journals relevant to their research may be less obvious and may often change over time. To help researchers quickly identify appropriate journals to read and publish in, we developed a web application for finding related journals based on the analysis of PubMed log data. Availability: http://www.ncbi.nlm.nih.gov/IRET/Journals Contact: luzh@ncbi.nlm.nih.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19734155

  9. IDENTIFYING PERFORMANCE ASSURANCE CHALLENGES FOR SMART MANUFACTURING.

    PubMed

    Helu, Moneer; Morris, Katherine; Jung, Kiwook; Lyons, Kevin; Leong, Swee

    2015-10-01

    Smart manufacturing has the potential to address many of the challenges faced by industry. However, the manufacturing community often needs assistance to leverage available technologies to improve their systems. To assure the performance of these technologies, this paper proposes a shared knowledge base that collects problem areas, solutions, and best practices for manufacturing technology. An Implementation Risk Assessment Framework (IRAF) is also described to identify the primary weaknesses of technologies in specific manufacturing contexts. Such approaches have the potential to stimulate new ideas and drive standardization activities critical to scale up and deploy smart manufacturing technologies successfully and quickly.

  10. IDENTIFYING PERFORMANCE ASSURANCE CHALLENGES FOR SMART MANUFACTURING

    PubMed Central

    Helu, Moneer; Morris, Katherine; Jung, Kiwook; Lyons, Kevin; Leong, Swee

    2015-01-01

    Smart manufacturing has the potential to address many of the challenges faced by industry. However, the manufacturing community often needs assistance to leverage available technologies to improve their systems. To assure the performance of these technologies, this paper proposes a shared knowledge base that collects problem areas, solutions, and best practices for manufacturing technology. An Implementation Risk Assessment Framework (IRAF) is also described to identify the primary weaknesses of technologies in specific manufacturing contexts. Such approaches have the potential to stimulate new ideas and drive standardization activities critical to scale up and deploy smart manufacturing technologies successfully and quickly. PMID:26783512

  11. Nocturnal Sleep Dynamics Identify Narcolepsy Type 1

    PubMed Central

    Pizza, Fabio; Vandi, Stefano; Iloti, Martina; Franceschini, Christian; Liguori, Rocco; Mignot, Emmanuel; Plazzi, Giuseppe

    2015-01-01

    Study Objectives: To evaluate the reliability of nocturnal sleep dynamics in the differential diagnosis of central disorders of hypersomnolence. Design: Cross-sectional. Setting: Sleep laboratory. Patients: One hundred seventy-five patients with hypocretin-deficient narcolepsy type 1 (NT1, n = 79), narcolepsy type 2 (NT2, n = 22), idiopathic hypersomnia (IH, n = 22), and “subjective” hypersomnolence (sHS, n = 52). Interventions: None. Methods: Polysomnographic (PSG) work-up included 48 h of continuous PSG recording. From nocturnal PSG conventional sleep macrostructure, occurrence of sleep onset rapid eye movement period (SOREMP), sleep stages distribution, and sleep stage transitions were calculated. Patient groups were compared, and receiver operating characteristic (ROC) curve analysis was used to test the diagnostic utility of nocturnal PSG data to identify NT1. Results: Sleep macrostructure was substantially stable in the 2 nights of each diagnostic group. NT1 and NT2 patients had lower latency to rapid eye movement (REM) sleep, and NT1 patients showed the highest number of awakenings, sleep stage transitions, and more time spent in N1 sleep, as well as most SOREMPs at daytime PSG and at multiple sleep latency test (MSLT) than all other groups. ROC curve analysis showed that nocturnal SOREMP (area under the curve of 0.724 ± 0.041, P < 0.0001), percent of total sleep time spent in N1 (0.896 ± 0.023, P < 0.0001), and the wakefulness-sleep transition index (0.796 ± 0.034, P < 0.0001) had a good sensitivity and specificity profile to identify NT1 sleep, especially when used in combination (0.903 ± 0.023, P < 0.0001), similarly to SOREMP number at continuous daytime PSG (0.899 ± 0.026, P < 0.0001) and at MSLT (0.956 ± 0.015, P < 0.0001). Conclusions: Sleep macrostructure (i.e. SOREMP, N1 timing) including stage transitions reliably identifies hypocretin-deficient narcolepsy type 1 among central disorders of hypersomnolence. Citation: Pizza F, Vandi S

  12. Identifying inference attacks against healthcare data repositories

    PubMed Central

    Vaidya, Jaideep; Shafiq, Basit; Jiang, Xiaoqian; Ohno-Machado, Lucila

    Health care data repositories play an important role in driving progress in medical research. Finding new pathways to discovery requires having adequate data and relevant analysis. However, it is critical to ensure the privacy and security of the stored data. In this paper, we identify a dangerous inference attack against naive suppression based approaches that are used to protect sensitive information. We base our attack on the querying system provided by the Healthcare Cost and Utilization Project, though it applies in general to any medical database providing a query capability. We also discuss potential solutions to this problem. PMID:24303279

  13. Identifying bacterial predictors of honey bee health.

    PubMed

    Budge, Giles E; Adams, Ian; Thwaites, Richard; Pietravalle, Stéphane; Drew, Georgia C; Hurst, Gregory D D; Tomkies, Victoria; Boonham, Neil; Brown, Mike

    2016-11-01

    Non-targeted approaches are useful tools to identify new or emerging issues in bee health. Here, we utilise next generation sequencing to highlight bacteria associated with healthy and unhealthy honey bee colonies, and then use targeted methods to screen a wider pool of colonies with known health status. Our results provide the first evidence that bacteria from the genus Arsenophonus are associated with poor health in honey bee colonies. We also discovered Lactobacillus and Leuconostoc spp. were associated with healthier honey bee colonies. Our results highlight the importance of understanding how the wider microbial population relates to honey bee colony health.

  14. Method of identifying defective particle coatings

    DOEpatents

    Cohen, Mark E.; Whiting, Carlton D.

    1986-01-01

    A method for identifying coated particles having defective coatings desig to retain therewithin a build-up of gaseous materials including: (a) Pulling a vacuum on the particles; (b) Backfilling the particles at atmospheric pressure with a liquid capable of wetting the exterior surface of the coated particles, said liquid being a compound which includes an element having an atomic number higher than the highest atomic number of any element in the composition which forms the exterior surface of the particle coating; (c) Drying the particles; and (d) Radiographing the particles. By television monitoring, examination of the radiographs is substantially enhanced.

  15. Identifying tandem Ankyrin repeats in protein structures.

    PubMed

    Chakrabarty, Broto; Parekh, Nita

    2014-12-30

    Tandem repetition of structural motifs in proteins is frequently observed across all forms of life. Topology of repeating unit and its frequency of occurrence are associated to a wide range of structural and functional roles in diverse proteins, and defects in repeat proteins have been associated with a number of diseases. It is thus desirable to accurately identify specific repeat type and its copy number. Weak evolutionary constraints on repeat units and insertions/deletions between them make their identification difficult at the sequence level and structure based approaches are desired. The proposed graph spectral approach is based on protein structure represented as a graph for detecting one of the most frequently observed structural repeats, Ankyrin repeat. It has been shown in a large number of studies that 3-dimensional topology of a protein structure is well captured by a graph, making it possible to analyze a complex protein structure as a mathematical entity. In this study we show that eigen spectra profile of a protein structure graph exhibits a unique repetitive profile for contiguous repeating units enabling the detection of the repeat region and the repeat type. The proposed approach uses a non-redundant set of 58 Ankyrin proteins to define rules for the detection of Ankyrin repeat motifs. It is evaluated on a set of 370 proteins comprising 125 known Ankyrin proteins and remaining non-solenoid proteins and the prediction compared with UniProt annotation, sequence-based approach, RADAR, and structure-based approach, ConSole. To show the efficacy of the approach, we analyzed the complete PDB structural database and identified 641 previously unrecognized Ankyrin repeat proteins. We observe a unique eigen spectra profile for different repeat types and show that the method can be easily extended to detect other repeat types. It is implemented as a web server, AnkPred. It is freely available at 'bioinf.iiit.ac.in/AnkPred'. AnkPred provides an elegant and

  16. EVA: Visual Analytics to Identify Fraudulent Events.

    PubMed

    Leite, Roger A; Gschwandtner, Theresia; Miksch, Silvia; Kriglstein, Simone; Pohl, Margit; Gstrein, Erich; Kuntner, Johannes

    2017-08-31

    Financial institutions are interested in ensuring security and quality for their customers. Banks, for instance, need to identify and stop harmful transactions in a timely manner. In order to detect fraudulent operations, data mining techniques and customer profile analysis are commonly used. However, these approaches are not supported by Visual Analytics techniques yet. Visual Analytics techniques have potential to considerably enhance the knowledge discovery process and increase the detection and prediction accuracy of financial fraud detection systems. Thus, we propose EVA, a Visual Analytics approach for supporting fraud investigation, fine-tuning fraud detection algorithms, and thus, reducing false positive alarms.

  17. Subject Matter Expert Workshop to Identify Cybersecurity ...

    EPA Pesticide Factsheets

    Report In recognition of the growing need to better address cyber risk and cyber management, the U.S. Environmental Protection Agency’s (EPA) National Homeland Security Research Center (NHSRC) held a Subject Matter Expert Workshop to Identify Cybersecurity Research Gaps and Needs of the Nation’s Water and Wastewater Systems Sector on March 30th and 31st, 2016, at the Ronald Reagan Building in Washington, D.C. The workshop was designed to create a forum for subject matter experts (SMEs) to exchange ideas and address important cybersecurity challenges facing the water sector.

  18. Identifying discourse connectives in biomedical text.

    PubMed

    Ramesh, Balaji Polepalli; Yu, Hong

    2010-11-13

    Discourse connectives are words or phrases that connect or relate two coherent sentences or phrases and indicate the presence of discourse relations. Automatic recognition of discourse connectives may benefit many natural language processing applications. In this pilot study, we report the development of the supervised machine-learning classifiers with conditional random fields (CRFs) for automatically identifying discourse connectives in full-text biomedical articles. Our first classifier was trained on the open-domain 1 million token Penn Discourse Tree Bank (PDTB). We performed cross validation on biomedical articles (approximately 100K word tokens) that we annotated. The results show that the classifier trained on PDTB data attained a 0.55 F1-score for identifying discourse connectives in biomedical text, while the cross-validation results in the biomedical text attained a 0.69 F1-score, a much better performance despite a much smaller training size. Our preliminary analysis suggests the existence of domain-specific features, and we speculate that domain-adaption approaches may further improve performance.

  19. White dwarfs identified in LAMOST DR 2

    NASA Astrophysics Data System (ADS)

    Guo, Jincheng; Zhao, Jingkun; Tziamtzis, Anestis; Liu, Jifeng; Li, Lifang; Zhang, Yong; Hou, Yonghui; Wang, Yuefei

    2015-12-01

    Here we present a catalogue of 1056 spectroscopically identified hydrogen-dominated white dwarfs (DAWDs), 34 helium-dominated white dwarfs (DBWDs) and 276 white dwarf main sequence (WDMS) binaries from the Large sky Area Multi-Object Fiber Spectroscopic Telescope (LAMOST) survey data release 2 (DR2). 383 DAWDs, 4 DBWDs and 138 WDMSs are new identifications after cross-match with literature. There are ˜4100 k spectra in total from DR 2. The low ratio of white dwarfs found in LAMOST is attributed to biased selection of LAMOST input catalogue and much brighter targets relative to stars observed in Sloan Digital Sky Survey. In this paper, a new DAWD selection method is adopted as a new attempt and supplement to the traditional methods. The effective temperature, surface gravity, mass, cooling age and distance of high signal-to-noise DAWDs are estimated. The peak of the mass distribution is found to be ˜0.6 M⊙, which is consistent with previous work. The parameters of WDMS binaries are also provided in this paper. As the foundation of our future work, which is to identify more WDs with debris disc, WDs found in LAMOST showed a lot of potential. Interesting infrared-excess WDs will be reported in our forthcoming paper.

  20. Using Dissimilarity Metrics to Identify Interesting Designs

    NASA Technical Reports Server (NTRS)

    Feather, Martin; Kiper, James

    2006-01-01

    A computer program helps to blend the power of automated-search software, which is able to generate large numbers of design solutions, with the insight of expert designers, who are able to identify preferred designs but do not have time to examine all the solutions. From among the many automated solutions to a given design problem, the program selects a smaller number of solutions that are worthy of scrutiny by the experts in the sense that they are sufficiently dissimilar from each other. The program makes the selection in an interactive process that involves a sequence of data-mining steps interspersed with visual displays of results of these steps to the experts. At crucial points between steps, the experts provide directives to guide the process. The program uses heuristic search techniques to identify nearly optimal design solutions and uses dissimilarity metrics defined by the experts to characterize the degree to which solutions are interestingly different. The search, data-mining, and visualization features of the program were derived from previously developed risk-management software used to support a risk-centric design methodology