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Sample records for enables quantitative phosphoproteomics

  1. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research.

    PubMed

    Chan, Chi Yuet X'avia; Gritsenko, Marina A; Smith, Richard D; Qian, Wei-Jun

    2016-01-01

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been implicated in various human diseases. Kinases and their cognate inhibitors have been considered as hotspots for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for diseases of interest. This review highlights recent advances in phosphoproteomics, the current state of the art of the technologies and the challenges and future perspectives of this research area. Finally, some exemplary applications of phosphoproteomics in diabetes research are underscored.

  2. The current state of the art of quantitative phosphoproteomics and its applications to diabetes research

    SciTech Connect

    Chan, Chi Yuet X’avia; Gritsenko, Marina A.; Smith, Richard D.; Qian, Wei-Jun

    2016-03-17

    Protein phosphorylation is a fundamental regulatory mechanism in many cellular processes and aberrant perturbation of phosphorylation has been revealed in various human diseases. Kinases and their cognate inhibitors have been hotspot for drug development. Therefore, the emerging tools, which enable a system-wide quantitative profiling of phosphoproteome, would offer a powerful impetus in unveiling novel signaling pathways, drug targets and/or biomarkers for the disease of interest. In this review, we will highlight recent advances in phosphoproteomics, the current state-of-the-art of the technologies, and the challenges and future perspectives of this research area. Finally, we will underscore some exemplary applications of phosphoproteomics in diabetes research.

  3. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

    PubMed

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  4. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    PubMed Central

    Jacobs, Kris; Klammer, Martin; Jordan, Nicole; Elschenbroich, Sarah; Parade, Marc; Jacoby, Edgar; Linders, Joannes T. M.; Brehmer, Dirk; Cools, Jan; Daub, Henrik

    2016-01-01

    The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies. PMID:26745281

  5. Quantitative phosphoproteomics by mass spectrometry: Past, present, and future

    PubMed Central

    Nita-Lazar, Aleksandra; Saito-Benz, Hideshiro; White, Forest M.

    2009-01-01

    Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight. PMID:18846511

  6. Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs.

    PubMed

    Soderblom, Erik J; Philipp, Melanie; Thompson, J Will; Caron, Marc G; Moseley, M Arthur

    2011-05-15

    Protein phosphorylation is a critical regulator of signaling in nearly all eukaryotic cellular pathways and dysregulated phosphorylation has been implicated in an array of diseases. The majority of MS-based quantitative phosphorylation studies are currently performed from transformed cell lines because of the ability to generate large amounts of starting material with incorporated isotopically labeled amino acids during cell culture. Here we describe a general label-free quantitative phosphoproteomic strategy capable of directly analyzing relatively small amounts of virtually any biological matrix, including human tissue and biological fluids. The strategy utilizes a TiO(2) enrichment protocol in which the selectivity and recovery of phosphopeptides were optimized by assessing a twenty-point condition matrix of binding modifier concentrations and peptide-to-resin capacity ratios. The quantitative reproducibility of the TiO(2) enrichment was determined to be 16% RSD through replicate enrichments of a wild-type Danio rerio (zebrafish) lysate. Measured phosphopeptide fold-changes from alpha-casein spiked into wild-type zebrafish lysate backgrounds were within 5% of the theoretical value. Application to a morpholino induced knock-down of G protein-coupled receptor kinase 5 (GRK5) in zebrafish embryos resulted in the quantitation of 719 phosphorylated peptides corresponding to 449 phosphorylated proteins from 200 μg of zebrafish embryo lysates.

  7. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis*

    PubMed Central

    Queiroz, Rayner M. L.; Charneau, Sébastien; Mandacaru, Samuel C.; Schwämmle, Veit; Lima, Beatriz D.; Roepstorff, Peter; Ricart, Carlos A. O.

    2014-01-01

    Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets. PMID:25225356

  8. Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

    PubMed

    Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

    2015-05-01

    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research.

  9. Quantitative Proteomic and Phosphoproteomic Comparison of 2D and 3D Colon Cancer Cell Culture Models.

    PubMed

    Yue, Xiaoshan; Lukowski, Jessica K; Weaver, Eric M; Skube, Susan B; Hummon, Amanda B

    2016-12-02

    Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.

  10. SPECHT – Single-stage phosphopeptide enrichment and stable-isotope chemical tagging: Quantitative phosphoproteomics of insulin action in muscle

    PubMed Central

    Kettenbach, Arminja N.; Sano, Hiroyuki; Keller, Susanna R.; Lienhard, Gustav E.; Gerber, Scott A.

    2014-01-01

    The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. PMID:25463755

  11. Integration of conventional quantitative and phospho-proteomics reveals new elements in activated Jurkat T-cell receptor pathway maintenance.

    PubMed

    Jouy, Florent; Müller, Stephan A; Wagner, Juliane; Otto, Wolfgang; von Bergen, Martin; Tomm, Janina M

    2015-01-01

    Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T-cell receptor (TCR) pathway in a T-cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T-cells and phosphopeptides enriched via a TiO2-based chromatographic step. Both phosphopeptide-enriched and flow-through fractions were analyzed by LC-MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow-through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR-related function were detected. A kinase-substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR-related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T-cells.

  12. Machine learning of global phosphoproteomic profiles enables discrimination of direct versus indirect kinase substrates.

    PubMed

    Kanshin, Evgeny; Giguere, Sebastien; Cheng, Jing; Tyers, Michael D; Thibault, Pierre

    2017-03-06

    Mass spectrometry allows quantification of tens of thousands of phosphorylation sites from minute amounts of cellular material. Despite this wealth of information, our understanding of phosphorylation-based signaling is limited, in part because it is not possible to deconvolute substrate phosphorylation that is directly mediated by a particular kinase versus phosphorylation that is mediated by downstream kinases. Here, we describe a framework for assignment of direct in-vivo kinase substrates using a combination of selective chemical inhibition, quantitative phosphoproteomics, and machine learning techniques. Our workflow allows classification of phosphorylation events following inhibition of an analog-sensitive kinase into kinase-independent effects of the inhibitor, direct effects on cognate substrates and indirect effects mediated by downstream kinases or phosphatases. We applied this method to identify many direct targets of Cdc28 and Snf1 kinases in the budding yeast S. cerevisiae. Global phosphoproteome analysis of acute time-series demonstrated that dephosphorylation of direct kinase substrates occurs more rapidly compared to indirect substrates, both after inhibitor treatment and under a physiological nutrient shift in wild-type cells. Mutagenesis experiments revealed a high proportion of functionally relevant phosphorylation sites on Snf1 targets. For example, Snf1 itself was inhibited through autophosphorylation on S391 and new phosphosites were discovered that modulate the activity of the Reg1 regulatory subunit of the Glc7 phosphatase and the Gal83 β-subunit of SNF1 complex. This methodology applies to any kinase for which a functional analog sensitive version can be constructed to facilitate the dissection of the global phosphorylation network.

  13. Quantitative phosphoproteomic analysis of early seed development in rice (Oryza sativa L.).

    PubMed

    Qiu, Jiehua; Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Lin, Haiyan; Liu, Qing; Zhang, Wen; Li, Zhiyong; Nallamilli, Babi R; Zhang, Jian

    2016-02-01

    Rice (Oryza sativa L.) seed serves as a major food source for over half of the global population. Though it has been long recognized that phosphorylation plays an essential role in rice seed development, the phosphorylation events and dynamics in this process remain largely unknown so far. Here, we report the first large scale identification of rice seed phosphoproteins and phosphosites by using a quantitative phosphoproteomic approach. Thorough proteomic studies in pistils and seeds at 3, 7 days after pollination resulted in the successful identification of 3885, 4313 and 4135 phosphopeptides respectively. A total of 2487 proteins were differentially phosphorylated among the three stages, including Kip related protein 1, Rice basic leucine zipper factor 1, Rice prolamin box binding factor and numerous other master regulators of rice seed development. Moreover, differentially phosphorylated proteins may be extensively involved in the biosynthesis and signaling pathways of phytohormones such as auxin, gibberellin, abscisic acid and brassinosteroid. Our results strongly indicated that protein phosphorylation is a key mechanism regulating cell proliferation and enlargement, phytohormone biosynthesis and signaling, grain filling and grain quality during rice seed development. Overall, the current study enhanced our understanding of the rice phosphoproteome and shed novel insight into the regulatory mechanism of rice seed development.

  14. Quantitative Phosphoproteomics Identifies Filaggrin and other Targets of Ionizing Radiation in a Human Skin Model

    SciTech Connect

    Yang, Feng; Waters, Katrina M.; Webb-Robertson, Bobbie-Jo M.; Sowa, Marianne B.; Freiin von Neubeck, Claere H.; Aldrich, Joshua T.; Markillie, Lye Meng; Wirgau, Rachel M.; Gristenko, Marina A.; Zhao, Rui; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2012-04-17

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low and high dose ionizing radiation dependent signaling in a complex 3-dimensional setting. Application of an isobaric labeling strategy using sham and 3 radiation doses (3, 10, 200 cGy) resulted in the identification of 1113 unique phosphopeptides. Statistical analyses identified 151 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1 and 2,) had altered phosphorylation levels following exposure to both low and high doses of radiation. A phosphorylation site present in multiple copies in the linker regions of human profilaggrin underwent the largest fold change. Increased phosphorylation of these sites coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain skin barrier functions and minimize the damaging consequences of radiation exposure.

  15. Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide.

    PubMed

    Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

    2017-02-10

    Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

  16. Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

    PubMed

    Mauriat, Mélanie; Leplé, Jean-Charles; Claverol, Stéphane; Bartholomé, Jérôme; Negroni, Luc; Richet, Nicolas; Lalanne, Céline; Bonneu, Marc; Coutand, Catherine; Plomion, Christophe

    2015-08-07

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

  17. Urinary proteomic and non-prefractionation quantitative phosphoproteomic analysis during pregnancy and non-pregnancy

    PubMed Central

    2013-01-01

    Background Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine has become an ideal and rich source of biomarkers in clinical proteomics. Here we performed a proteomic analysis of urine samples from pregnant and non-pregnant patients using gel electrophoresis and high-resolution mass spectrometry. Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic approach using mTRAQ labeling to evaluate the expression of specific phosphoproteins during pregnancy comparison with non-pregnancy. Results In total, 2579 proteins (10429 unique peptides) were identified, including 1408 from the urine of pregnant volunteers and 1985 from the urine of non-pregnant volunteers. One thousand and twenty-three proteins were not reported in previous studies at the proteome level and were unique to our study. Furthermore, we obtained 237 phosphopeptides, representing 105 phosphoproteins. Among these phosphoproteins, 16 of them were found to be significantly differentially expressed, of which 14 were up-regulated and two were down-regulated in urine samples from women just before vaginal delivery. Conclusion Taken together, these results offer a comprehensive urinary proteomic profile of healthy women during before and after vaginal delivery and novel information on the phosphoproteins that are differentially regulated during the maintenance of normal pregnancy. Our results may provide a better understanding of the mechanisms of pregnancy maintenance, potentially leading to the development of biomarker-based sensitive assays for understanding pregnancy. PMID:24215720

  18. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells

    PubMed Central

    Rusin, Scott F.; Schlosser, Kate A.; Adamo, Mark E.; Kettenbach, Arminja N.

    2017-01-01

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c–dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. PMID:26462736

  19. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  20. Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.

    PubMed

    Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei; Nakagami, Hirofumi; Somers, David E

    2015-08-01

    The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.

  1. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition

    PubMed Central

    Beck, Florian; Geiger, Jörg; Gambaryan, Stepan; Solari, Fiorella A.; Dell’Aica, Margherita; Loroch, Stefan; Mattheij, Nadine J.; Mindukshev, Igor; Pötz, Oliver; Jurk, Kerstin; Burkhart, Julia M.; Fufezan, Christian; Heemskerk, Johan W. M.; Walter, Ulrich

    2017-01-01

    Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein–coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbβ3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. PMID:28060719

  2. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  3. Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

    PubMed

    Verma, Renu; Pinto, Sneha Maria; Patil, Arun Hanumana; Advani, Jayshree; Subba, Pratigya; Kumar, Manish; Sharma, Jyoti; Dey, Gourav; Ravikumar, Raju; Buggi, Shashidhar; Satishchandra, Parthasarathy; Sharma, Kusum; Suar, Mrutyunjay; Tripathy, Srikanth Prasad; Chauhan, Devendra Singh; Gowda, Harsha; Pandey, Akhilesh; Gandotra, Sheetal; Prasad, Thottethodi Subrahmanya Keshava

    2017-03-20

    Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

  4. Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

    SciTech Connect

    Nguyen, Tran H.; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese RW; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolic, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-11-11

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e., roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag 8-plex ITRAQ, enriched using Ni-NTA magnetic beads and subjected to nRPLC-MS/MS analysis using HCD and decision tree guided CID/ETD strategy. A total of 1,625 unique phosphopeptides, spanning 1,659 non-redundant phosphorylation sites, were detected from 1,126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5 fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  5. Discovery of mouse spleen signaling responses to anthrax using label-free quantitative phosphoproteomics via mass spectrometry.

    PubMed

    Manes, Nathan P; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C; Choi, Dahan; Bailey, Charles L; Petricoin, Emanuel F; Liotta, Lance A; Popov, Serguei G

    2011-03-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24-48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48-72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  6. Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry*

    PubMed Central

    Manes, Nathan P.; Dong, Li; Zhou, Weidong; Du, Xiuxia; Reghu, Nikitha; Kool, Arjan C.; Choi, Dahan; Bailey, Charles L.; Petricoin, Emanuel F.; Liotta, Lance A.; Popov, Serguei G.

    2011-01-01

    Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24–48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48–72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic

  7. Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid

    PubMed Central

    Wu, Liuji; Hu, Xiuli; Wang, Shunxi; Tian, Lei; Pang, Yanjie; Han, Zanping; Wu, Liancheng; Chen, Yanhui

    2015-01-01

    Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA. PMID:26659305

  8. Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-05

    Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype.

  9. Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid.

    PubMed

    Wu, Liuji; Hu, Xiuli; Wang, Shunxi; Tian, Lei; Pang, Yanjie; Han, Zanping; Wu, Liancheng; Chen, Yanhui

    2015-12-11

    Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA.

  10. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

    PubMed Central

    Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

  11. Quantitative phosphoproteomic analyses of the inferior parietal lobule from three different pathological stages of Alzheimer's disease.

    PubMed

    Triplett, Judy C; Swomley, Aaron M; Cai, Jian; Klein, Jon B; Butterfield, D Allan

    2016-01-01

    Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between 'normal' cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as 'asymptomatic' or 'preclinical' AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain.

  12. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation on Keratin-8 in skin squamous cell carcinoma derived cell- line.

    PubMed

    Tiwari, Richa; Sahu, Indrajit; Soni, Bihari Lal; Sathe, Gajanan J; Datta, Keshava K; Thapa, Pankaj; Sinha, Shruti; Vadivel, Chella Krishna; Dhaka, Bharti; Gowda, Harsha; Vaidya, Milind M

    2017-02-07

    Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine(73) (head-domain) and Serine(431) (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed TMT-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1(T14,Y15) , EIF4EBP1(T46,T50) , EIF4B(S422) , AKT1S1T246,S247, CTTN1(T401,S405,) Y421 & CAP1(S307/309) in K8-S73A/D mutant and CTTN1(T401,S405,Y421) , BUB1B(S1043) & CARHSP1(S30,S32) in K8-S431A/D mutants as well as some anonymous phosphosites including MYC(S176) , ZYX(S344) and PNN(S692) could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site- specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies. This article is protected by copyright. All rights reserved.

  13. Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

    PubMed

    Gioia, Romain; Leroy, Cédric; Drullion, Claire; Lagarde, Valérie; Etienne, Gabriel; Dulucq, Stéphanie; Lippert, Eric; Roche, Serge; Mahon, François-Xavier; Pasquet, Jean-Max

    2011-08-25

    In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML.

  14. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    PubMed

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-02-02

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

  15. Quantitative phosphoproteomics of murine Fmr1-KO cell lines provides new insights into FMRP-dependent signal transduction mechanisms.

    PubMed

    Matic, Katarina; Eninger, Timo; Bardoni, Barbara; Davidovic, Laetitia; Macek, Boris

    2014-10-03

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS.

  16. Phosphoproteomics in Cancer

    PubMed Central

    Harsha, H. C.; Pandey, Akhilesh

    2010-01-01

    Reversible protein phosphorylation serves as a basis for regulating a number of cellular processes. Aberrant activation of kinase signaling pathways is commonly associated with several cancers. Recent developments in phosphoprotein/phosphopeptide enrichment strategies and quantitative mass spectrometry have resulted in robust pipelines for high-throughput characterization of phosphorylation in a global fashion. Today, it is possible to profile site-specific phosphorylation events on thousands of proteins in a single experiment. The potential of this approach is already being realized to characterize signaling pathways that govern oncogenesis. In addition, chemical proteomic strategies have been used to unravel targets of kinase inhibitors, which are otherwise difficult to characterize. This review summarizes various approaches used for analysis of the phosphoproteome in general, and protein kinases in particular, highlighting key cancer phosphoproteomic studies. PMID:20937571

  17. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

    PubMed Central

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang

    2013-01-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

  18. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  19. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  20. Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.

    PubMed

    Rinalducci, Sara; Longo, Valentina; Ceci, Luigi R; Zolla, Lello

    2015-02-01

    One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.

  1. Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

    PubMed

    Nguyen, Elizabeth V; Imanishi, Susumu Y; Haapaniemi, Pekka; Yadav, Avinash; Saloheimo, Markku; Corthals, Garry L; Pakula, Tiina M

    2016-02-05

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

  2. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.

    PubMed

    Mertins, Philipp; Udeshi, Namrata D; Clauser, Karl R; Mani, D R; Patel, Jinal; Ong, Shao-en; Jaffe, Jacob D; Carr, Steven A

    2012-06-01

    Labeling of primary amines on peptides with reagents containing stable isotopes is a commonly used technique in quantitative mass spectrometry. Isobaric labeling techniques such as iTRAQ™ or TMT™ allow for relative quantification of peptides based on ratios of reporter ions in the low m/z region of spectra produced by precursor ion fragmentation. In contrast, nonisobaric labeling with mTRAQ™ yields precursors with different masses that can be directly quantified in MS1 spectra. In this study, we compare iTRAQ- and mTRAQ-based quantification of peptides and phosphopeptides derived from EGF-stimulated HeLa cells. Both labels have identical chemical structures, therefore precursor ion- and fragment ion-based quantification can be directly compared. Our results indicate that iTRAQ labeling has an additive effect on precursor intensities, whereas mTRAQ labeling leads to more redundant MS2 scanning events caused by triggering on the same peptide with different mTRAQ labels. We found that iTRAQ labeling quantified nearly threefold more phosphopeptides (12,129 versus 4,448) and nearly twofold more proteins (2,699 versus 1,597) than mTRAQ labeling. Although most key proteins in the EGFR signaling network were quantified with both techniques, iTRAQ labeling allowed quantification of twice as many kinases. Accuracy of reporter ion quantification by iTRAQ is adversely affected by peptides that are cofragmented in the same precursor isolation window, dampening observed ratios toward unity. However, because of tighter overall iTRAQ ratio distributions, the percentage of statistically significantly regulated phosphopeptides and proteins detected by iTRAQ and mTRAQ was similar. We observed a linear correlation of logarithmic iTRAQ to mTRAQ ratios over two orders of magnitude, indicating a possibility to correct iTRAQ ratios by an average compression factor. Spike-in experiments using peptides of defined ratios in a background of nonregulated peptides show that i

  3. Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Wang, Lianshui; Renzi, Emily; Radivojac, Predrag; Tang, Haixu; Arnold, Randy; Zhu, Jian-Kang; Tao, W. Andy

    2013-01-01

    Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants. PMID:23660473

  4. Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family.

    PubMed

    Resjö, Svante; Ali, Ashfaq; Meijer, Harold J G; Seidl, Michael F; Snel, Berend; Sandin, Marianne; Levander, Fredrik; Govers, Francine; Andreasson, Erik

    2014-04-04

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

  5. Phosphoproteomics and molecular cardiology: techniques, applications and challenges.

    PubMed

    Sun, Zeyu; Hamilton, Karyn L; Reardon, Kenneth F

    2012-09-01

    Protein phosphorylation has been widely documented as a key regulatory and signaling mechanism associated with many cardiac diseases. Recent advances in phosphoproteomic technologies such as phosphopeptide enrichment, novel mass spectrometry applications, and bioinformatic tools have resulted in high-throughput identification and quantitation of protein phosphorylation in a global manner. This review summarizes mainstream phosphoproteomic workflows and highlights the most recent applications of phosphoproteomics used in a range of molecular cardiology research.

  6. Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

    PubMed Central

    Ravikumar, Vaishnavi; Shi, Lei; Krug, Karsten; Derouiche, Abderahmane; Jers, Carsten; Cousin, Charlotte; Kobir, Ahasanul; Mijakovic, Ivan; Macek, Boris

    2014-01-01

    Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates. PMID:24390483

  7. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling

    PubMed Central

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y.

    2015-01-01

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays. PMID:26278961

  8. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease.

    PubMed

    Triplett, Judy C; Zhang, Zhaoshu; Sultana, Rukhsana; Cai, Jian; Klein, Jon B; Büeler, Hansruedi; Butterfield, David Allan

    2015-06-01

    Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70

  9. Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*

    PubMed Central

    Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl

    2015-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis). PMID:25561503

  10. Quantitative Phosphoproteomics Reveals Pathways for Coordination of Cell Growth and Division by the Conserved Fission Yeast Kinase Pom1*

    PubMed Central

    Kettenbach, Arminja N.; Deng, Lin; Wu, Youjun; Baldissard, Suzanne; Adamo, Mark E.; Gerber, Scott A.; Moseley, James B.

    2015-01-01

    Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets. PMID:25720772

  11. Informatics Methods to Enable Sharing of Quantitative Imaging Research Data

    PubMed Central

    Levy, Mia A.; Freymann, John B.; Kirby, Justin S.; Fedorov, Andriy; Fennessy, Fiona M.; Eschrich, Steven A.; Berglund, Anders E.; Fenstermacher, David A.; Tan, Yongqiang; Guo, Xiaotao; Casavant, Thomas L.; Brown, Bartley J.; Braun, Terry A.; Dekker, Andre; Roelofs, Erik; Mountz, James M.; Boada, Fernando; Laymon, Charles; Oborski, Matt; Rubin, Daniel L

    2012-01-01

    Introduction The National Cancer Institute (NCI) Quantitative Research Network (QIN) is a collaborative research network whose goal is to share data, algorithms and research tools to accelerate quantitative imaging research. A challenge is the variability in tools and analysis platforms used in quantitative imaging. Our goal was to understand the extent of this variation and to develop an approach to enable sharing data and to promote reuse of quantitative imaging data in the community. Methods We performed a survey of the current tools in use by the QIN member sites for representation and storage of their QIN research data including images, image meta-data and clinical data. We identified existing systems and standards for data sharing and their gaps for the QIN use case. We then proposed a system architecture to enable data sharing and collaborative experimentation within the QIN. Results There area variety of tools currently used by each QIN institution. We developed a general information system architecture to support the QIN goals. We also describe the remaining architecture gaps we are developing to enable members to share research images and image meta-data across the network. Conclusions As a research network, the QIN will stimulate quantitative imaging research by pooling data, algorithms and research tools. However, there are gaps in current functional requirements that will need to be met by future informatics development. Special attention must be given to the technical requirements needed to translate these methods into the clinical research workflow to enable validation and qualification of these novel imaging biomarkers. PMID:22770688

  12. SILAC for global phosphoproteomic analysis.

    PubMed

    Pimienta, Genaro; Chaerkady, Raghothama; Pandey, Akhilesh

    2009-01-01

    Establishing the phosphorylation pattern of proteins in a comprehensive fashion is an important goal of a majority of cell signaling projects. Phosphoproteomic strategies should be designed in such a manner as to identify sites of phosphorylation as well as to provide quantitative information about the extent of phosphorylation at the sites. In this chapter, we describe an experimental strategy that outlines such an approach using stable isotope labeling with amino acids in cell culture (SILAC) coupled to LC-MS/MS. We highlight the importance of quantitative strategies in signal transduction as a platform for a systematic and global elucidation of biological processes.

  13. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells

    PubMed Central

    FANG, YI; ZHANG, QIAN; WANG, XIN; YANG, XUE; WANG, XIANGYU; HUANG, ZHEN; JIAO, YUCHEN; WANG, JING

    2016-01-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  14. Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

    PubMed Central

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-01-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. PMID:25693802

  15. Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

    PubMed

    Qing, Dongjin; Yang, Zhu; Li, Mingzhe; Wong, Wai Shing; Guo, Guangyu; Liu, Shichang; Guo, Hongwei; Li, Ning

    2016-01-04

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

  16. Quantitative phosphoproteomics after auxin-stimulated lateral root induction identifies an SNX1 protein phosphorylation site required for growth.

    PubMed

    Zhang, Hongtao; Zhou, Houjiang; Berke, Lidija; Heck, Albert J R; Mohammed, Shabaz; Scheres, Ben; Menke, Frank L H

    2013-05-01

    Protein phosphorylation is instrumental to early signaling events. Studying system-wide phosphorylation in relation to processes under investigation requires a quantitative proteomics approach. In Arabidopsis, auxin application can induce pericycle cell divisions and lateral root formation. Initiation of lateral root formation requires transcriptional reprogramming following auxin-mediated degradation of transcriptional repressors. The immediate early signaling events prior to this derepression are virtually uncharacterized. To identify the signal molecules responding to auxin application, we used a lateral root-inducible system that was previously developed to trigger synchronous division of pericycle cells. To identify and quantify the early signaling events following this induction, we combined (15)N-based metabolic labeling and phosphopeptide enrichment and applied a mass spectrometry-based approach. In total, 3068 phosphopeptides were identified from auxin-treated root tissue. This root proteome dataset contains largely phosphopeptides not previously reported and represents one of the largest quantitative phosphoprotein datasets from Arabidopsis to date. Key proteins responding to auxin treatment included the multidrug resistance-like and PIN2 auxin carriers, auxin response factor2 (ARF2), suppressor of auxin resistance 3 (SAR3), and sorting nexin1 (SNX1). Mutational analysis of serine 16 of SNX1 showed that overexpression of the mutated forms of SNX1 led to retarded growth and reduction of lateral root formation due to the reduced outgrowth of the primordium, showing proof of principle for our approach.

  17. Databases for plant phosphoproteomics.

    PubMed

    Schulze, Waltraud X; Yao, Qiuming; Xu, Dong

    2015-01-01

    Phosphorylation is the most studied posttranslational modification involved in signal transduction in stress responses, development, and growth. In the recent years large-scale phosphoproteomic studies were carried out using various model plants and several growth and stress conditions. Here we present an overview of online resources for plant phosphoproteomic databases: PhosPhAt as a resource for Arabidopsis phosphoproteins, P3DB as a resource expanding to crop plants, and Medicago PhosphoProtein Database as a resource for the model plant Medicago trunculata.

  18. Phosphoproteomics in cereals.

    PubMed

    Yang, Pingfang

    2015-01-01

    Cereals are the most important crop plant supplying staple food throughout the world. The economic importance and continued breeding of crop plants such as rice, maize, wheat, or barley require a detailed scientific understanding of adaptive and developmental processes. Protein phosphorylation is one of the most important regulatory posttranslational modifications and its analysis allows deriving functional and regulatory principles in plants. This minireview summarizes the current knowledge of phosphoproteomic studies in cereals.

  19. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

    PubMed Central

    Nukarinen, Ella; Nägele, Thomas; Pedrotti, Lorenzo; Wurzinger, Bernhard; Mair, Andrea; Landgraf, Ramona; Börnke, Frederik; Hanson, Johannes; Teige, Markus; Baena-Gonzalez, Elena; Dröge-Laser, Wolfgang; Weckwerth, Wolfram

    2016-01-01

    Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants. PMID:27545962

  20. Phosphoproteomic profiling of the myocyte.

    PubMed

    Edwards, Alistair V G; Cordwell, Stuart J; White, Melanie Y

    2011-10-01

    Protein phosphorylation underpins major cellular processes including energy metabolism, signal transduction, excitation-contraction coupling, apoptosis, and cell survival mechanisms and is thus critical to the myocyte. Targeted approaches, whereby a handful of phosphoproteins are investigated, can suffer from a relatively narrow view of cellular phosphorylation. In contrast, recent technical advances have allowed for the comprehensive documentation of phosphorylation events in complex biological environments, providing a deeper view of the "phosphoproteome." A global, high-throughput characterization of the myocardial phosphoproteome, however, has not yet been achieved. Efficient analysis of phosphorylated proteins and their roles in a dynamic cellular environment requires high-resolution strategies that can identify, localize, and quantify many thousands of phosphorylation sites in a single experiment. Such an approach requires specific enrichment and purification techniques, developed to align with high-end instrumentation for analysis. Cutting-edge phosphoproteomics is no longer restricted to gel-based technology, instead focusing on affinity enrichment prior to liquid chromatography and mass spectrometry. We will describe the best current methods and how they can be applied, as well as the challenges associated with them. We also present current phosphoproteomic investigations in the myocyte and its subcompartments. Although the techniques and instrumentation required to achieve the goal of a myocardial phosphoprotein catalog in physiological and diseased states are highly specialized, the potential biological insight provided by such an approach makes phosphoproteomics an important new avenue of investigation for the cardiovascular researcher.

  1. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization

    PubMed Central

    Ficarro, Scott B.; Hutchinson, John N.; Csepanyi-Komi, Roland; Nguyen, Phi T.; Wisniewski, Eva; Sullivan, Jessica; Hofmann, Oliver; Ligeti, Erzsebet; Marto, Jarrod A.; Wagers, Amy J.

    2016-01-01

    Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization. PMID:27365422

  2. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    PubMed Central

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

  3. Cross-Species PTM Mapping from Phosphoproteomic Data.

    PubMed

    Chaudhuri, Rima; Yang, Jean Yee Hwa

    2017-01-01

    Protein post-translational modifications (PTMs) are crucial for signal transduction in cells. In order to understand key cell signaling events, identification of functionally important PTMs, which are more likely to be evolutionarily conserved, is necessary. In recent times, high-throughput mass spectrometry (MS) has made quantitative datasets in diverse species readily available, which has led to a growing need for tools to facilitate cross-species comparison of PTM data. Cross-species comparison of PTM sites is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on previously annotated orthologous phosphosites and do not enable cross-species mapping of newly identified modification sites. Here, we describe an automated web-based tool, PhosphOrtholog, that accurately maps annotated and novel orthologous PTM sites from high-throughput MS-based experimental data obtained from different species without relying on existing PTM databases. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of evolutionarily conserved and functional PTM sites that influence most biological processes. In this Chapter, we illustrate with examples how to use PhosphOrtholog to map novel PTM sites from cross-species MS-based phosphoproteomics data.

  4. Toward quantitative prediction of charge mobility in organic semiconductors: tunneling enabled hopping model.

    PubMed

    Geng, Hua; Peng, Qian; Wang, Linjun; Li, Haijiao; Liao, Yi; Ma, Zhiying; Shuai, Zhigang

    2012-07-10

    A tunneling-enabled hopping mechanism is proposed, providing a pratical tool to quantitatively assess charge mobility in organic semiconductors. The paradoxical phenomena in TIPS-pentacene is well explained in that the optical probe indicates localized charges while transport measurements show bands of charge.

  5. Insights Regarding Fungal Phosphoproteomic Analysis.

    PubMed

    Ribeiro, Liliane F C; Chelius, Cynthia L; Harris, Steven D; Marten, Mark R

    2017-03-10

    Protein phosphorylation is a major means of regulation for cellular processes, and is important in cell signaling, growth, and cell proliferation. To study phosphorylated proteins, high throughput phosphoproteomic technologies, such as reverse phase protein array, phospho-specific flow cytometry, and mass spectrometry (MS) based technologies, have been developed. Among them, mass spectrometry has become the primary tool employed for the identification of phosphoproteins and phosphosites in fungi, leading to an improved understanding of a number of signaling pathways. Using mass spectrometry techniques, researchers have discovered new kinase substrates, established connections between kinases and fungal pathogenicity, and studied the evolutionary lineage of kinases between different fungal species. Further, many specific phosphorylation sites recognized by individual kinases have been described. In this review, we will focus on recent discoveries made in yeast and filamentous fungi using phosphoproteomic analysis.

  6. Functional phosphoproteomic mass spectrometry-based approaches

    PubMed Central

    2012-01-01

    Mass Spectrometry (MS)-based phosphoproteomics tools are crucial for understanding the structure and dynamics of signaling networks. Approaches such as affinity purification followed by MS have also been used to elucidate relevant biological questions in health and disease. The study of proteomes and phosphoproteomes as linked systems, rather than research studies of individual proteins, are necessary to understand the functions of phosphorylated and un-phosphorylated proteins under spatial and temporal conditions. Phosphoproteome studies also facilitate drug target protein identification which may be clinically useful in the near future. Here, we provide an overview of general principles of signaling pathways versus phosphorylation. Likewise, we detail chemical phosphoproteomic tools, including pros and cons with examples where these methods have been applied. In addition, basic clues of electrospray ionization and collision induced dissociation fragmentation are detailed in a simple manner for successful phosphoproteomic clinical studies. PMID:23369623

  7. Method for collecting and immobilizing individual cumulus cells enabling quantitative immunofluorescence analysis of proteins.

    PubMed

    Appeltant, R; Maes, D; Van Soom, A

    2015-07-01

    Most immunofluorescence methods rely on techniques dealing with a very large number of cells. However, when the number of cells in a sample is low (e.g., when cumulus cells must be analyzed from individual cumulus-oocyte complexes), specific techniques are required to conserve, fix, and analyze cells individually. We established and validated a simple and effective method for collecting and immobilizing low numbers of cumulus cells that enables easy and quick quantitative immunofluorescence analysis of proteins from individual cells. To illustrate this technique, we stained proprotein of a disintegrin and metalloproteinase with thrombospondin-like repeats-1 (proADAMTS-1) and analyzed its levels in individual porcine cumulus cells.

  8. Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

    Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enables comprehensive profiling of the phosphoproteome and facilitates deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009–2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis. PMID:24687451

  9. Portable SERS-enabled micropipettes for microarea sampling and reliably quantitative detection of surface organic residues.

    PubMed

    Fang, Wei; Zhang, Xinwei; Chen, Yong; Wan, Liang; Huang, Weihua; Shen, Aiguo; Hu, Jiming

    2015-09-15

    We report the first microsampling device for reliably quantitative, label-free and separation-free detection of multicomponents of surface organic residues (SORs) by means of a quality controllable surface-enhanced Raman scattering (SERS)-enabled micropipette. The micropipette is comprised of a drawn glass capillary with a tiny orifice (∼50 μm) at the distal tip, where the specially designed nanorattles (NRs) are compactly coated on the inner wall surface. SERS signals of 4-mercapto benzoic acid (MBA) anchored inside the internal gap of NRs could be used to evaluate and control the quality of micropipettes and, therefore, allow us to overcome the limitations of a reliably quantitative SERS assay using traditional substrates without an internal standard. By dropping a trace extraction agent on targeting SORs located on a narrow surface, the capillary and SERS functionalities of these micropipettes allow on-site microsampling via capillary action and subsequent multiplex distinction/detection due to their molecularly narrow Raman peaks. For example, 8 nM thiram (TMTD), 8 nM malachite green (MG), and 1.5 μM (400 ppb) methyl parathion (MPT) on pepper and cucumber peels have been simultaneously detected in a wide detection range. The portable SERS-enabled device could potentially be facilely incorporated with liquid-liquid or solid phase micro-extracting devices for a broader range of applications in rapid and field analysis of food/public/environment security related SORs.

  10. Glucose-regulated and drug-perturbed phosphoproteome reveals molecular mechanisms controlling insulin secretion

    PubMed Central

    Sacco, Francesca; Humphrey, Sean J.; Cox, Jürgen; Mischnik, Marcel; Schulte, Anke; Klabunde, Thomas; Schäfer, Matthias; Mann, Matthias

    2016-01-01

    Insulin-secreting beta cells play an essential role in maintaining physiological blood glucose levels, and their dysfunction leads to the development of diabetes. To elucidate the signalling events regulating insulin secretion, we applied a recently developed phosphoproteomics workflow. We quantified the time-resolved phosphoproteome of murine pancreatic cells following their exposure to glucose and in combination with small molecule compounds that promote insulin secretion. The quantitative phosphoproteome of 30,000 sites clustered into three main groups in concordance with the modulation of the three key kinases: PKA, PKC and CK2A. A high-resolution time course revealed key novel regulatory sites, revealing the importance of methyltransferase DNMT3A phosphorylation in the glucose response. Remarkably a significant proportion of these novel regulatory sites is significantly downregulated in diabetic islets. Control of insulin secretion is embedded in an unexpectedly broad and complex range of cellular functions, which are perturbed by drugs in multiple ways. PMID:27841257

  11. Phosphoproteomics for the masses

    PubMed Central

    Grimsrud, Paul A.; Swaney, Danielle L.; Wenger, Craig D.; Beauchene, Nicole A.; Coon, Joshua J.

    2010-01-01

    Protein phosphorylation serves as a primary mechanism of signal transduction in the cells of biological organisms. Technical advancements over the last several years in mass spectrometry now allow for the large-scale identification and quantitation of in vivo phosphorylation at unprecedented levels. These developments have occurred in the areas of sample preparation, instrumentation, quantitative methodology, and informatics so that today, ten to twenty thousand phosphorylation sites can be identified and quantified within a few weeks. With the rapid development and widespread availability of such data, its translation into biological insight and knowledge is a current obstacle. Here we present an overview of how this technology came to be and is currently applied, as well as future challenges for the field. PMID:20047291

  12. Phosphoproteome Discovery in Human Biological Fluids

    PubMed Central

    Giorgianni, Francesco; Beranova-Giorgianni, Sarka

    2016-01-01

    Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented. PMID:28248247

  13. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  14. Accuracy of the Morphology Enabled Dipole Inversion (MEDI) Algorithm for Quantitative Susceptibility Mapping in MRI

    PubMed Central

    Liu, Tian; Xu, Weiyu; Spincemaille, Pascal; Avestimehr, A. Salman

    2013-01-01

    Determining the susceptibility distribution from the magnetic field measured in a magnetic resonance (MR) scanner is an ill-posed inverse problem, because of the presence of zeroes in the convolution kernel in the forward problem. An algorithm called morphology enabled dipole inversion (MEDI), which incorporates spatial prior information, has been proposed to generate a quantitative susceptibility map (QSM). The accuracy of QSM can be validated experimentally. However, there is not yet a rigorous mathematical demonstration of accuracy for a general regularized approach or for MEDI specifically. The error in the susceptibility map reconstructed by MEDI is expressed in terms of the acquisition noise and the error in the spatial prior information. A detailed analysis demonstrates that the error in the susceptibility map reconstructed by MEDI is bounded by a linear function of these two error sources. Numerical analysis confirms that the error of the susceptibility map reconstructed by MEDI is on the same order of the noise in the original MRI data, and comprehensive edge detection will lead to reduced model error in MEDI. Additional phantom validation and human brain imaging demonstrated the practicality of the MEDI method. PMID:22231170

  15. Phosphoproteome Dynamics Upon Changes in Plant Water Status Reveal Early Events Associated With Rapid Growth Adjustment in Maize Leaves*

    PubMed Central

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-01-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  16. Phosphoproteome dynamics upon changes in plant water status reveal early events associated with rapid growth adjustment in maize leaves.

    PubMed

    Bonhomme, Ludovic; Valot, Benoît; Tardieu, François; Zivy, Michel

    2012-10-01

    Plant growth adjustment during water deficit is a crucial adaptive response. The rapid fine-tuned control achieved at the post-translational level is believed to be of considerable importance for regulating early changes in plant growth reprogramming. Aiming at a better understanding of early responses to contrasting plant water statuses, we carried out a survey of the protein phosphorylation events in the growing zone of maize leaves upon a range of water regimes. In this study, the impact of mild and severe water deficits were evaluated in comparison with constant optimal watering and with recovery periods lasting 5, 10, 20, 30, 45, and 60 min. Using four biological replicates per treatment and a robust quantitative phosphoproteomic methodology based on stable-isotope labeling, we identified 3664 unique phosphorylation sites on 2496 proteins. The abundance of nearly 1250 phosphorylated peptides was reproducibly quantified and profiled with high confidence among treatments. A total of 138 phosphopeptides displayed highly significant changes according to water regimes and enabled to identify specific patterns of response to changing plant water statuses. Further quantification of protein amounts emphasized that most phosphorylation changes did not reflect protein abundance variation. During water deficit and recovery, extensive changes in phosphorylation status occurred in critical regulators directly or indirectly involved in plant growth and development. These included proteins influencing epigenetic control, gene expression, cell cycle-dependent processes and phytohormone-mediated responses. Some of the changes depended on stress intensity whereas others depended on rehydration duration, including rapid recoveries that occurred as early as 5 or 10 mins after rewatering. By combining a physiological approach and a quantitative phosphoproteomic analysis, this work provides new insights into the in vivo early phosphorylation events triggered by rapid changes in

  17. Combining Metabolic ¹⁵N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

    PubMed

    Thomas, Martin; Huck, Nicola; Hoehenwarter, Wolfgang; Conrath, Uwe; Beckers, Gerold J M

    2015-01-01

    that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

  18. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  19. Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis.

    PubMed

    Polat, Ayse Nur; Karayel, Özge; Giese, Sven H; Harmanda, Büşra; Sanal, Erdem; Hu, Chi-Kuo; Renard, Bernhard Y; Özlü, Nurhan

    2015-09-04

    Cytokinesis is the last step of the cell cycle that requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton, and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (stable isotope labeling with amino acids in cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680- or AZD1152-mediated aurora kinase inhibition. In total, our analysis quantified over 20 000 phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.

  20. Characterization of the Phosphoproteome in SLE Patients

    PubMed Central

    Huang, Jianrong; Dai, Yong

    2012-01-01

    Protein phosphorylation is a complex regulatory event that is involved in the signaling networks that affect virtually every cellular process. The protein phosphorylation may be a novel source for discovering biomarkers and drug targets. However, a systematic analysis of the phosphoproteome in patients with SLE has not been performed. To clarify the pathogenesis of systemic lupus erythematosus (SLE), we compared phosphoprotein expression in PBMCs from SLE patients and normal subjects using proteomics analyses. Phosphopeptides were enriched using TiO2 from PBMCs isolated from 15 SLE patients and 15 healthy subjects and then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. A total of 1035 phosphorylation sites corresponding to 618 NCBI-annotated genes were identified in SLE patients compared with normal subjects. Differentially expressed proteins, peptides and phosphorylation sites were then subjected to bioinformatics analyses. Gene ontology(GO) and pathway analyses showed that nucleic acid metabolism, cellular component organization, transport and multicellular organismal development pathways made up the largest proportions of the differentially expressed genes. Pathway analyses showed that the mitogen-activated protein kinase (MAPK) signaling pathway and actin cytoskeleton regulators made up the largest proportions of the metabolic pathways. Network analysis showed that rous sarcoma oncogene (SRC), v-rel reticuloendotheliosis viral oncogene homolog A (RELA), histone deacetylase (HDA1C) and protein kinase C, delta (PRKCD) play important roles in the stability of the network. These data suggest that aberrant protein phosphorylation may contribute to SLE pathogenesis. PMID:23285258

  1. Phosphoproteomic Analysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma

    PubMed Central

    Parker, Robert; Vella, Laura J.; Xavier, Dylan; Amirkhani, Ardeshir; Parker, Jimmy; Cebon, Jonathan; Molloy, Mark P.

    2015-01-01

    The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype. PMID:26029660

  2. 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

  3. Nanoparticle-mediated photothermal effect enables a new method for quantitative biochemical analysis using a thermometer.

    PubMed

    Fu, Guanglei; Sanjay, Sharma T; Dou, Maowei; Li, XiuJun

    2016-03-14

    A new biomolecular quantitation method, nanoparticle-mediated photothermal bioassay, using a common thermometer as the signal reader was developed. Using an immunoassay as a proof of concept, iron oxide nanoparticles (NPs) captured in the sandwich-type assay system were transformed into a near-infrared (NIR) laser-driven photothermal agent, Prussian blue (PB) NPs, which acted as a photothermal probe to convert the assay signal into heat through the photothermal effect, thus allowing sensitive biomolecular quantitation using a thermometer. This is the first report of biomolecular quantitation using a thermometer and also serves as the first attempt to introduce the nanoparticle-mediated photothermal effect for bioassays.

  4. Enabling Quantitative Analysis in Ambient Ionization Mass Spectrometry: Internal Standard Coated Capillary Samplers

    PubMed Central

    Liu, Jiangjiang; Cooks, R. Graham; Ouyang, Zheng

    2013-01-01

    We describe a sampling method using glass capillaries for quantitative analysis of trace analytes in small volumes of complex mixtures (~1 μL) using ambient ionization mass spectrometry. The internal surface of a sampling glass capillary was coated with internal standard then used to draw liquid sample and so transfer both the analyte and internal standard in a single fixed volume onto a substrate for analysis. The internal standard was automatically mixed into the sample during this process and the volumes of the internal standard solution and sample are both fixed by the capillary volume. Precision in quantitation is insensitive to variations in length of the capillary, making the preparation of the sampling capillary simple and providing a robust sampling protocol. Significant improvements in quantitation accuracy were obtained for analysis of 1 μL samples using various ambient ionization methods. PMID:23731380

  5. Nanotag-enabled photonic crystal fiber as quantitative surface-enhanced Raman scattering optofluidic platform

    SciTech Connect

    Pinkhasova, Polina; Chen, Hui; Du, Henry; Kanka, Jiri; Mergo, Pawel

    2015-02-16

    Core-shell nanotags that are active in surface-enhanced Raman scattering (SERS) and entrapped with thiocyanate (SCN) label molecules were immobilized in the air channels of suspended-core photonic crystal fiber (PCF) to impart quantitative capacity to SERS-based PCF optofluidic sensing platform. The Raman intensity of Rhodamine 6G increases with concentration, whereas the intensity of SCN remains constant when measured using this platform. The signal from the SCN label can be used as an internal reference to establish calibration for quantitative measurements of analytes of unknown concentrations. The long optical path-length PCF optofluidic platform integrated with SERS-active core-shell nanotags holds significant promise for sensitive quantitative chem/bio measurements with the added benefit of small sampling volume. The dependence of SERS intensity on the nanotag coverage density and PCF length was interpreted based on numerical-analytical simulations.

  6. Nanoparticle-mediated photothermal effect enables a new method for quantitative biochemical analysis using a thermometer

    NASA Astrophysics Data System (ADS)

    Fu, Guanglei; Sanjay, Sharma T.; Dou, Maowei; Li, Xiujun

    2016-03-01

    A new biomolecular quantitation method, nanoparticle-mediated photothermal bioassay, using a common thermometer as the signal reader was developed. Using an immunoassay as a proof of concept, iron oxide nanoparticles (NPs) captured in the sandwich-type assay system were transformed into a near-infrared (NIR) laser-driven photothermal agent, Prussian blue (PB) NPs, which acted as a photothermal probe to convert the assay signal into heat through the photothermal effect, thus allowing sensitive biomolecular quantitation using a thermometer. This is the first report of biomolecular quantitation using a thermometer and also serves as the first attempt to introduce the nanoparticle-mediated photothermal effect for bioassays.A new biomolecular quantitation method, nanoparticle-mediated photothermal bioassay, using a common thermometer as the signal reader was developed. Using an immunoassay as a proof of concept, iron oxide nanoparticles (NPs) captured in the sandwich-type assay system were transformed into a near-infrared (NIR) laser-driven photothermal agent, Prussian blue (PB) NPs, which acted as a photothermal probe to convert the assay signal into heat through the photothermal effect, thus allowing sensitive biomolecular quantitation using a thermometer. This is the first report of biomolecular quantitation using a thermometer and also serves as the first attempt to introduce the nanoparticle-mediated photothermal effect for bioassays. Electronic supplementary information (ESI) available: Additional information on FTIR characterization (Fig. S1), photothermal immunoassay of PSA in human serum samples (Table S1), and the Experimental section, including preparation of antibody-conjugated iron oxide NPs, sandwich-type immunoassay, characterization, and photothermal detection protocol. See DOI: 10.1039/c5nr09051b

  7. TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics*

    PubMed Central

    Zhong, Jun; Kim, Min-Sik; Chaerkady, Raghothama; Wu, Xinyan; Huang, Tai-Chung; Getnet, Derese; Mitchell, Christopher J.; Palapetta, Shyam M.; Sharma, Jyoti; O'Meally, Robert N.; Cole, Robert N.; Yoda, Akinori; Moritz, Albrecht; Loriaux, Marc M.; Rush, John; Weinstock, David M.; Tyner, Jeffrey W.; Pandey, Akhilesh

    2012-01-01

    Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets

  8. A peptide-retrieval strategy enables significant improvement of quantitative performance without compromising confidence of identification.

    PubMed

    Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

    2017-01-30

    Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches.

  9. High-Resolution Enabled 12-Plex DiLeu Isobaric Tags for Quantitative Proteomics

    PubMed Central

    2015-01-01

    Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer. PMID:25405479

  10. Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

    PubMed Central

    Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

    2017-01-01

    Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well. PMID:28358111

  11. Advances in Surface Plasmon Resonance Imaging enable quantitative measurement of laterally heterogeneous coatings of nanoscale thickness

    NASA Astrophysics Data System (ADS)

    Raegen, Adam; Reiter, Kyle; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

    2013-03-01

    The Surface Plasmon Resonance (SPR) phenomenon is routinely exploited to qualitatively probe changes to the optical properties of nanoscale coatings on thin metallic surfaces, for use in probes and sensors. Unfortunately, extracting truly quantitative information is usually limited to a select few cases - uniform absorption/desorption of small biomolecules and films, in which a continuous ``slab'' model is a good approximation. We present advancements in the SPR technique that expand the number of cases for which the technique can provide meaningful results. Use of a custom, angle-scanning SPR imaging system, together with a refined data analysis method, allow for quantitative kinetic measurements of laterally heterogeneous systems. We first demonstrate the directionally heterogeneous nature of the SPR phenomenon using a directionally ordered sample, then show how this allows for the calculation of the average coverage of a heterogeneous sample. Finally, the degradation of cellulose microfibrils and bundles of microfibrils due to the action of cellulolytic enzymes will be presented as an excellent example of the capabilities of the SPR imaging system.

  12. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

  13. Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories.

    PubMed

    Wu, C-H; Kong, L; Bialecka-Fornal, M; Park, S; Thompson, A L; Kulkarni, G; Conway, S J; Newman, D K

    2015-07-01

    external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.

  14. Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories

    PubMed Central

    Wu, C-H; Kong, L; Bialecka-Fornal, M; Park, S; Thompson, A L; Kulkarni, G; Conway, S J; Newman, D K

    2015-01-01

    external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples. PMID:25865768

  15. Phosphoproteomics technologies and applications in plant biology research

    PubMed Central

    Li, Jinna; Silva-Sanchez, Cecilia; Zhang, Tong; Chen, Sixue; Li, Haiying

    2015-01-01

    Protein phosphorylation has long been recognized as an essential mechanism to regulate many important processes of plant life. However, studies on phosphorylation mediated signaling events in plants are challenged with low stoichiometry and dynamic nature of phosphorylated proteins. Significant advances in mass spectrometry based phosphoproteomics have taken place in recent decade, including phosphoprotein/phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. This review describes a variety of separation and enrichment methods for phosphoproteins and phosphopeptides, the applications of technological innovations in plant phosphoproteomics, and highlights significant achievement of phosphoproteomics in the areas of plant signal transduction, growth and development. PMID:26136758

  16. Laboratory reflectance spectra of clay minerals mixed with Mars analog materials: Toward enabling quantitative clay abundances from Mars spectra

    NASA Astrophysics Data System (ADS)

    Roush, Ted L.; Bishop, Janice L.; Brown, Adrian J.; Blake, David F.; Bristow, Thomas F.

    2015-09-01

    Quantitative estimates of clay minerals on the martian surface, via remote sensing observations, provide constraints on activity, timing, duration, and extent of aqueous processes and the geochemical environment in martian history. We describe an analytical study to begin enabling quantitative estimates of phyllosilicates when mixed with martian analog materials. We characterize the chemistry, mineralogy, particle size distribution, and reflectance spectra of the end-member materials: saponite, montmorillonite, pyroxene, and palagonitic soil. Reflectance spectra were obtained for physical mixtures of saponite and montmorillonite with pyroxene, and saponite with palagonitic soil. We analyzed the diagnostic phyllosilicate spectral signatures in the 2.2-2.4 μm wavelength region in detail for the mixtures. This involved fitting the observed ∼2.3 or ∼2.2 μm band depth, associated with the presence of saponite and montmorillonite, respectively, as a function of the abundance of these materials in the mixtures. Based upon the band depth of the spectral features we find that 3-5 wt.% of the clay minerals in the mixture with pyroxene can be recognized and at 25 wt.% their presence is indisputable in the mixtures. When the saponite is mixed with the lower albedo palagonitic soil, its presence is clearly distinguishable via the 1.4 and 2.3 μm features at 25 wt.% abundance. These relationships, between abundance and band depth, provide an ability to quantitatively address the amount of these materials in mixtures. The trends described here provide guidance for estimating the presence of phyllosilicates in matrices on the martian surface.

  17. TiSH--a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC.

    PubMed

    Engholm-Keller, Kasper; Birck, Pernille; Størling, Joachim; Pociot, Flemming; Mandrup-Poulsen, Thomas; Larsen, Martin R

    2012-10-22

    Large scale quantitative phosphoproteomics depends upon multidimensional strategies for peptide fractionation, phosphopeptide enrichment, and mass spectrometric analysis. Previously, most robust comprehensive large-scale phosphoproteomics strategies have relied on milligram amounts of protein. We have set up a multi-dimensional phosphoproteomics strategy combining a number of well-established enrichment and fraction methods: An initial TiO(2) phosphopeptide pre-enrichment step is followed by post-fractionation using sequential elution from IMAC (SIMAC) to separate multi- and mono-phosphorylated peptides, and hydrophilic interaction liquid chromatography (HILIC) of the mono-phosphorylated peptides (collectively abbreviated "TiSH"). The advantages of the strategy include a high specificity and sample preparation workload reduction due to the TiO(2) pre-enrichment step, as well as low adsorptive losses. We demonstrate the capability of this strategy by quantitative investigation of early interferon-γ signaling in low quantities of insulinoma cells. We identified ~6600 unique phosphopeptides from 300 μg of peptides/condition (22 unique phosphopeptides/μg) in a duplex dimethyl labeling experiment, with an enrichment specificity>94%. When doing network analysis of putative phosphorylation changes it could be noted that the identified protein interaction network centered upon proteins known to be affected by the interferon-γ pathway, thereby supporting the utility of this global phosphoproteomics strategy. This strategy thus shows great potential for interrogating signaling networks from low amounts of sample with high sensitivity and specificity.

  18. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  19. Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress.

    PubMed

    Hu, Xiuli; Wu, Liuji; Zhao, Feiyun; Zhang, Dayong; Li, Nana; Zhu, Guohui; Li, Chaohao; Wang, Wei

    2015-01-01

    Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149, and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors.

  20. Phosphoproteomic analysis of Her2/neu signaling and inhibition

    PubMed Central

    Bose, Ron; Molina, Henrik; Patterson, A. Scott; Bitok, John K.; Periaswamy, Balamurugan; Bader, Joel S.; Pandey, Akhilesh; Cole, Philip A.

    2006-01-01

    Her2/neu (Her2) is a tyrosine kinase belonging to the EGF receptor (EGFR)/ErbB family and is overexpressed in 20–30% of human breast cancers. We sought to characterize Her2 signal transduction pathways further by using MS-based quantitative proteomics. Stably transfected cell lines overexpressing Her2 or empty vector were generated, and the effect of an EGFR and Her2 selective tyrosine kinase inhibitor, PD168393, on these cells was characterized. Quantitative measurements were obtained on 462 proteins by using the SILAC (stable isotope labeling with amino acids in cell culture) method to monitor three conditions simultaneously. Of these proteins, 198 showed a significant increase in tyrosine phosphorylation in Her2-overexpressing cells, and 81 showed a significant decrease in phosphorylation. Treatment of Her2-overexpressing cells with PD168393 showed rapid reversibility of the majority of the Her2-triggered phosphorylation events. Phosphoproteins that were identified included many known Her2 signaling molecules as well as known EGFR signaling proteins that had not been previously linked to Her2, such as Stat1, Dok1, and δ-catenin. Importantly, several previously uncharacterized Her2 signaling proteins were identified, including Axl tyrosine kinase, the adaptor protein Fyb, and the calcium-binding protein Pdcd-6/Alg-2. We also identified a phosphorylation site in Her2, Y877, which is located in the activation loop of the kinase domain, is distinct from the known C-terminal tail autophosphorylation sites, and may have important implications for regulation of Her2 signaling. Network modeling, which combined phosphoproteomic results with literature-curated protein–protein interaction data, was used to suggest roles for some of the previously unidentified Her2 signaling proteins. PMID:16785428

  1. Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

    PubMed

    Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

    2014-08-01

    Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the β-actin gene (actb), β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

  2. Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

    SciTech Connect

    Sirenko, Oksana; Cromwell, Evan F.; Crittenden, Carole; Wignall, Jessica A.; Wright, Fred A.; Rusyn, Ivan

    2013-12-15

    Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca{sup 2+} flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive “cardiosafety” ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development. - Highlights: • Induced pluripotent stem cell-derived cardiomyocytes are promising in vitro models. • We tested if evaluation

  3. Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

    PubMed Central

    Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.

    2015-01-01

    Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499

  4. Deep Phosphoproteomic Measurements Pinpointing Drug Induced Protective Mechanisms in Neuronal Cells

    PubMed Central

    Yu, Chengli; Gao, Jing; Zhou, Yanting; Chen, Xiangling; Xiao, Ruoxuan; Zheng, Jing; Liu, Yansheng; Zhou, Hu

    2016-01-01

    Alzheimer's disease (AD) is a progressive and irreversible neurological disorder that impairs the living quality of old population and even life spans. New compounds have shown potential inneuroprotective effects in AD, such as GFKP-19, a 2-pyrrolidone derivative which has been proved to enhance the memory of dysmnesia mouse. The molecular mechanisms remain to be established for these drug candidates. Large-scale phosphoproteomic approach has been evolved rapidly in the last several years, which holds the potential to provide a useful toolkit to understand cellular signaling underlying drug effects. To establish and test such a method, we accurately analyzed the deep quantitative phosphoproteome of the neuro-2a cells treated with and without GFKP-19 using triple SILAC labeling. A total of 14,761 Class I phosphosites were quantified between controls, damaged, and protected conditions using the high resolution mass spectrometry, with a decent inter-mass spectrometer reproducibility for even subtle regulatory events. Our data suggests that GFKP-19 can reverse Aβ25−35 induced phosphorylation change in neuro-2a cells, and might protect the neuron system in two ways: firstly, it may decrease oxidative damage and inflammation induced by NO via down regulating the phosphorylation of nitric oxide synthase NOS1 at S847; Secondly, it may decrease tau protein phosphorylation through down-regulating the phosphorylation level of MAPK14 at T180. All mass spectrometry data are available via ProteomeXchange with identifier PXD005312. PMID:28066266

  5. Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats.

    PubMed

    Nirujogi, Raja Sekhar; Wright, James D; Manda, Srikanth S; Zhong, Jun; Na, Chan Hyun; Meyerhoff, James; Benton, Bernard; Jabbour, Rabih; Willis, Kristen; Kim, Min-Sik; Pandey, Akhilesh; Sekowski, Jennifer W

    2015-01-01

    To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

  6. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana

    PubMed Central

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  7. Shotguns in the front line: phosphoproteomics in plants.

    PubMed

    Nakagami, Hirofumi; Sugiyama, Naoyuki; Ishihama, Yasushi; Shirasu, Ken

    2012-01-01

    The emergence of 'shotgun proteomics' has paved the way for high-throughput proteome analysis, by which thousands of proteins can be identified simultaneously from complex samples. Although the shotgun approach has the potential to monitor many different post-translational modifications, further technological development is needed to enrich each post-translational 'modificome'. Large-scale in vivo phosphorylation site mapping, so-called shotgun phosphoproteomics, has become feasible in various organisms, including plants, owing to recent technological breakthroughs. Shotgun phosphoproteomics is not a mature technology, but progress has been rapid. In this review, we highlight the scope and limitations of current methods, and some key technological issues in this field.

  8. Versatile nanocomposites in phosphoproteomics: a review.

    PubMed

    Najam-ul-Haq, Muhammad; Jabeen, Fahmida; Hussain, Dilshad; Saeed, Adeela; Musharraf, Syed Ghulam; Huck, Christian W; Bonn, Günther K

    2012-10-17

    Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment.

  9. Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.

    PubMed

    Qian, Wei-Jun; Liu, Tao; Petyuk, Vladislav A; Gritsenko, Marina A; Petritis, Brianne O; Polpitiya, Ashoka D; Kaushal, Amit; Xiao, Wenzhong; Finnerty, Celeste C; Jeschke, Marc G; Jaitly, Navdeep; Monroe, Matthew E; Moore, Ronald J; Moldawer, Lyle L; Davis, Ronald W; Tompkins, Ronald G; Herndon, David N; Camp, David G; Smith, Richard D

    2009-01-01

    The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope (18)O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the (18)O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on (16)O/(18)O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free (16)O-MS intensity data extracted from the same data sets. The results showed that the (18)O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the (18)O-reference for accurate quantitation makes this

  10. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  11. Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

    PubMed

    Salih, Erdjan

    2005-01-01

    spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

  12. Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane

    SciTech Connect

    Huang, Zhenlie; Ichihara, Sahoko; Oikawa, Shinji; Chang, Jie; Zhang, Lingyi; Hu, Shijie; Huang, Hanlin; Ichihara, Gaku

    2015-01-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn{sup 2+})-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p < 0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn{sup 2+}-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity. - Highlights: • 1-BP modified hippocampal phosphoproteome in rat and 23 altered proteins were identified. • 1-BP changed phosphorylation

  13. Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

    PubMed Central

    2014-01-01

    Background Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. Results Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. Conclusions The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration. PMID:24987309

  14. Battle through Signaling between Wheat and the Fungal Pathogen Septoria tritici Revealed by Proteomics and Phosphoproteomics*

    PubMed Central

    Yang, Fen; Melo-Braga, Marcella N.; Larsen, Martin R.; Jørgensen, Hans J. L.; Palmisano, Giuseppe

    2013-01-01

    The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat–S. tritici interaction, we performed a time-course study of S. tritici infection in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger accumulation of signal molecules, including calcium, H2O2, NO, and sugars, in the resistant than in the susceptible cultivar in response to the infection. Additionally, 31 proteins and 5 phosphoproteins from the pathogen were identified, including metabolic proteins and signaling proteins such as GTP-binding proteins, 14–3-3 proteins, and calcium-binding proteins. Quantitative PCR analysis showed the expression of fungal signaling genes and genes encoding a superoxide dismutase and cell-wall degrading enzymes. These results indicate roles of signaling, antioxidative stress mechanisms, and nutrient acquisition in facilitating the initial symptomless growth. Taken in its entirety, our dataset suggests interplay between the plant and S. tritici through complex signaling networks and downstream molecular events. Resistance is likely related to several rapidly and intensively triggered signal transduction cascades resulting in a multiple-level activation of transcription and translation processes of defense responses. Our sensitive approaches and model provide a

  15. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus.

    PubMed

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y L; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  16. Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus

    PubMed Central

    Chiang, Cheng-Kang; Xu, Bo; Mehta, Neel; Mayne, Janice; Sun, Warren Y. L.; Cheng, Kai; Ning, Zhibin; Dong, Jing; Zou, Hanfa; Cheng, Hai-Ying Mary; Figeys, Daniel

    2017-01-01

    The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription–translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short- to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of

  17. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    PubMed

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T; Herbert, Alex; Lando, David; Etheridge, Thomas J; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M; Klenerman, David; Lee, Steven F

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.

  18. Quantitative Imaging of Turbulent Mixing Dynamics in High-Pressure Fuel Injection to Enable Predictive Simulations of Engine Combustion

    SciTech Connect

    Frank, Jonathan H.; Pickett, Lyle M.; Bisson, Scott E.; Patterson, Brian D.; Ruggles, Adam J.; Skeen, Scott A.; Manin, Julien Luc; Huang, Erxiong; Cicone, Dave J.; Sphicas, Panos

    2015-09-01

    In this LDRD project, we developed a capability for quantitative high - speed imaging measurements of high - pressure fuel injection dynamics to advance understanding of turbulent mixing in transcritical flows, ignition, and flame stabilization mechanisms, and to provide e ssential validation data for developing predictive tools for engine combustion simulations. Advanced, fuel - efficient engine technologies rely on fuel injection into a high - pressure, high - temperature environment for mixture preparation and com bustion. Howe ver, the dynamics of fuel injection are not well understood and pose significant experimental and modeling challenges. To address the need for quantitative high - speed measurements, we developed a Nd:YAG laser that provides a 5ms burst of pulses at 100 kHz o n a robust mobile platform . Using this laser, we demonstrated s patially and temporally resolved Rayleigh scattering imaging and particle image velocimetry measurements of turbulent mixing in high - pressure gas - phase flows and vaporizing sprays . Quantitativ e interpretation of high - pressure measurements was advanced by reducing and correcting interferences and imaging artifacts.

  19. HistoFlex--a microfluidic device providing uniform flow conditions enabling highly sensitive, reproducible and quantitative in situ hybridizations.

    PubMed

    Søe, Martin Jensen; Okkels, Fridolin; Sabourin, David; Alberti, Massimo; Holmstrøm, Kim; Dufva, Martin

    2011-11-21

    A microfluidic device (the HistoFlex) designed to perform and monitor molecular biological assays under dynamic flow conditions on microscope slide-substrates, with special emphasis on analyzing histological tissue sections, is presented. Microscope slides were reversibly sealed onto a cast polydimethylsiloxane (PDMS) insert, patterned with distribution channels and reaction chambers. Topology optimization was used to design reaction chambers with uniform flow conditions. The HistoFlex provided uniform hybridization conditions, across the reaction chamber, as determined by hybridization to microscope slides of spotted DNA microarrays when applying probe concentrations generally used in in situ hybridization (ISH) assays. The HistoFlex's novel ability in online monitoring of an in situ hybridization assay was demonstrated using direct fluorescent detection of hybridization to 18S rRNA. Tissue sections were not visually damaged during assaying, which enabled adapting a complete ISH assay for detection of microRNAs (miRNA). The effects of flow based incubations on hybridization, antibody incubation and Tyramide Signal Amplification (TSA) steps were investigated upon adapting the ISH assay for performing in the HistoFlex. The hybridization step was significantly enhanced using flow based incubations due to improved hybridization efficiency. The HistoFlex device enabled a fast miRNA ISH assay (3 hours) which provided higher hybridization signal intensity compared to using conventional techniques (5 h 40 min). We further demonstrate that the improved hybridization efficiency using the HistoFlex permits more complex assays e.g. those comprising sequential hybridization and detection of two miRNAs to be performed with significantly increased sensitivity. The HistoFlex provides a new histological analysis platform that will allow multiple and sequential assays to be performed under their individual optimum assay conditions. Images can subsequently be recorded either in

  20. Data set from a comprehensive phosphoproteomic analysis of rice variety IRBB5 in response to bacterial blight.

    PubMed

    Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Zhang, Wen; Huang, Shiwen; Zhang, Jian

    2016-03-01

    Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has become one of the most devastating diseases for rice, a major food source for over half of the world populations. To investigate the roles of protein phosphorylation in rice bacterial blight resistance, a quantitative phosphoproteomic study was conducted in rice variety IRBB5 at 0 h and 24 h after Xoo infection. 2367 and 2223 phosphosites on 1334 and 1297 representative proteins were identified in 0 h and 24 h after Xoo infection, respectively, out of which 762 proteins were found to be differentially phosphorylated. In associated with the published article "A comprehensive quantitative phosphoproteome analysis of rice in response to bacterial blight" in BMC Plant Biology (Hou et al., 2015) [1], this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of mass spectrometry (MS) identification. The MS proteomics data could be fully accessed from the ProteomeXchange Consortium with the dataset identifier PXD002222.

  1. A Novel Image-Analysis Toolbox Enabling Quantitative Analysis of Root System Architecture1[W][OA

    PubMed Central

    Lobet, Guillaume; Pagès, Loïc; Draye, Xavier

    2011-01-01

    We present in this paper a novel, semiautomated image-analysis software to streamline the quantitative analysis of root growth and architecture of complex root systems. The software combines a vectorial representation of root objects with a powerful tracing algorithm that accommodates a wide range of image sources and quality. The root system is treated as a collection of roots (possibly connected) that are individually represented as parsimonious sets of connected segments. Pixel coordinates and gray level are therefore turned into intuitive biological attributes such as segment diameter and orientation as well as distance to any other segment or topological position. As a consequence, user interaction and data analysis directly operate on biological entities (roots) and are not hampered by the spatially discrete, pixel-based nature of the original image. The software supports a sampling-based analysis of root system images, in which detailed information is collected on a limited number of roots selected by the user according to specific research requirements. The use of the software is illustrated with a time-lapse analysis of cluster root formation in lupin (Lupinus albus) and an architectural analysis of the maize (Zea mays) root system. The software, SmartRoot, is an operating system-independent freeware based on ImageJ and relies on cross-platform standards for communication with data-analysis software. PMID:21771915

  2. In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing

    PubMed Central

    Aguilar, Carlos A.; Shcherbina, Anna; Ricke, Darrell O.; Pop, Ramona; Carrigan, Christopher T.; Gifford, Casey A.; Urso, Maria L.; Kottke, Melissa A.; Meissner, Alexander

    2015-01-01

    Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual’s muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing. PMID:26381351

  3. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  4. Up-to-Date Workflow for Plant (Phospho)proteomics Identifies Differential Drought-Responsive Phosphorylation Events in Maize Leaves.

    PubMed

    Vu, Lam Dai; Stes, Elisabeth; Van Bel, Michiel; Nelissen, Hilde; Maddelein, Davy; Inzé, Dirk; Coppens, Frederik; Martens, Lennart; Gevaert, Kris; De Smet, Ive

    2016-12-02

    Protein phosphorylation is one of the most common post-translational modifications (PTMs), which can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes. Phosphopeptide enrichment techniques enable plant researchers to acquire insight into phosphorylation-controlled signaling networks in various plant species. Most phosphoproteome analyses of plant samples still involve stable isotope labeling, peptide fractionation, and demand a lot of mass spectrometry (MS) time. Here, we present a simple workflow to probe, map, and catalogue plant phosphoproteomes, requiring relatively low amounts of starting material, no labeling, no fractionation, and no excessive analysis time. Following optimization of the different experimental steps on Arabidopsis thaliana samples, we transferred our workflow to maize, a major monocot crop, to study signaling upon drought stress. In addition, we included normalization to protein abundance to identify true phosphorylation changes. Overall, we identified a set of new phosphosites in both Arabidopsis thaliana and maize, some of which are differentially phosphorylated upon drought. All data are available via ProteomeXchange with identifier PXD003634, but to provide easy access to our model plant and crop data sets, we created an online database, Plant PTM Viewer ( bioinformatics.psb.ugent.be/webtools/ptm_viewer/ ), where all phosphosites identified in our study can be consulted.

  5. Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics.

    PubMed

    Refsgaard, Jan C; Munk, Stephanie; Jensen, Lars J

    2016-01-01

    Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weaknesses that must be considered for the individual needs. These are reviewed in this chapter. Equally critical for generating highly confident output datasets is the application of sound statistical criteria to limit the inclusion of incorrect peptide identifications from database searches. Additionally, careful filtering and use of appropriate statistical tests on the output datasets affects the quality of all downstream analyses and interpretation of the data. Our considerations and general practices on these aspects of phosphoproteomics data processing are presented here.

  6. Microfluidic device enabled quantitative time-lapse microscopic-photography for phenotyping vegetative and reproductive phases in Fusarium virguliforme, which is pathogenic to soybean

    PubMed Central

    Marshall, Jill; Qiao, Xuan; Baumbach, Jordan; Xie, Jingyu; Dong, Liang; Bhattacharyya, Madan K.

    2017-01-01

    Time-lapse microscopic-photography allows in-depth phenotyping of microorganisms. Here we report development of such a system using a microfluidic device, generated from polydimethylsiloxane and glass slide, placed on a motorized stage of a microscope for conducting time-lapse microphotography of multiple observations in 20 channels simultaneously. We have demonstrated the utility of the device in studying growth, germination and sporulation in Fusarium virguliforme that causes sudden death syndrome in soybean. To measure the growth differences, we developed a polyamine oxidase fvpo1 mutant in this fungus that fails to grow in minimal medium containing polyamines as the sole nitrogen source. Using this system, we demonstrated that the conidiospores of the pathogen take an average of five hours to germinate. During sporulation, it takes an average of 10.5 h for a conidiospore to mature and get detached from its conidiophore for the first time. Conidiospores are developed in a single conidiophore one after another. The microfluidic device enabled quantitative time-lapse microphotography reported here should be suitable for screening compounds, peptides, micro-organisms to identify fungitoxic or antimicrobial agents for controlling serious plant pathogens. The device could also be applied in identifying suitable target genes for host-induced gene silencing in pathogens for generating novel disease resistance in crop plants. PMID:28295054

  7. Microfluidic device enabled quantitative time-lapse microscopic-photography for phenotyping vegetative and reproductive phases in Fusarium virguliforme, which is pathogenic to soybean.

    PubMed

    Marshall, Jill; Qiao, Xuan; Baumbach, Jordan; Xie, Jingyu; Dong, Liang; Bhattacharyya, Madan K

    2017-03-15

    Time-lapse microscopic-photography allows in-depth phenotyping of microorganisms. Here we report development of such a system using a microfluidic device, generated from polydimethylsiloxane and glass slide, placed on a motorized stage of a microscope for conducting time-lapse microphotography of multiple observations in 20 channels simultaneously. We have demonstrated the utility of the device in studying growth, germination and sporulation in Fusarium virguliforme that causes sudden death syndrome in soybean. To measure the growth differences, we developed a polyamine oxidase fvpo1 mutant in this fungus that fails to grow in minimal medium containing polyamines as the sole nitrogen source. Using this system, we demonstrated that the conidiospores of the pathogen take an average of five hours to germinate. During sporulation, it takes an average of 10.5 h for a conidiospore to mature and get detached from its conidiophore for the first time. Conidiospores are developed in a single conidiophore one after another. The microfluidic device enabled quantitative time-lapse microphotography reported here should be suitable for screening compounds, peptides, micro-organisms to identify fungitoxic or antimicrobial agents for controlling serious plant pathogens. The device could also be applied in identifying suitable target genes for host-induced gene silencing in pathogens for generating novel disease resistance in crop plants.

  8. Quantitative fluorescence imaging enabled by spatial frequency domain optical-property mapping in the sub-diffusive regime for surgical guidance

    NASA Astrophysics Data System (ADS)

    Sibai, Mira; Veilleux, Israel; Elliott, Jonathan T.; Leblond, Frederic; Roberts, David W.; Wilson, Brian C.

    2015-03-01

    Intraoperative fluorescence guidance enables maximum safe resection of, for example, glioblastomas by providing surgeons with real-time tumor optical contrast. Specifically, 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence guided resection can improve surgical outcomes by better defining tumor margins and identifying satellite tumor foci. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation (absorption and scattering) by tissue and background tissue autofluorescence. We have previously shown, using a point fluorescence-reflectance fiberoptic probe, that non-invasive measurement of the absolute PpIX concentration, [PpIX], further improves sensitivity and specificity, leading to the demonstration that the technique can also detect low-grade gliomas as well as otherwise undetectable residual tumor foci in high-grade disease. Here, we extend this approach to wide-field quantitative fluorescence imaging (qFI) by implementing spatial frequency domain imaging (SFDI) to recover the tissue optical absorption and transport scattering coefficients across the field of view. We report on the performance of this approach to determine the intrinsic fluorescence intensity in tissue-simulating phantoms in both the fully diffusive (i.e. scatter-dominated) and sub-diffusive (low transport albedo) regimes, for which higher spatial frequencies are used. The performance of qFI is compared to a Born- normalization correction scheme, as well as to the values obtained using the fiberoptic probe on homogeneous tissue phantoms containing PpIX.

  9. In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species.

    PubMed

    Han, Bin; Fang, Yu; Feng, Mao; Lu, Xiaoshan; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

    2014-12-05

    The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. Although the importance of post-translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C

  10. Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars*

    PubMed Central

    Pi, Erxu; Qu, Liqun; Hu, Jianwen; Huang, Yingying; Qiu, Lijuan; Lu, Hongfei; Jiang, Bo; Liu, Cong; Peng, Tingting; Zhao, Ying; Wang, Huizhong; Tsai, Sau-Na; Ngai, Saiming; Du, Liqun

    2016-01-01

    Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by

  11. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro *

    PubMed Central

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-01-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

  12. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

    PubMed

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

  13. Phosphoproteomics Identifies CK2 as a Negative Regulator of Beige Adipocyte Thermogenesis and Energy Expenditure.

    PubMed

    Shinoda, Kosaku; Ohyama, Kana; Hasegawa, Yutaka; Chang, Hsin-Yi; Ogura, Mayu; Sato, Ayaka; Hong, Haemin; Hosono, Takashi; Sharp, Louis Z; Scheel, David W; Graham, Mark; Ishihama, Yasushi; Kajimura, Shingo

    2015-12-01

    Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under β3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the β3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes.

  14. Phosphoproteomic analysis reveals interconnected system-wide responses to perturbations of kinases and phosphatases in yeast.

    PubMed

    Bodenmiller, Bernd; Wanka, Stefanie; Kraft, Claudine; Urban, Jörg; Campbell, David; Pedrioli, Patrick G; Gerrits, Bertran; Picotti, Paola; Lam, Henry; Vitek, Olga; Brusniak, Mi-Youn; Roschitzki, Bernd; Zhang, Chao; Shokat, Kevan M; Schlapbach, Ralph; Colman-Lerner, Alejandro; Nolan, Garry P; Nesvizhskii, Alexey I; Peter, Matthias; Loewith, Robbie; von Mering, Christian; Aebersold, Ruedi

    2010-12-21

    The phosphorylation and dephosphorylation of proteins by kinases and phosphatases constitute an essential regulatory network in eukaryotic cells. This network supports the flow of information from sensors through signaling systems to effector molecules and ultimately drives the phenotype and function of cells, tissues, and organisms. Dysregulation of this process has severe consequences and is one of the main factors in the emergence and progression of diseases, including cancer. Thus, major efforts have been invested in developing specific inhibitors that modulate the activity of individual kinases or phosphatases; however, it has been difficult to assess how such pharmacological interventions would affect the cellular signaling network as a whole. Here, we used label-free, quantitative phosphoproteomics in a systematically perturbed model organism (Saccharomyces cerevisiae) to determine the relationships between 97 kinases, 27 phosphatases, and more than 1000 phosphoproteins. We identified 8814 regulated phosphorylation events, describing the first system-wide protein phosphorylation network in vivo. Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery-and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis.

  15. The Quantitative Criteria Based on the Fractal Dimensions, Entropy, and Lacunarity for the Spatial Distribution of Cancer Cell Nuclei Enable Identification of Low or High Aggressive Prostate Carcinomas

    PubMed Central

    Waliszewski, Przemyslaw

    2016-01-01

    relevant information. Two novel quantitative criteria based on the complexity and the diversity measures enabled the identification of low or high aggressive prostate carcinomas and should be verified in the future multicenter, randomized studies. PMID:26903883

  16. Phosphorylation of proteins during human myometrial contractions: A phosphoproteomic approach.

    PubMed

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

    Phasic myometrial contractility is a key component of human parturition and the contractions are driven by reversible phosphorylation of myosin light chains catalyzed by the calcium (Ca(2+))-dependent enzyme myosin light chain kinase (MYLK). Other yet unknown phosphorylation or de-phosphorylation events may contribute to myometrial contraction and relaxation. In this study we have performed a global phosphoproteomic analysis of human myometrial tissue using tandem mass tagging to detect changes in the phosphorylation status of individual myometrial proteins during spontaneous and oxytocin-driven phasic contractions. We were able to detect 22 individual phosphopeptides whose relative ratio changed (fold > 2 or < 0.5) in response to spontaneous or oxytocin-stimulated contraction. The most significant changes in phosphorylation were to MYLK on serine 1760, a site associated with reductions in calmodulin binding and subsequent kinase activity. Phosphorylated MYLK (ser1760) increased significantly during spontaneous (9.83 ± 3.27 fold, P < 0.05) and oxytocin -induced (18.56 ± 8.18 fold, P < 0.01) contractions and we were able to validate these data using immunoblotting. Pathway analysis suggested additional proteins involved in calcium signalling, cGMP-PRKG signalling, adrenergic signalling and oxytocin signalling were also phosphorylated during contractions. This study demonstrates that a global phosphoproteomic analysis of myometrial tissue is a sensitive approach to detect changes in the phosphorylation of proteins during myometrial contractions, and provides a platform for further validation of these changes and for identification of their functional significance.

  17. Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

    PubMed

    Raijmakers, Reinout; Kraiczek, Karsten; de Jong, Ad P; Mohammed, Shabaz; Heck, Albert J R

    2010-02-01

    The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

  18. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation.

    PubMed

    Tan, Haiyan; Yang, Kai; Li, Yuxin; Shaw, Timothy I; Wang, Yanyan; Blanco, Daniel Bastardo; Wang, Xusheng; Cho, Ji-Hoon; Wang, Hong; Rankin, Sherri; Guy, Cliff; Peng, Junmin; Chi, Hongbo

    2017-03-21

    The molecular circuits by which antigens activate quiescent T cells remain poorly understood. We combined temporal profiling of the whole proteome and phosphoproteome via multiplexed isobaric labeling proteomics technology, computational pipelines for integrating multi-omics datasets, and functional perturbation to systemically reconstruct regulatory networks underlying T cell activation. T cell receptors activated the T cell proteome and phosphoproteome with discrete kinetics, marked by early dynamics of phosphorylation and delayed ribosome biogenesis and mitochondrial activation. Systems biology analyses identified multiple functional modules, active kinases, transcription factors and connectivity between them, and mitochondrial pathways including mitoribosomes and complex IV. Genetic perturbation revealed physiological roles for mitochondrial enzyme COX10-mediated oxidative phosphorylation in T cell quiescence exit. Our multi-layer proteomics profiling, integrative network analysis, and functional studies define landscapes of the T cell proteome and phosphoproteome and reveal signaling and bioenergetics pathways that mediate lymphocyte exit from quiescence.

  19. Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

    PubMed Central

    Gonzales, Patricia A.; Pisitkun, Trairak; Hoffert, Jason D.; Tchapyjnikov, Dmitry; Star, Robert A.; Kleta, Robert; Wang, Nam Sun; Knepper, Mark A.

    2009-01-01

    Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/. PMID:19056867

  20. Integration of phosphoproteomic, chemical, and biological strategies for the functional analysis of targeted protein phosphorylation.

    PubMed

    Guo, Mingquan; Huang, Bill X

    2013-02-01

    Reversible phosphorylation, tightly controlled by protein kinases and phosphatases, plays a central role in mediating biological processes, such as protein-protein interactions, subcellular translocation, and activation of cellular enzymes. MS-based phosphoproteomics has now allowed the detection and quantification of tens of thousands of phosphorylation sites from a typical biological sample in a single experiment, which has posed new challenges in functional analysis of each and every phosphorylation site on specific signaling phosphoproteins of interest. In this article, we review recent advances in the functional analysis of targeted phosphorylation carried out by various chemical and biological approaches in combination with the MS-based phosphoproteomics. This review focuses on three types of strategies, including forward functional analysis, defined for the result-driven phosphoproteomics efforts in determining the substrates of a specific protein kinase; reverse functional analysis, defined for tracking the kinase(s) for specific phosphosite(s) derived from the discovery-driven phosphoproteomics efforts; and MS-based analysis on the structure-function relationship of phosphoproteins. It is expected that this review will provide a state-of-the-art overview of functional analysis of site-specific phosphorylation and explore new perspectives and outline future challenges.

  1. Synaptic activity bidirectionally regulates a novel sequence-specific S-Q phosphoproteome in neurons

    PubMed Central

    Siddoway, Benjamin; Hou, Hailong; Yang, Hongtian; Petralia, Ronald; Xia, Houhui

    2013-01-01

    Protein phosphorylation plays a critical role in neuronal transcription, translation, cell viability, and synaptic plasticity. In neurons, phospho-enzymes and specific substrates directly link glutamate release and post-synaptic depolarization to these cellular functions; however, many of these enzymes and their protein substrates remain uncharacterized or unidentified. In this article, we identify a novel, synaptically-driven neuronal phosphoproteome characterized by a specific motif of serine/threonine-glutamine ([S/T]-Q, abbreviated as SQ). These SQ-containing substrates are predominantly localized to dendrites, synapses, the soma; and activation of this SQ phosphoproteome by bicuculline application is induced via calcium influx through L-type calcium channels. On the other hand, acute application of NMDA can inactivate this SQ phosphoproteome. We demonstrate that the SQ motif kinase Ataxia-telangiectasia mutated (ATM) can also localize to dendrites and dendritic spines, in addition to other subcellular compartments, and is activated by bicuculline application. Pharmacology studies indicate that ATM and its sister kinase ATR up-regulate these neuronal SQ substrates. Phosphoproteomics identified over 150 SQ-containing substrates whose phosphorylation is bidirectionally-regulated by synaptic activity. PMID:24117848

  2. Semi-quantitative analysis of solid waste flows from nano-enabled consumer products in Europe, Denmark and the United Kingdom - Abundance, distribution and management.

    PubMed

    Heggelund, Laura; Hansen, Steffen Foss; Astrup, Thomas Fruergaard; Boldrin, Alessio

    2016-10-01

    Many nano-enabled consumer products are known to be in the global market. At the same, little is known about the quantity, type, location etc. of the engineered nanomaterials (ENMs) inside the products. This limits the scientific investigations of potential environmental effects of these materials, and especially the knowledge of ENM behaviour and potential effects at the end-of-life stage of the products is scarce. To gain a better understanding of the end-of-life waste treatment of nano-enabled consumer product, we provide an overview of the ENMs flowing into and throughout waste systems in Europe, Denmark and the United Kingdom. Using a nanoproduct inventory (nanodb.dk), we performed a four-step analysis to estimate the most abundant ENMs and in which waste fractions they are present. We found that in terms of number of products: (i) nano silver is the most used ENM in consumer products, and (ii) plastic from used product containers is the largest waste fraction also comprising a large variety of ENMs, though possibly in very small masses. Also, we showed that the local waste management system can influence the distribution of ENMs. It is recommended that future research focus on recycling and landfilling of nano-enabled products since these compartments represent hot spots for end-of-life nanoproducts.

  3. Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

    PubMed Central

    Grimes, Mark L.; Lee, Wan-Jui; van der Maaten, Laurens; Shannon, Paul

    2013-01-01

    The interpretation of biological data sets is essential for generating hypotheses that guide research, yet modern methods of global analysis challenge our ability to discern meaningful patterns and then convey results in a way that can be easily appreciated. Proteomic data is especially challenging because mass spectrometry detectors often miss peptides in complex samples, resulting in sparsely populated data sets. Using the R programming language and techniques from the field of pattern recognition, we have devised methods to resolve and evaluate clusters of proteins related by their pattern of expression in different samples in proteomic data sets. We examined tyrosine phosphoproteomic data from lung cancer samples. We calculated dissimilarities between the proteins based on Pearson or Spearman correlations and on Euclidean distances, whilst dealing with large amounts of missing data. The dissimilarities were then used as feature vectors in clustering and visualization algorithms. The quality of the clusterings and visualizations were evaluated internally based on the primary data and externally based on gene ontology and protein interaction networks. The results show that t-distributed stochastic neighbor embedding (t-SNE) followed by minimum spanning tree methods groups sparse proteomic data into meaningful clusters more effectively than other methods such as k-means and classical multidimensional scaling. Furthermore, our results show that using a combination of Spearman correlation and Euclidean distance as a dissimilarity representation increases the resolution of clusters. Our analyses show that many clusters contain one or more tyrosine kinases and include known effectors as well as proteins with no known interactions. Visualizing these clusters as networks elucidated previously unknown tyrosine kinase signal transduction pathways that drive cancer. Our approach can be applied to other data types, and can be easily adopted because open source software

  4. Phosphoproteomic Profiling of Selenate-Treated Alzheimer's Disease Model Cells

    PubMed Central

    Wang, Yong; Li, Shuiming; Shen, Liming; Liu, Qiong; Ni, Jiazuan

    2014-01-01

    The reversible phosphorylation of proteins regulates most biological processes, while abnormal phosphorylation is a cause or consequence of many diseases including Alzheimer's disease (AD). One of the hallmarks of AD is the formation of neurofibrillary tangles (NFTs), which is composed of hyperphosphorylated tau proteins. Sodium selenate has been recently found to reduce tau hyperphosphorylation and NFTs formation, and to improve spatial learning and motor performance in AD mice. In the current study, the phosphoproteomics of N2aSW cells treated with selenate were investigated. To avoid missing low-abundance phosphoproteins, both the total proteins of cells and the phosphor-enriched proteins were extracted and subjected to the two-dimensional gel electrophoresis with Pro-Q diamond staining and then LC-MS/MS analysis. A total of 65 proteins were altered in phosphorylation level, of which 39 were up-regulated and 26 were down-regulated. All identified phosphoproteins were bioinformatically annotated according to their physiochemical features, subcellular location, and biological function. Most of these significantly changed phosphoproteins are involved in crucial neural processes such as protesome activity, oxidative stress, cysteine and methionine metabolism, and energy metabolism. Furthermore, decreases were found in homocysteine, phosphor-tau and amyloid β upon selenate treatment. Our results suggest that selenate may intervene in the pathological process of AD by altering the phosphorylation of some key proteins involved in oxidative stress, energy metabolism and protein degradation, thus play important roles in maintaining redox homeostasis, generating ATP, and clearing misfolded proteins and aggregates. The present paper provides some new clues to the mechanism of selenate in AD prevention. PMID:25485856

  5. Photodiode array to charged aerosol detector response ratio enables comprehensive quantitative monitoring of basic drugs in blood by ultra-high performance liquid chromatography.

    PubMed

    Viinamäki, Jenni; Ojanperä, Ilkka

    2015-03-20

    Quantitative screening for a broad range of drugs in blood is regularly required to assess drug abuse and poisoning within analytical toxicology. Mass spectrometry-based procedures suffer from the large amount of work required to maintain quantitative calibration in extensive multi-compound methods. In this study, a quantitative drug screening method for blood samples was developed based on ultra-high performance liquid chromatography with two consecutive detectors: a photodiode array detector and a corona charged aerosol detector (UHPLC-DAD-CAD). The 2.1 mm × 150 mm UHPLC column contained a high-strength silica C18 bonded phase material with a particle size of 1.8 μm, and the mobile phase consisted of methanol/0.1% trifluoroacetic acid in gradient mode. Identification was based on retention time, UV spectrum and the response ratio from the two detectors. Using historic calibration over a one-month period, the median precision (RSD) of retention times was 0.04% and the median accuracy (bias) of quantification 6.75%. The median precision of the detector response ratio over two orders of magnitude was 12%. The applicable linear ranges were generally 0.05-5 mg L(-1). The method was validated for 161 compounds, including antipsychotics, antidepressants, antihistamines, opioid analgesics, and adrenergic beta blocking drugs, among others. The main novelty of the method was the proven utility of the response ratio of DAD to CAD, which provided the additional identification efficiency required. Unlike with mass spectrometry, the high stability of identification and quantification allowed the use of facile historic calibration.

  6. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    PubMed

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  7. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    PubMed

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers.

  8. Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia

    PubMed Central

    Zahari, Muhammad Saddiq; Wu, Xinyan; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Kim, Min-Sik; Fetics, Barry; Philip, Mathew; Barnes, Sheri R.; Godfrey, Beverly; Gabrielson, Edward; Nevo, Erez; Pandey, Akhilesh

    2015-01-01

    Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues. PMID:26329039

  9. Dataset of the Botrytis cinerea phosphoproteome induced by different plant-based elicitors.

    PubMed

    Liñeiro, Eva; Chiva, Cristina; Cantoral, Jesús M; Sabido, Eduard; Fernández-Acero, Francisco Javier

    2016-06-01

    Phosphorylation is one of the main post-translational modification (PTM) involved in signaling network in the ascomycete Botrytis cinerea, one of the most relevant phytopathogenic fungus. The data presented in this article provided a differential mass spectrometry-based analysis of the phosphoproteome of B. cinerea under two different phenotypical conditions induced by the use of two different elicitors: glucose and deproteinized Tomate Cell Walls (TCW). A total 1138 and 733 phosphoproteins were identified for glucose and TCW culture conditions respectively. Raw data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier (PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD003099). Further interpretation and discussion of these data are provided in our research article entitled "Phosphoproteome analysis of B.cinerea in response to different plant-based elicitors" (Liñeiro et al., 2016) [1].

  10. Application of a Web-Enabled Leg Training System for the Objective Monitoring and Quantitative Analysis of Exercise-Induced Fatigue

    PubMed Central

    Dedova, Irina V

    2016-01-01

    Background Sustained cardiac rehabilitation is the key intervention in the prevention and treatment of many human diseases. However, implementation of exercise programs can be challenging because of early fatigability in patients with chronic diseases, overweight individuals, and aged people. Current methods of fatigability assessment are based on subjective self-reporting such as rating of perceived exertion or require specialized laboratory conditions and sophisticated equipment. A practical approach allowing objective measurement of exercise-induced fatigue would be useful for the optimization of sustained delivery of cardiac rehabilitation to improve patient outcomes. Objectives The objective of this study is to develop and validate an innovative approach, allowing for the objective assessment of exercise-induced fatigue using the Web-enabled leg rehabilitation system. Methods MedExercise training devices were equipped with wireless temperature sensors in order to monitor their usage by temperature rise in the resistance unit (Δt°). Since Δt° correlated with the intensity and duration of exercise, this parameter was used to characterize participants’ leg work output (LWO). Personal smart devices such as laptop computers with wireless gateways and relevant software were used for monitoring of self-control training. Connection of smart devices to the Internet and cloud-based software allowed remote monitoring of LWO in participants training at home. Heart rates (HRs) were measured by fingertip pulse oximeters simultaneously with Δt° in 7 healthy volunteers. Results Exercise-induced fatigue manifested as the decline of LWO and/or rising HR, which could be observed in real-time. Conversely, training at the steady-state LWO and HR for the entire duration of exercise bout was considered as fatigue-free. The amounts of recommended daily physical activity were expressed as the individual Δt° values reached during 30-minute fatigue-free exercise of moderate

  11. StageTip-based HAMMOC, an efficient and inexpensive phosphopeptide enrichment method for plant shotgun phosphoproteomics.

    PubMed

    Nakagami, Hirofumi

    2014-01-01

    Phosphopeptide enrichment is the most critical step for successful LC-MS/MS-based shotgun phosphoproteomics. Recent technological improvements have made selective phosphopeptide enrichment from non-fractionated whole cell lysate digests with a single-step procedure possible. Here, a handy protocol is described for phosphopeptide enrichment from plant materials using hydroxy acid-modified metal oxide chromatography (HAMMMOC) with a stop-and-go-extraction tip (StageTip).

  12. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation

    PubMed Central

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria. PMID:25873915

  13. Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

    PubMed

    Tan, Haiyan; Wu, Zhiping; Wang, Hong; Bai, Bing; Li, Yuxin; Wang, Xusheng; Zhai, Bo; Beach, Thomas G; Peng, Junmin

    2015-01-01

    Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180).

  14. The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

    PubMed

    Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

  15. Application of Phosphoproteomics to Find Targets of Casein Kinase 1 in the Flagellum of Chlamydomonas

    PubMed Central

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum. PMID:23316220

  16. Application of phosphoproteomics to find targets of casein kinase 1 in the flagellum of chlamydomonas.

    PubMed

    Boesger, Jens; Wagner, Volker; Weisheit, Wolfram; Mittag, Maria

    2012-01-01

    The green biflagellate alga Chlamydomonas reinhardtii serves as model for studying structural and functional features of flagella. The axoneme of C. reinhardtii anchors a network of kinases and phosphatases that control motility. One of them, Casein Kinase 1 (CK1), is known to phosphorylate the Inner Dynein Arm I1 Intermediate Chain 138 (IC138), thereby regulating motility. CK1 is also involved in regulating the circadian rhythm of phototaxis and is relevant for the formation of flagella. By a comparative phosphoproteome approach, we determined phosphoproteins in the flagellum that are targets of CK1. Thereby, we applied the specific CK1 inhibitor CKI-7 that causes significant changes in the flagellum phosphoproteome and reduces the swimming velocity of the cells. In the CKI-7-treated cells, 14 phosphoproteins were missing compared to the phosphoproteome of untreated cells, including IC138, and four additional phosphoproteins had a reduced number of phosphorylation sites. Notably, inhibition of CK1 causes also novel phosphorylation events, indicating that it is part of a kinase network. Among them, Glycogen Synthase Kinase 3 is of special interest, because it is involved in the phosphorylation of key clock components in flies and mammals and in parallel plays an important role in the regulation of assembly in the flagellum.

  17. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network

    PubMed Central

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-01

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism. PMID:28106078

  18. Comparative Phosphoproteomics Reveals an Important Role of MKK2 in Banana (Musa spp.) Cold Signal Network.

    PubMed

    Gao, Jie; Zhang, Sheng; He, Wei-Di; Shao, Xiu-Hong; Li, Chun-Yu; Wei, Yue-Rong; Deng, Gui-Ming; Kuang, Rui-Bin; Hu, Chun-Hua; Yi, Gan-Jun; Yang, Qiao-Song

    2017-01-20

    Low temperature is one of the key environmental stresses, which greatly affects global banana production. However, little is known about the global phosphoproteomes in Musa spp. and their regulatory roles in response to cold stress. In this study, we conducted a comparative phosphoproteomic profiling of cold-sensitive Cavendish Banana and relatively cold tolerant Dajiao under cold stress. Phosphopeptide abundances of five phosphoproteins involved in MKK2 interaction network, including MKK2, HY5, CaSR, STN7 and kinesin-like protein, show a remarkable difference between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated peptide verified the phosphoproteomic results of increased T31 phosphopeptide abundance with decreased MKK2 abundance in Daojiao for a time course of cold stress. Meanwhile increased expression of MKK2 with no detectable T31 phosphorylation was found in Cavendish Banana. These results suggest that the MKK2 pathway in Dajiao, along with other cold-specific phosphoproteins, appears to be associated with the molecular mechanisms of high tolerance to cold stress in Dajiao. The results also provide new evidence that the signaling pathway of cellular MKK2 phosphorylation plays an important role in abiotic stress tolerance that likely serves as a universal plant cold tolerance mechanism.

  19. Phosphoproteomic analysis of protein phosphorylation networks in Tetrahymena thermophila, a model single-celled organism.

    PubMed

    Tian, Miao; Chen, Xiulan; Xiong, Qian; Xiong, Jie; Xiao, Chuanle; Ge, Feng; Yang, Fuquan; Miao, Wei

    2014-02-01

    Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase-substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

  20. Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development.

    PubMed

    Franck, William L; Gokce, Emine; Randall, Shan M; Oh, Yeonyee; Eyre, Alex; Muddiman, David C; Dean, Ralph A

    2015-06-05

    The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings, and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic, and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics, and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.

  1. Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

    PubMed Central

    Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia; Biswas, Sudipta; Graeber, Thomas G.; Podrez, Eugene A.

    2014-01-01

    Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. PMID:24400094

  2. Phosphoproteomic Analysis of Protein Kinase C Signaling in Saccharomyces cerevisiae Reveals Slt2 Mitogen-activated Protein Kinase (MAPK)-dependent Phosphorylation of Eisosome Core Components*

    PubMed Central

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J.; Molina, María

    2013-01-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1–cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module. PMID:23221999

  3. Identification of the PLK2-Dependent Phosphopeptidome by Quantitative Proteomics

    PubMed Central

    Franchin, Cinzia; Cesaro, Luca; Pinna, Lorenzo A.; Arrigoni, Giorgio; Salvi, Mauro

    2014-01-01

    Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites. PMID:25338102

  4. Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics.

    PubMed

    Diez Fernández, Silvia; Sugishama, Naoki; Ruiz Encinar, Jorge; Sanz-Medel, Alfredo

    2012-07-17

    It is clear that sensitive and interference-free quantification of ICP-detectable elements naturally present in proteins will boost the role of ICPMS in proteomics. In this study, a completely new way of polyatomic interference removal in ICPMS for detection of sulfur (present in the majority of proteins as methionine or cysteine) and phosphorus (present in phosphorylated proteins) is presented. It is based on the concept of tandem mass spectrometry (QQQ) typically used in molecular MS. Briefly, the first quadrupole can be operated as 1 amu window band-pass mass filter to select target analyte ions ((31)P, (32)S, and their on-mass polyatomic interferences). In this way, only selected ions enter the cell and react with O(2), reducing the interferences produced by matrix ions as well as background noise. After optimization of the cell conditions, product ions formed for the targets, (47)PO(+) and (48)SO(+), could be detected with enhanced sensitivity and selectivity. The coupling to capillary HPLC allowed analysis of S- and P-containing species with the lowest detection limits ever published (11 and 6.6 fmol, respectively). The potential of the approach for proteomics studies was demonstrated for the highly sensitive simultaneous absolute quantification of different S-containing peptides and phosphopeptides.

  5. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  6. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome*

    PubMed Central

    Solari, Fiorella A.; Mattheij, Nadine J.A.; Burkhart, Julia M.; Swieringa, Frauke; Collins, Peter W.; Cosemans, Judith M.E.M.; Sickmann, Albert; Heemskerk, Johan W.M.; Zahedi, René P.

    2016-01-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. PMID:27535140

  7. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

    PubMed

    Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

    2015-08-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

  8. Concerted Changes in the Phosphoproteome and Metabolome Under Different CO2/O2 Gaseous Conditions in Arabidopsis Rosettes.

    PubMed

    Abadie, Cyril; Mainguet, Samuel; Davanture, Marlène; Hodges, Michael; Zivy, Michel; Tcherkez, Guillaume

    2016-07-01

    Considerable efforts are currently devoted to understanding the regulation of primary carbon metabolism in plant leaves, which is known to change dramatically with environmental conditions, e.g. during light/dark transitions. Protein phosphorylation is believed to be a key factor in such a metabolic control. In fact, some studies have suggested modifications in the phosphorylation status of key enzymes in the dark compared with the light, or when photosynthesis varies. However, a general view of the phosphoproteome and reciprocal alterations in both the phosphoproteome and metabolome under a wide spectrum of CO2 and O2 conditions so as to vary both gross photosynthesis and photorespiration is currently lacking. Here, we used an instant sampling system and strictly controlled gaseous conditions to examine short-term metabolome and phosphoproteome changes in Arabidopsis rosettes. We show that light/dark, CO2 and O2 mole fraction have differential effects on enzyme phosphorylation. Phosphorylation events that appear to be the most important to regulate metabolite contents when photosynthesis varies are those associated with sugar and pyruvate metabolism: sucrose and starch synthesis are major phosphorylation-controlled steps but pyruvate utilization (by phosphoenolpyruvate carboxylase and pyruvate dehydrogenase) and pyruvate reformation (by pyruvate orthophosphate dikinase) are also subjected to phosphorylation control. Our results thus show that the phosphoproteome response to light/dark transition and gaseous conditions (CO2, O2) contributes to the rapid adjustment of major pathways of primary C metabolism.

  9. SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets

    PubMed Central

    Petsalaki, Evangelia; Helbig, Andreas O.; Gopal, Anjali; Pasculescu, Adrian; Roth, Frederick P.; Pawson, Tony

    2015-01-01

    While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific ‘network wiring’. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib—a tyrosine kinase inhibitor (TKI)—in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI. PMID:25948583

  10. HOPE-fixation of lung tissue allows retrospective proteome and phosphoproteome studies.

    PubMed

    Shevchuk, Olga; Abidi, Nada; Klawonn, Frank; Wissing, Josef; Nimtz, Manfred; Kugler, Christian; Steinert, Michael; Goldmann, Torsten; Jänsch, Lothar

    2014-11-07

    Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, the suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins constitute a prime resource for drug research and can give valuable insights into the activity status of signaling pathways. In this study, we extracted proteins from human lung tissue and tested HOPE-treated and snap-frozen tissues comparatively by proteome and phosphoproteome analyses. High confident data from accurate mass spectrometry allowed the identification of 2603 proteins and 3036 phosphorylation sites. HOPE-fixation did not hinder the representative extraction of proteins, and investigating their biochemical properties, covered subcellular localizations, and cellular processes revealed no bias caused by the type of fixation. In conclusion, proteome as well as phosphoproteome data of HOPE lung samples were qualitatively equivalent to results obtained from snap-frozen tissues. Thus, HOPE-treated tissues match clinical demands in both histology and retrospective proteome analyses of patient samples by proteomics.

  11. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

    PubMed Central

    Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

    2015-01-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

  12. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

    PubMed

    Rhoads, Timothy W; Prasad, Aman; Kwiecien, Nicholas W; Merrill, Anna E; Zawack, Kelson; Westphall, Michael S; Schroeder, Frank C; Kimble, Judith; Coon, Joshua J

    2015-11-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

  13. Phosphoproteomic differences in major depressive disorder postmortem brains indicate effects on synaptic function.

    PubMed

    Martins-de-Souza, Daniel; Guest, Paul C; Vanattou-Saifoudine, Natacha; Rahmoune, Hassan; Bahn, Sabine

    2012-12-01

    There is still a lack in the molecular comprehension of major depressive disorder (MDD) although this condition affects approximately 10% of the world population. Protein phosphorylation is a posttranslational modification that regulates approximately one-third of the human proteins involved in a range of cellular and biological processes such as cellular signaling. Whereas phosphoproteome studies have been carried out extensively in cancer research, few such investigations have been carried out in studies of psychiatric disorders. Here, we present a comparative phosphoproteome analysis of postmortem dorsolateral prefrontal cortex tissues from 24 MDD patients and 12 control donors. Tissue extracts were analyzed using liquid chromatography mass spectrometry in a data-independent manner (LC-MS(E)). Our analyses resulted in the identification of 5,195 phosphopeptides, corresponding to 802 non-redundant proteins. Ninety of these proteins showed differential levels of phosphorylation in tissues from MDD subjects compared to controls, being 20 differentially phosphorylated in at least 2 peptides. The majority of these phosphorylated proteins were associated with synaptic transmission and cellular architecture not only pointing out potential biomarker candidates but mainly shedding light to the comprehension of MDD pathobiology.

  14. Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern

    PubMed Central

    Stone, Matthew D.; Chen, Xiaobing; McGowan, Thomas; Bandhakavi, Sricharan; Cheng, Bin; Rhodus, Nelson L.; Griffin, Timothy J.

    2011-01-01

    In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in “extracellular” and “plasma membrane” categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation. PMID:21299198

  15. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.

    PubMed

    McNulty, Dean E; Annan, Roland S

    2008-05-01

    The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-microg equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

  16. The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvation.

    PubMed

    Rampitsch, Christof; Subramaniam, Rajagopal; Djuric-Ciganovic, Slavica; Bykova, Natalia V

    2010-01-01

    Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEFxSDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO(2) enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12 h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum.

  17. Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin

    PubMed Central

    Domanski, Dominik; Helbing, Caren C

    2007-01-01

    Background Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently

  18. Novel aspects of grapevine response to phytoplasma infection investigated by a proteomic and phospho-proteomic approach with data integration into functional networks

    PubMed Central

    2013-01-01

    Background Translational and post-translational protein modifications play a key role in the response of plants to pathogen infection. Among the latter, phosphorylation is critical in modulating protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D gels to study quantitative changes in the proteome and phosphoproteome of Flavescence dorée-affected and recovered ‘Barbera’ grapevines, compared to healthy plants. Results We identified 48 proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17 proteins not detected in infected plants. Some enzymes involved in the antioxidant response that were up-regulated in infected plants, such as isocitrate dehydrogenase and glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (oxidoreductase GLYR1 and ascorbate peroxidase). Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from infection. Fifteen differentially phosphorylated proteins (| ratio | > 2, p < 0.05) were identified in infected compared to healthy plants, including proteins involved in photosynthesis, response to stress and the antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical

  19. Phosphoproteomics Reveals HMGA1, a CK2 Substrate, as a Drug-Resistant Target in Non-Small Cell Lung Cancer

    PubMed Central

    Wang, Yi-Ting; Pan, Szu-Hua; Tsai, Chia-Feng; Kuo, Ting-Chun; Hsu, Yuan-Ling; Yen, Hsin-Yung; Choong, Wai-Kok; Wu, Hsin-Yi; Liao, Yen-Chen; Hong, Tse-Ming; Sung, Ting-Yi; Yang, Pan-Chyr; Chen, Yu-Ju

    2017-01-01

    Although EGFR tyrosine kinase inhibitors (TKIs) have demonstrated good efficacy in non-small-cell lung cancer (NSCLC) patients harboring EGFR mutations, most patients develop intrinsic and acquired resistance. We quantitatively profiled the phosphoproteome and proteome of drug-sensitive and drug-resistant NSCLC cells under gefitinib treatment. The construction of a dose-dependent responsive kinase-substrate network of 1548 phosphoproteins and 3834 proteins revealed CK2-centric modules as the dominant core network for the potential gefitinib resistance-associated proteins. CK2 knockdown decreased cell survival in gefitinib-resistant NSCLCs. Using motif analysis to identify the CK2 core sub-network, we verified that elevated phosphorylation level of a CK2 substrate, HMGA1 was a critical node contributing to EGFR-TKI resistance in NSCLC cell. Both HMGA1 knockdown or mutation of the CK2 phosphorylation site, S102, of HMGA1 reinforced the efficacy of gefitinib in resistant NSCLC cells through reactivation of the downstream signaling of EGFR. Our results delineate the TKI resistance-associated kinase-substrate network, suggesting a potential therapeutic strategy for overcoming TKI-induced resistance in NSCLC. PMID:28290473

  20. Unraveling the phosphoproteome dynamics in mammal mitochondria from a network perspective.

    PubMed

    Padrão, Ana Isabel; Vitorino, Rui; Duarte, José Alberto; Ferreira, Rita; Amado, Francisco

    2013-10-04

    With mitochondrion garnering more attention for its inextricable involvement in pathophysiological conditions, it seems imperative to understand the means by which the molecular pathways harbored in this organelle are regulated. Protein phosphorylation has been considered a central event in cellular signaling and, more recently, in the modulation of mitochondrial activity. Efforts have been made to understand the molecular mechanisms by which protein phosphorylation regulates mitochondrial signaling. With the advances in mass-spectrometry-based proteomics, there is a substantial hope and expectation in the increased knowledge of protein phosphorylation profile and its mode of regulation. On the basis of phosphorylation profiles, attempts have been made to disclose the kinases involved and how they control the molecular processes in mitochondria and, consequently, the cellular outcomes. Still, few studies have focused on mitochondrial phosphoproteome profiling, particularly in diseases. The present study reviews current data on protein phosphorylation profiling in mitochondria, the potential kinases involved and how pathophysiological conditions modulate the mitochondrial phosphoproteome. To integrate data from distinct research papers, we performed network analysis, with bioinformatic tools like Cytoscape, String, and PANTHER taking into consideration variables such as tissue specificity, biological processes, molecular functions, and pathophysiological conditions. For instance, data retrieved from these analyses evidence some homology in the mitochondrial phosphoproteome among liver and heart, with proteins from transport and oxidative phosphorylation clusters particularly susceptible to phosphorylation. A distinct profile was noticed for adipocytes, with proteins form metabolic processes, namely, triglycerides metabolism, as the main targets of phosphorylation. Regarding disease conditions, more phosphorylated proteins were observed in diabetics with some

  1. Acetylome and phosphoproteome modifications in imatinib resistant chronic myeloid leukaemia cells treated with valproic acid.

    PubMed

    Buchi, Francesca; Pastorelli, Roberta; Ferrari, Germano; Spinelli, Elena; Gozzini, Antonella; Sassolini, Francesca; Bosi, Alberto; Tombaccini, Donatella; Santini, Valeria

    2011-07-01

    Chronic myeloid leukaemia has a specific therapy: BCR/ABL inhibitor imatinib. Resistance due to BCR/ABL dependent and independent mechanisms is partially reversible by histone deacetylase inhibitors. We analysed by 2D-electrophoresis and anti-pan-acetylated and anti-phosphotyrosine immunoblots, followed by spot-matching and MALDI-TOF mass spectrometry, which proteome modifications would parallel restoration of sensitivity to imatinib by valproic acid (VPA). VPA plus imatinib significantly increased acetylation of HSP90 and hnRNP L and decreased phosphorylation of HSPs and hnRNPs in imatinib resistant cells. VPA was able to modify profoundly acetylome and phosphoproteome of CML cells, while reverting resistance to imatinib.

  2. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    PubMed

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-05

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

  3. Phosphoproteomics as an emerging weapon to develop new antibiotics against carbapenem resistant strain of Acinetobacter baumannii.

    PubMed

    Tiwari, Vishvanath; Tiwari, Monalisa

    2015-01-01

    Acinetobacter baumannii causes pneumonia, bloodstream infections, urinary tract infections, respiratory infections and meningitis. A. baumannii has developed resistance against most of the antibiotics including carbapenem. Therefore, to battle carbapenem resistance, there is a need to develop antimicrobial drugs with new modes of action. Phosphoproteomics will help identify the differentially phosphorylated protein and its crucial phosphosites which facilitate the elucidation of molecular mechanism of signaling and regulation of carbapenem resistant strain of A. baumannii as compared to carbapenem sensitive strain. This understanding might be useful for the development of new antibiotics against kinases involved in the phosphorylation of identified phosphosites in carbapenem resistant strain of A. baumannii. The proposed antibiotics selectively inhibit carbapenem resistant strain which further avoids its excessive use against carbapenem sensitive strain and thereafter reduces emergence of resistance.

  4. Mitochondrial tyrosine phosphoproteome: new insights from an up-to-date analysis.

    PubMed

    Cesaro, Luca; Salvi, Mauro

    2010-01-01

    Tyrosine phosphorylation is a newcomer in the mitochondrial signaling and is currently emerging as an important mechanism for regulating mitochondrial processes. But to what extent? By analyzing an updated draft of the mitochondrial tyrosine phosphoproteome, the following observations can be drawn: more than a hundred mitochondrial proteins undergo tyrosine phosphorylation, phosphotyrosine proteins are distributed in each of the submitochondrial compartments, and mitochondrial tyrosine phosphorylated proteins are involved in a variety of functions as metabolism (electron transport chain, Krebs cycle, fatty acid and amino acid metabolism), solute and protein transport, mitochondrial translation machinery, quality protein assessment, oxidative stress, apoptosis, fission, and other. This large and varied collection suggests that tyrosine phosphorylation could be a widespread mechanism in modulating mitochondrial functions. Moreover the in silico model is here used to explore potential effects of tyrosine phosphorylation on selected mitochondrial proteins pointing out some future perspectives in this field.

  5. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  6. Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

    PubMed Central

    Marabotti, Anna; Lepretti, Marilena; Salzano, Anna Maria; Scaloni, Andrea; Vitale, Monica; Zambrano, Nicola; Sblattero, Daniele; Esposito, Carla

    2013-01-01

    Background Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. Methods and Principal Findings We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. Conclusions Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study

  7. Phosphoproteomic profiling of human myocardial tissues distinguishes ischemic from non-ischemic end stage heart failure.

    PubMed

    Schechter, Matthew A; Hsieh, Michael K H; Njoroge, Linda W; Thompson, J Will; Soderblom, Erik J; Feger, Bryan J; Troupes, Constantine D; Hershberger, Kathleen A; Ilkayeva, Olga R; Nagel, Whitney L; Landinez, Gina P; Shah, Kishan M; Burns, Virginia A; Santacruz, Lucia; Hirschey, Matthew D; Foster, Matthew W; Milano, Carmelo A; Moseley, M Arthur; Piacentino, Valentino; Bowles, Dawn E

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥ 2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure.

  8. Deciphering the acute cellular phosphoproteome response to irradiation with X-rays, protons and carbon ions.

    PubMed

    Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

    2017-03-16

    Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Since radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of

  9. Phosphoproteomic Profiling of Human Myocardial Tissues Distinguishes Ischemic from Non-Ischemic End Stage Heart Failure

    PubMed Central

    Njoroge, Linda W.; Thompson, J. Will; Soderblom, Erik J.; Feger, Bryan J.; Troupes, Constantine D.; Hershberger, Kathleen A.; Ilkayeva, Olga R.; Nagel, Whitney L.; Landinez, Gina P.; Shah, Kishan M.; Burns, Virginia A.; Santacruz, Lucia; Hirschey, Matthew D.; Foster, Matthew W.; Milano, Carmelo A.; Moseley, M. Arthur; Piacentino, Valentino; Bowles, Dawn E.

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure. PMID:25117565

  10. Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis*

    PubMed Central

    Qi, Lin; Liu, Zexian; Wang, Jing; Cui, Yiqiang; Guo, Yueshuai; Zhou, Tao; Zhou, Zuomin; Guo, Xuejiang; Xue, Yu; Sha, Jiahao

    2014-01-01

    Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO2, both the phosphoprotein data sets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinase-substrate relations were computationally predicted for reconstructing kinase-substrate phosphorylation networks. A core sub-kinase-substrate phosphorylation networks among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2, and CDC2 with statistically more site-specific kinase-substrate relations might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of POLO-like kinases decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic

  11. Phosphatase of Regenerating Liver 3 (PRL3) Provokes a Tyrosine Phosphoproteome to Drive Prometastatic Signal Transduction*

    PubMed Central

    Walls, Chad D.; Iliuk, Anton; Bai, Yunpeng; Wang, Mu; Tao, W. Andy; Zhang, Zhong-Yin

    2013-01-01

    Phosphatase of regenerating liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when in excess. To date, the molecular basis for PRL3 function remains an enigma, making efforts at distilling a concerted mechanism for PRL3-mediated metastatic dissemination very difficult. We previously discovered that PRL3 expressing cells exhibit a pronounced increase in protein tyrosine phosphorylation. Here we take an unbiased mass spectrometry-based approach toward identifying the phosphoproteins exhibiting enhanced levels of tyrosine phosphorylation with a goal to define the “PRL3-mediated signaling network.” Phosphoproteomic data support intracellular activation of an extensive signaling network normally governed by extracellular ligand-activated transmembrane growth factor, cytokine, and integrin receptors in the PRL3 cells. Additionally, data implicate the Src tyrosine kinase as the major intracellular kinase responsible for “hijacking” this network and provide strong evidence that aberrant Src activation is a major consequence of PRL3 overexpression. Importantly, the data support a PDGF(α/β)-, Eph (A2/B3/B4)-, and Integrin (β1/β5)-receptor array as being the predominant network coordinator in the PRL3 cells, corroborating a PRL3-induced mesenchymal-state. Within this network, we find that tyrosine phosphorylation is increased on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, STAT, and ERK activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives prometastatic molecular events through Src activation. PMID:24030100

  12. Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

    PubMed

    Locard-Paulet, Marie; Lim, Lindsay; Veluscek, Giulia; McMahon, Kelly; Sinclair, John; van Weverwijk, Antoinette; Worboys, Jonathan D; Yuan, Yinyin; Isacke, Clare M; Jørgensen, Claus

    2016-02-09

    The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.

  13. Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

    PubMed Central

    Hernandez-Valladares, Maria; Aasebø, Elise; Selheim, Frode; Berven, Frode S.; Bruserud, Øystein

    2016-01-01

    Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility. PMID:28248234

  14. Mass spectrometric phosphoproteome analysis of HIV-infected brain reveals novel phosphorylation sites and differential phosphorylation patterns

    PubMed Central

    Uzasci, Lerna; Auh, Sungyoung; Cotter, Robert J.; Nath, Avindra

    2016-01-01

    Purpose To map the phosphoproteome and identify changes in the phosphorylation patterns in the HIV-infected and uninfected brain using high-resolution mass spectrometry. Experimental Design Parietal cortex from brain of individuals with and without HIV infection were lysed and trypsinized. The peptides were labeled with iTRAQ reagents, combined, phospho-enriched by titanium dioxide chromatography, and analyzed by LC-MS/MS with high-resolution. Results Our phosphoproteomic workflow resulted in the identification of 112 phosphorylated proteins and 17 novel phosphorylation sites in all the samples that were analyzed. The phosphopeptide sequences were searched for kinase substrate motifs which revealed potential kinases involved in important signaling pathways. The site-specific phosphopeptide quantification showed that peptides from neurofilament medium polypeptide, myelin basic protein, and 2′–3′-cyclic nucleotide-3′ phosphodiesterase have relatively higher phosphorylation levels during HIV infection. Clinical Relevance This study has enriched the global phosphoproteome knowledge of the human brain by detecting novel phosphorylation sites on neuronal proteins and identifying differentially phosphorylated brain proteins during HIV infection. Kinases that lead to unusual phosphorylations could be therapeutic targets for the treatment of HIV-associated neurocognitive disorders (HAND). PMID:26033855

  15. Comparative Phosphoproteomic Analysis under High-Nitrogen Fertilizer Reveals Central Phosphoproteins Promoting Wheat Grain Starch and Protein Synthesis

    PubMed Central

    Zhen, Shoumin; Deng, Xiong; Zhang, Ming; Zhu, Gengrui; Lv, Dongwen; Wang, Yaping; Zhu, Dong; Yan, Yueming

    2017-01-01

    Nitrogen (N) is a macronutrient important for plant growth and development. It also strongly influences starch and protein synthesis, closely related to grain yield and quality. We performed the first comparative phosphoproteomic analysis of developing wheat grains in response to high-N fertilizer. Physiological and biochemical analyses showed that application of high-N fertilizer resulted in significant increases in leaf length and area, chlorophyll content, the activity of key enzymes in leaves such as nitrate reductase (NR), and in grains such as sucrose phosphate synthase (SPS), sucrose synthase (SuSy), and ADP glucose pyrophosphorylase (AGPase). This enhanced enzyme activity led to significant improvements in starch content, grain yield, and ultimately, bread making quality. Comparative phosphoproteomic analysis of developing grains under the application of high-N fertilizer performed 15 and 25 days post-anthesis identified 2470 phosphosites among 1372 phosphoproteins, of which 411 unique proteins displayed significant changes in phosphorylation level (>2-fold or <0.5-fold). These phosphoproteins are involved mainly in signaling transduction, starch synthesis, energy metabolism. Pro-Q diamond staining and Western blotting confirmed our phosphoproteomic results. We propose a putative pathway to elucidate the important roles of the central phosphoproteins regulating grain starch and protein synthesis. Our results provide new insights into the molecular mechanisms of protein phosphorylation modifications involved in grain development, yield and quality formation. PMID:28194157

  16. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    SciTech Connect

    Matsumura, Takayuki; Oyama, Masaaki; Kozuka-Hata, Hiroko; Ishikawa, Kosuke; Inoue, Takafumi; Muta, Tatsushi; Semba, Kentaro; Inoue, Jun-ichiro

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  17. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

    PubMed

    Iwai, Leo K; Payne, Leo S; Luczynski, Maciej T; Chang, Francis; Xu, Huifang; Clinton, Ryan W; Paul, Angela; Esposito, Edward A; Gridley, Scott; Leitinger, Birgit; Naegle, Kristen M; Huang, Paul H

    2013-09-15

    Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

  18. Niobium(V) oxide (Nb2O5): application to phosphoproteomics.

    PubMed

    Ficarro, Scott B; Parikh, Jignesh R; Blank, Nathaniel C; Marto, Jarrod A

    2008-06-15

    Proteomics-based analysis of signaling cascades relies on a growing suite of affinity resins and methods aimed at efficient enrichment of phosphorylated peptides from complex biological mixtures. Given the heterogeneity of phosphopeptides and the overlap in chemical properties between phospho- and unmodified peptides, it is likely that the use of multiple resins will provide the best combination of specificity, yield, and coverage for large-scale proteomics studies. Recently titanium and zirconium dioxides have been used successfully for enrichment of phosphopeptides. Here we report the first demonstration that niobium pentoxide (Nb 2O 5) provides for efficient enrichment and recovery ( approximately 50-100%) of phosphopeptides from simple mixtures and facilitates identification of several hundred putative sites of phosphorylation from cell lysate. Comparison of phosphorylated peptides identified from Nb 2O 5 and TiO 2 with sequences in the PhosphoELM database suggests a useful degree of divergence in the selectivity of these metal oxide resins. Collectively our data indicate that Nb 2O 5 provides efficient enrichment for phosphopeptides and offers a complementary approach for large-scale phosphoproteomics studies.

  19. Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance.

    PubMed

    Pi, Zhi; Zhao, Mei-Ling; Peng, Xian-Jun; Shen, Shi-Hua

    2017-04-13

    Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement, and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 h, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 h, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes according to conserved sequence analysis. The results of Gene Ontology analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification, and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 h, respectively. On the basis of the protein-protein interaction network analysis, a protein kinase and phosphatases hub protein (P1959) were found to be involved in cross-talk between Ca(2+), BR, ABA, and ethylene-mediated signaling pathways. We also highlighted the phosphorylation of BpSIZ1 and BpICE1 possibly impacted on the CBF/DREB-responsive pathway. From these results, we developed a schematic for the chilling tolerance mechanism at phosphorylation level.

  20. Phosphoproteomic dynamics of chickpea (Cicer arietinum L.) reveals shared and distinct components of dehydration response.

    PubMed

    Subba, Pratigya; Barua, Pragya; Kumar, Rajiv; Datta, Asis; Soni, Kamlesh Kumar; Chakraborty, Subhra; Chakraborty, Niranjan

    2013-11-01

    Reversible protein phosphorylation is a ubiquitous regulatory mechanism that plays critical roles in transducing stress signals to bring about coordinated intracellular responses. To gain better understanding of dehydration response in plants, we have developed a differential phosphoproteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water, and the changes in the phosphorylation status of a large repertoire of proteins were monitored. The proteins were resolved by 2-DE and stained with phosphospecific fluorescent Pro-Q Diamond dye. Mass spectrometric analysis led to the identification of 91 putative phosphoproteins, presumably involved in a variety of functions including cell defense and rescue, photosynthesis and photorespiration, molecular chaperones, and ion transport, among others. Multiple sites of phosphorylation were predicted on several key elements, which include both the regulatory as well as the functional proteins. A critical survey of the phosphorylome revealed a DREPP (developmentally regulated plasma membrane protein) plasma membrane polypeptide family protein, henceforth designated CaDREPP1. The transcripts of CaDREPP1 were found to be differentially regulated under dehydration stress, further corroborating the proteomic results. This work provides new insights into the possible phosphorylation events triggered by the conditions of progressive water-deficit in plants.

  1. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

    PubMed

    Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2015-01-01

    Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

  2. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

    PubMed

    Siciliano, Angela; Turrini, Franco; Bertoldi, Mariarita; Matte, Alessandro; Pantaleo, Antonella; Olivieri, Oliviero; De Franceschi, Lucia

    2010-04-15

    Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

  3. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    PubMed Central

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco AS; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: http://dx.doi.org/10.7554/eLife.12813.001 PMID:26824392

  4. Newly fabricated magnetic lanthanide oxides core-shell nanoparticles in phosphoproteomics.

    PubMed

    Jabeen, Fahmida; Najam-Ul-Haq, Muhammad; Rainer, Matthias; Güzel, Yüksel; Huck, Christian W; Bonn, Guenther K

    2015-01-01

    Metal oxides show high selectivity and sensitivity toward mass spectrometry based enrichment strategies. Phosphopeptides/phosphoproteins enrichment from biological samples is cumbersome because of their low abundance. Phosphopeptides are of interest in enzymes and phosphorylation pathways which lead to the clinical links of a disease. Magnetic core-shell lanthanide oxide nanoparticles (Fe3O4@SiO2-La2O3 and Fe3O4@SiO2-Sm2O3) are fabricated, characterized by SEM, FTIR, and EDX and employed in the enrichment of phosphopeptides. The nanoparticles enrich phosphopeptides from casein variants, nonfat milk, egg yolk, human serum and HeLa cell extract. The materials and enrichment protocols are designed in a way that there are almost no nonspecific bindings. The selectivity is achieved up to 1:8500 using β-casein/BSA mixture and sensitivity down to 1 atto-mole. Batch-to-batch reproducibility is high with the reuse of core-shell nanoparticles up to four cycles. The enrichment followed by MALDI-MS analyses is carried out for the identification of phosphopeptides from serum digest and HeLa cell extract. Characteristic phosphopeptides of phosphoproteins are identified from human serum after the enrichment, which have the diagnostic potential toward prostate cancer. Thus, the lanthanide based magnetic core-shell materials offer a highly selective and sensitive workflow in phosphoproteomics.

  5. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    DOE PAGES

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; ...

    2014-05-29

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggestsmore » that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.« less

  6. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    SciTech Connect

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H. D.; Yang, Feng; Glass, N. Louise

    2014-05-29

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

  7. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines

    PubMed Central

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the “kinome”. Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression. PMID:27362937

  8. iPhos: a toolkit to streamline the alkaline phosphatase-assisted comprehensive LC-MS phosphoproteome investigation

    PubMed Central

    2014-01-01

    Background Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. Results We developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). Conclusions In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC

  9. Quantitative phosphoproteomic analysis identifies activation of the RET and IGF-1R/IR signaling pathways in neuroblastoma.

    PubMed

    DeNardo, Bradley D; Holloway, Michael P; Ji, Qinqin; Nguyen, Kevin T; Cheng, Yan; Valentine, Marcus B; Salomon, Arthur; Altura, Rachel A

    2013-01-01

    Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.

  10. Salt-induced changes in cardiac phosphoproteome in a rat model of chronic renal failure.

    PubMed

    Su, Zhengxiu; Zhu, Hongguo; Zhang, Menghuan; Wang, Liangliang; He, Hanchang; Jiang, Shaoling; Hou, Fan Fan; Li, Aiqing

    2014-01-01

    Heart damage is widely present in patients with chronic kidney disease. Salt diet is the most important environmental factor affecting development of chronic renal failure and cardiovascular diseases. The proteins involved in chronic kidney disease -induced heart damage, especially their posttranslational modifications, remain largely unknown to date. Sprague-Dawley rats underwent 5/6 nephrectomy (chronic renal failure model) or sham operation were treated for 2 weeks with a normal-(0.4% NaCl), or high-salt (4% NaCl) diet. We employed TiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for phosphoproteomic profiling of left ventricular free walls in these animals. A total of 1724 unique phosphopeptides representing 2551 non-redundant phosphorylation sites corresponding to 763 phosphoproteins were identified. During normal salt feeding, 89 (54%) phosphopeptides upregulated and 76 (46%) phosphopeptides downregulated in chronic renal failure rats relative to sham rats. In chronic renal failure rats, high salt intake induced upregulation of 84 (49%) phosphopeptides and downregulation of 88 (51%) phosphopeptides. Database searches revealed that most of the identified phospholproteins were important signaling molecules such as protein kinases, receptors and phosphatases. These phospholproteins were involved in energy metabolism, cell communication, cell differentiation, cell death and other biological processes. The Search Tool for the Retrieval of Interacting Genes analysis revealed functional links among 15 significantly regulated phosphoproteins in chronic renal failure rats compared to sham group, and 23 altered phosphoproteins induced by high salt intake. The altered phosphorylation levels of two proteins involved in heart damage, lamin A and phospholamban were validated. Expression of the downstream genes of these two proteins, desmin and SERCA2a, were also analyzed.

  11. Phosphoproteomic analysis of the non-seed vascular plant model Selaginella moellendorffii

    PubMed Central

    2014-01-01

    Background Selaginella (Selaginella moellendorffii) is a lycophyte which diverged from other vascular plants approximately 410 million years ago. As the first reported non-seed vascular plant genome, Selaginella genome data allow comparative analysis of genetic changes that may be associated with land plant evolution. Proteomics investigations on this lycophyte model have not been extensively reported. Phosphorylation represents the most common post-translational modifications and it is a ubiquitous regulatory mechanism controlling the functional expression of proteins inside living organisms. Results In this study, polyethylene glycol fractionation and immobilized metal ion affinity chromatography were employed to isolate phosphopeptides from wild-growing Selaginella. Using liquid chromatography-tandem mass spectrometry analysis, 1593 unique phosphopeptides spanning 1104 non-redundant phosphosites with confirmed localization on 716 phosphoproteins were identified. Analysis of the Selaginella dataset revealed features that are consistent with other plant phosphoproteomes, such as the relative proportions of phosphorylated Ser, Thr, and Tyr residues, the highest occurrence of phosphosites in the C-terminal regions of proteins, and the localization of phosphorylation events outside protein domains. In addition, a total of 97 highly conserved phosphosites in evolutionary conserved proteins were identified, indicating the conservation of phosphorylation-dependent regulatory mechanisms in phylogenetically distinct plant species. On the other hand, close examination of proteins involved in photosynthesis revealed phosphorylation events which may be unique to Selaginella evolution. Furthermore, phosphorylation motif analyses identified Pro-directed, acidic, and basic signatures which are recognized by typical protein kinases in plants. A group of Selaginella-specific phosphoproteins were found to be enriched in the Pro-directed motif class. Conclusions Our work provides

  12. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  13. Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-05

    Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

  14. Identifying drug effects via pathway alterations using an integer linear programming optimization formulation on phosphoproteomic data.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Siminelakis, Paraskeuas; Chairakaki, Aikaterini D; Saez-Rodriguez, Julio; Alexopoulos, Leonidas G

    2009-12-01

    Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1alpha) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on

  15. Chronic low-dose-rate ionising radiation affects the hippocampal phosphoproteome in the ApoE−/− Alzheimer's mouse model

    PubMed Central

    Kempf, Stefan J.; Janik, Dirk; Barjaktarovic, Zarko; Braga-Tanaka, Ignacia; Tanaka, Satoshi; Neff, Frauke; Saran, Anna; Larsen, Martin R.; Tapio, Soile

    2016-01-01

    Accruing data indicate that radiation-induced consequences resemble pathologies of neurodegenerative diseases such as Alzheimer's. The aim of this study was to elucidate the effect on hippocampus of chronic low-dose-rate radiation exposure (1 mGy/day or 20 mGy/day) given over 300 days with cumulative doses of 0.3 Gy and 6.0 Gy, respectively. ApoE deficient mutant C57Bl/6 mouse was used as an Alzheimer's model. Using mass spectrometry, a marked alteration in the phosphoproteome was found at both dose rates. The radiation-induced changes in the phosphoproteome were associated with the control of synaptic plasticity, calcium-dependent signalling and brain metabolism. An inhibition of CREB signalling was found at both dose rates whereas Rac1-Cofilin signalling was found activated only at the lower dose rate. Similarly, the reduction in the number of activated microglia in the molecular layer of hippocampus that paralleled with reduced levels of TNFα expression and lipid peroxidation was significant only at the lower dose rate. Adult neurogenesis, investigated by Ki67, GFAP and NeuN staining, and cell death (activated caspase-3) were not influenced at any dose or dose rate. This study shows that several molecular targets induced by chronic low-dose-rate radiation overlap with those of Alzheimer's pathology. It may suggest that ionising radiation functions as a contributing risk factor to this neurodegenerative disease. PMID:27708245

  16. Meta-Analysis of Arabidopsis thaliana Phospho-Proteomics Data Reveals Compartmentalization of Phosphorylation Motifs[C][W

    PubMed Central

    van Wijk, Klaas J.; Friso, Giulia; Walther, Dirk; Schulze, Waltraud X.

    2014-01-01

    Protein (de)phosphorylation plays an important role in plants. To provide a robust foundation for subcellular phosphorylation signaling network analysis and kinase-substrate relationships, we performed a meta-analysis of 27 published and unpublished in-house mass spectrometry–based phospho-proteome data sets for Arabidopsis thaliana covering a range of processes, (non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of 60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data for quality and consistency generated a set of medium and a set of high confidence phospho-proteins and their assigned phospho-sites. The relation between single and multiphosphorylated peptides is discussed. The distribution of p-proteins across cellular functions and subcellular compartments was determined and showed overrepresentation of protein kinases. Extensive differences in frequency of pY were found between individual studies due to proteomics and mass spectrometry workflows. Interestingly, pY was underrepresented in peroxisomes but overrepresented in mitochondria. Using motif-finding algorithms motif-x and MMFPh at high stringency, we identified compartmentalization of phosphorylation motifs likely reflecting localized kinase activity. The filtering of the data assembly improved signal/noise ratio for such motifs. Identified motifs were linked to kinases through (bioinformatic) enrichment analysis. This study also provides insight into the challenges/pitfalls of using large-scale phospho-proteomic data sets to nonexperts. PMID:24894044

  17. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

    PubMed Central

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  18. In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    PubMed Central

    Højlund, Kurt; Bowen, Benjamin P.; Hwang, Hyonson; Flynn, Charles R.; Madireddy, Lohith; Thangiah, Geetha; Langlais, Paul; Meyer, Christian; Mandarino, Lawrence J.; Yi, Zhengping

    2009-01-01

    Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca2+-cycle), glycolysis and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disc proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo. PMID:19764811

  19. Outcomes from Enabling Courses.

    ERIC Educational Resources Information Center

    Phan, Oanh; Ball, Katrina

    The outcomes of enabling courses offered in Australia's vocational education and training (VET) sector were examined. "Enabling course" was defined as lower-level preparatory and prevocational courses covering a wide range of areas, including remedial education, bridging courses, precertificate courses, and general employment preparation…

  20. Technology Enabled Learning. Symposium.

    ERIC Educational Resources Information Center

    2002

    This document contains three papers on technology-enabled learning and human resource development. Among results found in "Current State of Technology-enabled Learning Programs in Select Federal Government Organizations: a Case Study of Ten Organizations" (Letitia A. Combs) are the following: the dominant delivery method is traditional…

  1. Physician Enabling Skills Questionnaire

    PubMed Central

    Hudon, Catherine; Lambert, Mireille; Almirall, José

    2015-01-01

    Abstract Objective To evaluate the reliability and validity of the newly developed Physician Enabling Skills Questionnaire (PESQ) by assessing its internal consistency, test-retest reliability, concurrent validity with patient-centred care, and predictive validity with patient activation and patient enablement. Design Validation study. Setting Saguenay, Que. Participants One hundred patients with at least 1 chronic disease who presented in a waiting room of a regional health centre family medicine unit. Main outcome measures Family physicians’ enabling skills, measured with the PESQ at 2 points in time (ie, while in the waiting room at the family medicine unit and 2 weeks later through a mail survey); patient-centred care, assessed with the Patient Perception of Patient-Centredness instrument; patient activation, assessed with the Patient Activation Measure; and patient enablement, assessed with the Patient Enablement Instrument. Results The internal consistency of the 6 subscales of the PESQ was adequate (Cronbach α = .69 to .92). The test-retest reliability was very good (r = 0.90; 95% CI 0.84 to 0.93). Concurrent validity with the Patient Perception of Patient-Centredness instrument was good (r = −0.67; 95% CI −0.78 to −0.53; P < .001). The PESQ accounts for 11% of the total variance with the Patient Activation Measure (r2 = 0.11; P = .002) and 19% of the variance with the Patient Enablement Instrument (r2 = 0.19; P < .001). Conclusion The newly developed PESQ presents good psychometric properties, allowing for its use in practice and research. PMID:26889507

  2. Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

    PubMed

    Zarei, Mostafa; Sprenger, Adrian; Rackiewicz, Michal; Dengjel, Joern

    2016-01-01

    Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple and rapid strategy for phosphopeptide prefractionation. The combinatorial use of two distinct chromatographic techniques that address the inverse physicochemical properties of peptides allows for superior fractionation efficiency of multiple phosphorylated peptides. In the first step, multiphosphorylated peptides are separated according to the number of negatively charged phosphosites by electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). A subsequent strong cation exchange (SCX) step separates mostly singly phosphorylated peptides in the ERLIC flow-through according to their positive charge. The presented strategy is inexpensive and adaptable to large and small amounts of starting material, and it allows highly multiplexed sample preparation. Because of its implementation as solid-phase extraction, the entire workflow takes only 2 h to complete.

  3. Extracellular Matrix Proteome and Phosphoproteome of Potato Reveals Functionally Distinct and Diverse Canonical and Non-Canonical Proteoforms

    PubMed Central

    Elagamey, Eman; Narula, Kanika; Sinha, Arunima; Aggarwal, Pooja Rani; Ghosh, Sudip; Chakraborty, Niranjan; Chakraborty, Subhra

    2016-01-01

    The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling. PMID:28248230

  4. Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

    PubMed

    Islam, Md Shariful; Nolte, Hendrik; Jacob, Wright; Ziegler, Anna B; Pütz, Stefanie; Grosjean, Yael; Szczepanowska, Karolina; Trifunovic, Aleksandra; Braun, Thomas; Heumann, Hermann; Heumann, Rolf; Hovemann, Bernhard; Moore, Darren J; Krüger, Marcus

    2016-10-29

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson's disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

  5. Phosphoproteomes of Strongylocentrotus purpuratus shell and tooth matrix: identification of a major acidic sea urchin tooth phosphoprotein, phosphodontin

    PubMed Central

    2010-01-01

    Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. Conclusions The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental

  6. Microsystems Enabled Photovoltaics

    ScienceCinema

    Gupta, Vipin; Nielson, Greg; Okandan, Murat, Granata, Jennifer; Nelson, Jeff; Haney, Mike; Cruz-Campa, Jose Luiz

    2016-07-12

    Sandia's microsystems enabled photovoltaic advances combine mature technology and tools currently used in microsystem production with groundbreaking advances in photovoltaics cell design, decreasing production and system costs while improving energy conversion efficiency. The technology has potential applications in buildings, houses, clothing, portable electronics, vehicles, and other contoured structures.

  7. Liquid metal enabled pump

    PubMed Central

    Tang, Shi-Yang; Khoshmanesh, Khashayar; Sivan, Vijay; Petersen, Phred; O’Mullane, Anthony P.; Abbott, Derek; Mitchell, Arnan; Kalantar-zadeh, Kourosh

    2014-01-01

    Small-scale pumps will be the heartbeat of many future micro/nanoscale platforms. However, the integration of small-scale pumps is presently hampered by limited flow rate with respect to the input power, and their rather complicated fabrication processes. These issues arise as many conventional pumping effects require intricate moving elements. Here, we demonstrate a system that we call the liquid metal enabled pump, for driving a range of liquids without mechanical moving parts, upon the application of modest electric field. This pump incorporates a droplet of liquid metal, which induces liquid flow at high flow rates, yet with exceptionally low power consumption by electrowetting/deelectrowetting at the metal surface. We present theory explaining this pumping mechanism and show that the operation is fundamentally different from other existing pumps. The presented liquid metal enabled pump is both efficient and simple, and thus has the potential to fundamentally advance the field of microfluidics. PMID:24550485

  8. Nanotechnology - Enabled Sensing

    DTIC Science & Technology

    2009-05-07

    via subwavelength confinement of optical fields near metallic nanostructures, as shown in Figure 2.3. When a single cadmium selenide quantum dot is...optical modulator uses a coating of cadmium selenide quantum dots to convert two light beams into surface plasmon polaritons. (Reprinted by permission...helpful. Two- and three-dimensional photonic crystals can enable new sensing systems based on fluorescent molecules and/or quantum dots and

  9. Enabling Wind Power Nationwide

    SciTech Connect

    Jose, Zayas; Michael, Derby; Patrick, Gilman; Ananthan, Shreyas; Lantz, Eric; Cotrell, Jason; Beck, Fredic; Tusing, Richard

    2015-05-01

    Leveraging this experience, the U.S. Department of Energy’s (DOE’s) Wind and Water Power Technologies Office has evaluated the potential for wind power to generate electricity in all 50 states. This report analyzes and quantifies the geographic expansion that could be enabled by accessing higher above ground heights for wind turbines and considers the means by which this new potential could be responsibly developed.

  10. Grid-Enabled Quantitative Analysis of Breast Cancer

    DTIC Science & Technology

    2010-10-01

    large-scale, multi-modality computerized image analysis . The central hypothesis of this research is that large-scale image analysis for breast cancer...research, we designed a pilot study utilizing large scale parallel Grid computing harnessing nationwide infrastructure for medical image analysis . Also

  11. Grid-Enabled Quantitative Analysis of Breast Cancer

    DTIC Science & Technology

    2009-10-01

    large-scale, multi-modality computerized image analysis . The central hypothesis of this research is that large-scale image analysis for breast cancer...pilot study to utilize large scale parallel Grid computing to harness the nationwide cluster infrastructure for optimization of medical image ... analysis parameters. Additionally, we investigated the use of cutting edge dataanalysis/ mining techniques as applied to Ultrasound, FFDM, and DCE-MRI Breast

  12. Smart Grid Enabled EVSE

    SciTech Connect

    None, None

    2015-01-12

    The combined team of GE Global Research, Federal Express, National Renewable Energy Laboratory, and Consolidated Edison has successfully achieved the established goals contained within the Department of Energy’s Smart Grid Capable Electric Vehicle Supply Equipment funding opportunity. The final program product, shown charging two vehicles in Figure 1, reduces by nearly 50% the total installed system cost of the electric vehicle supply equipment (EVSE) as well as enabling a host of new Smart Grid enabled features. These include bi-directional communications, load control, utility message exchange and transaction management information. Using the new charging system, Utilities or energy service providers will now be able to monitor transportation related electrical loads on their distribution networks, send load control commands or preferences to individual systems, and then see measured responses. Installation owners will be able to authorize usage of the stations, monitor operations, and optimally control their electricity consumption. These features and cost reductions have been developed through a total system design solution.

  13. Enable, mediate, advocate.

    PubMed

    Saan, Hans; Wise, Marilyn

    2011-12-01

    The authors of the Ottawa Charter selected the words enable, mediate and advocate to describe the core activities in what was, in 1986, the new Public Health. This article considers these concepts and the values and ideas upon which they were based. We discuss their relevance in the current context within which health promotion is being conducted, and discuss the implications of changes in the health agenda, media and globalization for practice. We consider developments within health promotion since 1986: its central role in policy rhetoric, the increasing understanding of complexities and the interlinkage with many other societal processes. So the three core activities are reviewed: they still fit well with the main health promotion challenges, but should be refreshed by new ideas and values. As the role of health promotion in the political arena has grown we have become part of the policy establishment and that is a mixed blessing. Making way for community advocates is now our challenge. Enabling requires greater sensitivity to power relations involved and an understanding of the role of health literacy. Mediating keeps its central role as it bridges vital interests of parties. We conclude that these core concepts in the Ottawa Charter need no serious revision. There are, however, lessons from the last 25 years that point to ways to address present and future challenges with greater sensitivity and effectiveness. We invite the next generation to avoid canonizing this text: as is true of every heritage, the heirs must decide on its use.

  14. Enabling graphene nanoelectronics.

    SciTech Connect

    Pan, Wei; Ohta, Taisuke; Biedermann, Laura Butler; Gutierrez, Carlos; Nolen, C. M.; Howell, Stephen Wayne; Beechem Iii, Thomas Edwin; McCarty, Kevin F.; Ross, Anthony Joseph, III

    2011-09-01

    Recent work has shown that graphene, a 2D electronic material amenable to the planar semiconductor fabrication processing, possesses tunable electronic material properties potentially far superior to metals and other standard semiconductors. Despite its phenomenal electronic properties, focused research is still required to develop techniques for depositing and synthesizing graphene over large areas, thereby enabling the reproducible mass-fabrication of graphene-based devices. To address these issues, we combined an array of growth approaches and characterization resources to investigate several innovative and synergistic approaches for the synthesis of high quality graphene films on technologically relevant substrate (SiC and metals). Our work focused on developing the fundamental scientific understanding necessary to generate large-area graphene films that exhibit highly uniform electronic properties and record carrier mobility, as well as developing techniques to transfer graphene onto other substrates.

  15. Enabling immersive simulation.

    SciTech Connect

    McCoy, Josh; Mateas, Michael; Hart, Derek H.; Whetzel, Jonathan; Basilico, Justin Derrick; Glickman, Matthew R.; Abbott, Robert G.

    2009-02-01

    The object of the 'Enabling Immersive Simulation for Complex Systems Analysis and Training' LDRD has been to research, design, and engineer a capability to develop simulations which (1) provide a rich, immersive interface for participation by real humans (exploiting existing high-performance game-engine technology wherever possible), and (2) can leverage Sandia's substantial investment in high-fidelity physical and cognitive models implemented in the Umbra simulation framework. We report here on these efforts. First, we describe the integration of Sandia's Umbra modular simulation framework with the open-source Delta3D game engine. Next, we report on Umbra's integration with Sandia's Cognitive Foundry, specifically to provide for learning behaviors for 'virtual teammates' directly from observed human behavior. Finally, we describe the integration of Delta3D with the ABL behavior engine, and report on research into establishing the theoretical framework that will be required to make use of tools like ABL to scale up to increasingly rich and realistic virtual characters.

  16. Liquid metal enabled microfluidics.

    PubMed

    Khoshmanesh, Khashayar; Tang, Shi-Yang; Zhu, Jiu Yang; Schaefer, Samira; Mitchell, Arnan; Kalantar-Zadeh, Kourosh; Dickey, Michael D

    2017-03-14

    Several gallium-based liquid metal alloys are liquid at room temperature. As 'liquid', such alloys have a low viscosity and a high surface tension while as 'metal', they have high thermal and electrical conductivities, similar to mercury. However, unlike mercury, these liquid metal alloys have low toxicity and a negligible vapor pressure, rendering them much safer. In comparison to mercury, the distinguishing feature of these alloys is the rapid formation of a self-limiting atomically thin layer of gallium oxide over their surface when exposed to oxygen. This oxide layer changes many physical and chemical properties of gallium alloys, including their interfacial and rheological properties, which can be employed and modulated for various applications in microfluidics. Injecting liquid metal into microfluidic structures has been extensively used to pattern and encapsulate highly deformable and reconfigurable electronic devices including electrodes, sensors, antennas, and interconnects. Likewise, the unique features of liquid metals have been employed for fabricating miniaturized microfluidic components including pumps, valves, heaters, and electrodes. In this review, we discuss liquid metal enabled microfluidic components, and highlight their desirable attributes including simple fabrication, facile integration, stretchability, reconfigurability, and low power consumption, with promising applications for highly integrated microfluidic systems.

  17. Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.

    PubMed Central

    Romero-Rodríguez, M. Cristina; Abril, Nieves; Sánchez-Lucas, Rosa; Jorrín-Novo, Jesús V.

    2015-01-01

    As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes. PMID:26322061

  18. Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF)

    PubMed Central

    Reiland, Sonja; Finazzi, Giovanni; Endler, Anne; Willig, Adrian; Baerenfaller, Katja; Grossmann, Jonas; Gerrits, Bertran; Rutishauser, Dorothea; Gruissem, Wilhelm; Rochaix, Jean-David; Baginsky, Sacha

    2011-01-01

    Important aspects of photosynthetic electron transport efficiency in chloroplasts are controlled by protein phosphorylation. Two thylakoid-associated kinases, STN7 and STN8, have distinct roles in short- and long-term photosynthetic acclimation to changes in light quality and quantity. Although some substrates of STN7 and STN8 are known, the complexity of this regulatory kinase system implies that currently unknown substrates connect photosynthetic performance with the regulation of metabolic and regulatory functions. We performed an unbiased phosphoproteome-wide screen with Arabidopsis WT and stn8 mutant plants to identify unique STN8 targets. The phosphorylation status of STN7 was not affected in stn8, indicating that kinases other than STN8 phosphorylate STN7 under standard growth conditions. Among several putative STN8 substrates, PGRL1-A is of particular importance because of its possible role in the modulation of cyclic electron transfer. The STN8 phosphorylation site on PGRL1-A is absent in both monocotyledonous plants and algae. In dicots, spectroscopic measurements with Arabidopsis WT, stn7, stn8, and stn7/stn8 double-mutant plants indicate a STN8-mediated slowing down of the transition from cyclic to linear electron flow at the onset of illumination. This finding suggests a possible link between protein phosphorylation by STN8 and fine-tuning of cyclic electron flow during this critical step of photosynthesis, when the carbon assimilation is not commensurate to the electron flow capacity of the chloroplast. PMID:21768351

  19. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling.

    PubMed

    Alayev, Anya; Doubleday, Peter F; Berger, Sara Malka; Ballif, Bryan A; Holz, Marina K

    2014-12-05

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells.

  20. Large-Scale Phosphoproteome of Human Whole Saliva Using Disulphide-Thiol-Interchange Covalent Chromatography and Mass Spectrometry

    PubMed Central

    Salih, Erdjan; Siqueira, Walter L.; Helmerhorst, Eva J.; Oppenheim, Frank G.

    2010-01-01

    Thus far only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine containing peptides. The DTT-phosphopeptides were enriched by covalent disulphide-thiol-interchange chromatography and analysis by nano-flow LC-ESI-MS/MS. The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)2, blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples which were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS of which 28 were based on two or more peptide identification criteria with high confidence, and 37 were based on a single phosphopeptide identification. Most of the identified proteins, ~80%, were hitherto unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for both systemic and oral disease states. PMID:20659418

  1. Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

    PubMed

    Chidiac, Rony; Zhang, Ying; Tessier, Sylvain; Faubert, Denis; Delisle, Chantal; Gratton, Jean-Philippe

    2016-05-01

    VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.

  2. Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures.

    PubMed

    Davanture, Marlène; Dumur, Jérôme; Bataillé-Simoneau, Nelly; Campion, Claire; Valot, Benoît; Zivy, Michel; Simoneau, Philippe; Fillinger, Sabine

    2014-07-01

    This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

  3. Phosphoproteomics Reveals Resveratrol-Dependent Inhibition of Akt/mTORC1/S6K1 Signaling

    PubMed Central

    2015-01-01

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells. PMID:25311616

  4. PAPE (Prefractionation-Assisted Phosphoprotein Enrichment): A Novel Approach for Phosphoproteomic Analysis of Green Tissues from Plants

    PubMed Central

    Lassowskat, Ines; Naumann, Kai; Lee, Justin; Scheel, Dierk

    2013-01-01

    Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events. PMID:28250405

  5. Large-scale phosphoproteome of human whole saliva using disulfide-thiol interchange covalent chromatography and mass spectrometry.

    PubMed

    Salih, Erdjan; Siqueira, Walter L; Helmerhorst, Eva J; Oppenheim, Frank G

    2010-12-01

    To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)(2), blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples that were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS; of these, 28 were based on two or more peptide identification criteria with high confidence and 37 were based on a single phosphopeptide identification. Most of the identified proteins (∼80%) were previously unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for systemic and oral disease states.

  6. Dynamic Phosphoproteome Analysis of Seedling Leaves in Brachypodium distachyon L. Reveals Central Phosphorylated Proteins Involved in the Drought Stress Response

    PubMed Central

    Yuan, Lin-Lin; Zhang, Ming; Yan, Xing; Bian, Yan-Wei; Zhen, Shou-Min; Yan, Yue-Ming

    2016-01-01

    Drought stress is a major abiotic stress affecting plant growth and development. In this study, we performed the first dynamic phosphoproteome analysis of Brachypodium distachyon L. seedling leaves under drought stress for different times. A total of 4924 phosphopeptides, contained 6362 phosphosites belonging to 2748 phosphoproteins. Rigorous standards were imposed to screen 484 phosphorylation sites, representing 442 unique phosphoproteins. Comparative analyses revealed significant changes in phosphorylation levels at 0, 6, and 24 h under drought stress. The most phosphorylated proteins and the highest phosphorylation level occurred at 6 h. Venn analysis showed that the up-regulated phosphopeptides at 6 h were almost two-fold those at 24 h. Motif-X analysis identified the six motifs: [sP], [Rxxs], [LxRxxs], [sxD], [sF], and [TP], among which [LxRxxs] was also previously identified in B. distachyon. Results from molecular function and protein-protein interaction analyses suggested that phosphoproteins mainly participate in signal transduction, gene expression, drought response and defense, photosynthesis and energy metabolism, and material transmembrane transport. These phosphoproteins, which showed significant changes in phosphorylation levels, play important roles in signal transduction and material transmembrane transport in response to drought conditions. Our results provide new insights into the molecular mechanism of this plant’s abiotic stress response through phosphorylation modification. PMID:27748408

  7. Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

    PubMed Central

    Moniz, Larissa S.; Surinova, Silvia; Ghazaly, Essam; Velasco, Lorena Gonzalez; Haider, Syed; Rodríguez-Prados, Juan Carlos; Berenjeno, Inma M.; Chelala, Claude; Vanhaesebroeck, Bart

    2017-01-01

    To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3caH1047R) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3caH1047R and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing. We also identified novel PI3K effectors that were commonly-regulated, including putative AKT substrates. Validation of one of these hits, confirmed NT5C (5′,3′-Nucleotidase, Cytosolic) as a novel AKT substrate, with an unexpected role in actin cytoskeleton regulation via an interaction with the ARP2/3 complex. This study has produced a comprehensive data resource and identified a new link between PI3K pathway activation and actin regulation. PMID:28059163

  8. Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus

    PubMed Central

    Ren, Silin; Yang, Mingkun; Li, Yu; Zhang, Feng; Chen, Zhuo; Zhang, Jia; Yang, Guang; Yue, Yuewei; Li, Siting; Ge, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. PMID:27667718

  9. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation

    PubMed Central

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  10. Proteomic and phosphoproteomic analysis of renal cortex in a salt-load rat model of advanced kidney damage

    PubMed Central

    Jiang, Shaoling; He, Hanchang; Tan, Lishan; Wang, Liangliang; Su, Zhengxiu; Liu, Yufeng; Zhu, Hongguo; Zhang, Menghuan; Hou, Fan Fan; Li, Aiqing

    2016-01-01

    Salt plays an essential role in the progression of chronic kidney disease and hypertension. However, the mechanisms underlying pathogenesis of salt-induced kidney damage remain largely unknown. Here, Sprague-Dawley rats, that underwent 5/6 nephrectomy (5/6Nx, a model of advanced kidney damage) or sham operation, were treated for 2 weeks with a normal or high-salt diet. We employed aTiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for proteomic and phosphoproteomic profiling of the renal cortex. We found 318 proteins differentially expressed in 5/6Nx group relative to sham group, and 310 proteins significantly changed in response to salt load in 5/6Nx animals. Totally, 1810 unique phosphopeptides corresponding to 550 phosphoproteins were identified. We identified 113 upregulated and 84 downregulated phosphopeptides in 5/6Nx animals relative to sham animals. Salt load induced 78 upregulated and 91 downregulated phosphopeptides in 5/6Nx rats. The differentially expressed phospholproteins are important transporters, structural molecules, and receptors. Protein-protein interaction analysis revealed that the differentially phosphorylated proteins in 5/6Nx group, Polr2a, Srrm1, Gsta2 and Pxn were the most linked. Salt-induced differential phosphoproteins, Myh6, Lmna and Des were the most linked. Altered phosphorylation levels of lamin A and phospholamban were validated. This study will provide new insight into pathogenetic mechanisms of chronic kidney disease and salt sensitivity. PMID:27775022

  11. Enabling responsible public genomics.

    PubMed

    Conley, John M; Doerr, Adam K; Vorhaus, Daniel B

    2010-01-01

    As scientific understandings of genetics advance, researchers require increasingly rich datasets that combine genomic data from large numbers of individuals with medical and other personal information. Linking individuals' genetic data and personal information precludes anonymity and produces medically significant information--a result not contemplated by the established legal and ethical conventions governing human genomic research. To pursue the next generation of human genomic research and commerce in a responsible fashion, scientists, lawyers, and regulators must address substantial new issues, including researchers' duties with respect to clinically significant data, the challenges to privacy presented by genomic data, the boundary between genomic research and commerce, and the practice of medicine. This Article presents a new model for understanding and addressing these new challenges--a "public genomics" premised on the idea that ethically, legally, and socially responsible genomics research requires openness, not privacy, as its organizing principle. Responsible public genomics combines the data contributed by informed and fully consenting information altruists and the research potential of rich datasets in a genomic commons that is freely and globally available. This Article examines the risks and benefits of this public genomics model in the context of an ambitious genetic research project currently under way--the Personal Genome Project. This Article also (i) demonstrates that large-scale genomic projects are desirable, (ii) evaluates the risks and challenges presented by public genomics research, and (iii) determines that the current legal and regulatory regimes restrict beneficial and responsible scientific inquiry while failing to adequately protect participants. The Article concludes by proposing a modified normative and legal framework that embraces and enables a future of responsible public genomics.

  12. Enabling interstellar probe

    NASA Astrophysics Data System (ADS)

    McNutt, Ralph L.; Wimmer-Schweingruber, Robert F.; International Interstellar Probe Team

    2011-04-01

    The scientific community has advocated a scientific probe to the interstellar medium for over 30 years. While the Voyager spacecraft have passed through the termination shock of the solar wind, they have limited lifetimes as their radioisotope power supplies decay. It remains unclear whether they can reach the heliopause, the boundary between shocked solar wind and interstellar plasmas, and, in any case, they will not reach the undisturbed interstellar medium. As with most exploratory space missions, their ongoing observations continue to raise even more questions about the nature of the interaction of our heliosphere and the interstellar medium. Scientific questions including: What is the nature of the nearby interstellar medium? How do the Sun and galaxy affect the dynamics of the heliosphere? What is the structure of the heliosphere? How did matter in the solar system and interstellar medium originate and evolve? can only be answered by an "interstellar precursor" probe. Such a mission is required to make in situ measurements in the interaction region and interstellar medium itself at distances far from the Sun, but in a finite mission lifetime. By launching a probe toward the incoming "interstellar wind," whose direction is known, the distance to be traveled can be minimized but is still large. The current consensus is that a scientifically compelling mission must function to at least a distance of 200 astronomical units (AU) from the Sun and return a reasonable stream of data during the voyage. The central problem is that of providing a means of propulsion to accelerate a probe from the Solar System. Even with a low-mass payload and spacecraft, achieving the high speeds needed, even with gravity assists, have remained problematic. Voyager 1, the fastest object ever to leave the system is now traveling ˜3.6 AU/yr, and a credible probe must reach at least 2-3 times this speed. The use of an Ares V is an approach for enabling a fast interstellar precursor

  13. FOILFEST :community enabled security.

    SciTech Connect

    Moore, Judy Hennessey; Johnson, Curtis Martin; Whitley, John B.; Drayer, Darryl Donald; Cummings, John C., Jr.

    2005-09-01

    The Advanced Concepts Group of Sandia National Laboratories hosted a workshop, ''FOILFest: Community Enabled Security'', on July 18-21, 2005, in Albuquerque, NM. This was a far-reaching look into the future of physical protection consisting of a series of structured brainstorming sessions focused on preventing and foiling attacks on public places and soft targets such as airports, shopping malls, hotels, and public events. These facilities are difficult to protect using traditional security devices since they could easily be pushed out of business through the addition of arduous and expensive security measures. The idea behind this Fest was to explore how the public, which is vital to the function of these institutions, can be leveraged as part of a physical protection system. The workshop considered procedures, space design, and approaches for building community through technology. The workshop explored ways to make the ''good guys'' in public places feel safe and be vigilant while making potential perpetrators of harm feel exposed and convinced that they will not succeed. Participants in the Fest included operators of public places, social scientists, technology experts, representatives of government agencies including DHS and the intelligence community, writers and media experts. Many innovative ideas were explored during the fest with most of the time spent on airports, including consideration of the local airport, the Albuquerque Sunport. Some provocative ideas included: (1) sniffers installed in passage areas like revolving door, escalators, (2) a ''jumbotron'' showing current camera shots in the public space, (3) transparent portal screeners allowing viewing of the screening, (4) a layered open/funnel/open/funnel design where open spaces are used to encourage a sense of ''communitas'' and take advantage of citizen ''sensing'' and funnels are technological tunnels of sensors (the tunnels of truth), (5) curved benches with blast proof walls or backs, (6

  14. Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer

    PubMed Central

    2015-01-01

    As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides, corresponding to 11 000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628 000 binary comparisons collected in less than 48 h. PMID:25521595

  15. Discovery of Novel Virulence Factors of Biothreat Agents: Validation of the Phosphoproteome-Based Approach

    DTIC Science & Technology

    2007-06-01

    cell survival (1). However, AKT in response to LPS will phosphorylate glycogen synthase kinase beta . This phosphorylation leads to glycogen...status of multiple proteins simultaneously enables us to map the host cell response to infection with multiple strains and species of Francisella as...extracellular macromolecules, and host responses to individual proteins applied either extracellularly or intracellularly to the host cell . We will also

  16. Phosphoproteomic Analysis Reveals a Novel Mechanism of CaMKIIα Regulation Inversely Induced by Cocaine Memory Extinction versus Reconsolidation

    PubMed Central

    Rich, Matthew T.; Abbott, Thomas B.; Chung, Lisa; Gulcicek, Erol E.; Stone, Kathryn L.; Colangelo, Christopher M.; Lam, TuKiet T.; Nairn, Angus C.; Taylor, Jane R.

    2016-01-01

    Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance

  17. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  18. The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot Fraction Includes Key Proteins of the Light Signaling Pathway1[W

    PubMed Central

    Wagner, Volker; Ullmann, Katharina; Mollwo, Anne; Kaminski, Marc; Mittag, Maria; Kreimer, Georg

    2008-01-01

    Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca2+-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii. PMID:18065559

  19. Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture.

    PubMed

    Rinschen, Markus M; Pahmeyer, Caroline; Pisitkun, Trairak; Schnell, Nicole; Wu, Xiongwu; Maaß, Martina; Bartram, Malte P; Lamkemeyer, Tobias; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2015-04-01

    Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 (http://proteomecentral.proteomexchange.org/dataset/PXD001005).

  20. Quantitative research.

    PubMed

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  1. Relative quantification of phosphoproteomic changes in grapevine (Vitis vinifera L.) leaves in response to abscisic acid

    PubMed Central

    Rattanakan, Supakan; George, Iniga; Haynes, Paul A; Cramer, Grant R

    2016-01-01

    In a previous transcriptomic analysis, abscisic acid (ABA) was found to affect the abundance of a number of transcripts in leaves of Cabernet Sauvignon grapevines with roots that had been exposed to 10 μm ABA for 2 h. Other work has indicated that ABA affects protein abundance and protein phosphorylation as well. In this study we investigated changes in protein abundance and phosphorylation of Cabernet Sauvignon grapevine leaves. Protein abundance was assessed by both label-free and isobaric-label quantitive proteomic methods. Each identified common proteins, but also additional proteins not found with the other method. Overall, several thousand proteins were identified and several hundred were quantified. In addition, hundreds of phosphoproteins were identified. Tens of proteins were found to be affected in the leaf after the roots had been exposed to ABA for 2 h, more than half of them were phosphorylated proteins. Many phosphosites were confirmed and several new ones were identified. ABA increased the abundance of some proteins, but the majority of the proteins had their protein abundance decreased. Many of these proteins were involved in growth and plant organ development, including proteins involved in protein synthesis, photosynthesis, sugar and amino-acid metabolism. This study provides new insights into how ABA regulates plant responses and acclimation to water deficits. PMID:27366326

  2. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

    Abstract: In toxicology, the role of quantitative assessment of brain morphology can be understood in the context of two types of treatment-related alterations. One type of alteration is specifically associated with treatment and is not observed in control animals. Measurement ...

  3. Enabling Space Science and Exploration

    NASA Technical Reports Server (NTRS)

    Weber, William J.

    2006-01-01

    This viewgraph presentation on enabling space science and exploration covers the following topics: 1) Today s Deep Space Network; 2) Next Generation Deep Space Network; 3) Needed technologies; 4) Mission IT and networking; and 5) Multi-mission operations.

  4. Empowering versus Enabling in Academia.

    ERIC Educational Resources Information Center

    Espeland, Karen; Shanta, Linda

    2001-01-01

    Enabling behaviors that encourage dependence should be avoided by nursing faculty. An empowerment model that includes collegiality, communication, accountability, and autonomy is more suited to the professional preparation of nurses. (Contains 30 references.) (SK)

  5. Quantitative glycomics.

    PubMed

    Orlando, Ron

    2010-01-01

    The ability to quantitatively determine changes is an essential component of comparative glycomics. Multiple strategies are available by which this can be accomplished. These include label-free approaches and strategies where an isotopic label is incorporated into the glycans prior to analysis. The focus of this chapter is to describe each of these approaches while providing insight into their strengths and weaknesses, so that glycomic investigators can make an educated choice of the strategy that is best suited for their particular application.

  6. Comparative N-Glycoproteomic and Phosphoproteomic Profiling of Human Placental Plasma Membrane between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry

    PubMed Central

    Wang, Fuqiang; Wang, Ling; Shi, Zhonghua; Liang, Gaolin

    2013-01-01

    Preeclampsia is a serious complication of pregnancy, which affects 2–8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane. PMID:24260401

  7. Nanoplasmon-enabled macroscopic thermal management

    PubMed Central

    Jonsson, Gustav Edman; Miljkovic, Vladimir; Dmitriev, Alexandre

    2014-01-01

    In numerous applications of energy harvesting via transformation of light into heat the focus recently shifted towards highly absorptive nanoplasmonic materials. It is currently established that noble metals-based absorptive plasmonic platforms deliver significant light-capturing capability and can be viewed as super-absorbers of optical radiation. Naturally, approaches to the direct experimental probing of macroscopic temperature increase resulting from these absorbers are welcomed. Here we derive a general quantitative method of characterizing heat-generating properties of optically absorptive layers via macroscopic thermal imaging. We further monitor macroscopic areas that are homogeneously heated by several degrees with nanostructures that occupy a mere 8% of the surface, leaving it essentially transparent and evidencing significant heat generation capability of nanoplasmon-enabled light capture. This has a direct bearing to a large number of applications where thermal management is crucial. PMID:24870613

  8. Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17*

    PubMed Central

    Lai, Juo-Hsin; Yang, Jhih-Tian; Chern, Jeffy; Chen, Te-Li; Wu, Wan-Ling; Liao, Jiahn-Haur; Tsai, Shih-Feng; Liang, Suh-Yuen; Chou, Chi-Chi

    2016-01-01

    Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S88VS90K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. PMID:26499836

  9. Differences in Beef Quality between Angus (Bos taurus taurus) and Nellore (Bos taurus indicus) Cattle through a Proteomic and Phosphoproteomic Approach

    PubMed Central

    Chizzotti, Mario Luiz; Vital, Camilo Elber; Baracat-Pereira, Maria Cristina; Barros, Edvaldo; Busato, Karina Costa; Gomes, Rafael Aparecido; Ladeira, Márcio Machado; Martins, Taiane da Silva

    2017-01-01

    Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (P<0.05). Nellore had higher phosphorylation of myosin regulatory light chain-2, alpha actin-1, triosephosphate isomerase and 14-3-3 protein epsilon. However, Angus had greater phosphorylation of phosphoglucomutase-1 and troponin-T (P<0.05). Therefore, proteins involved in contraction and muscle organization, myofilaments expressed in fast or slow-twitch fibers and heat shock proteins localized in mitochondria or sarcoplasmic reticulum and involved in cell flux of calcium and apoptosis might be associated with differences in beef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force

  10. Secure Enclaves-Enabled Technologies

    DTIC Science & Technology

    2014-04-25

    solution. Recommendations There is the potential to exploit extremely lucrative opportunities utilizing our first- mover advantage in this...emerging market segment. However, there is still significant work to be completed. The SE Enabled browser extension application is still in the early

  11. Exogenous Attention Enables Perceptual Learning.

    PubMed

    Szpiro, Sarit F A; Carrasco, Marisa

    2015-12-01

    Practice can improve visual perception, and these improvements are considered to be a form of brain plasticity. Training-induced learning is time-consuming and requires hundreds of trials across multiple days. The process of learning acquisition is understudied. Can learning acquisition be potentiated by manipulating visual attentional cues? We developed a protocol in which we used task-irrelevant cues for between-groups manipulation of attention during training. We found that training with exogenous attention can enable the acquisition of learning. Remarkably, this learning was maintained even when observers were subsequently tested under neutral conditions, which indicates that a change in perception was involved. Our study is the first to isolate the effects of exogenous attention and to demonstrate its efficacy to enable learning. We propose that exogenous attention boosts perceptual learning by enhancing stimulus encoding.

  12. Echo-Enabled Harmonic Generation

    SciTech Connect

    Stupakov, Gennady; /SLAC

    2012-06-28

    A recently proposed concept of the Echo-Enabled Harmonic Generation (EEHG) FEL uses two laser modulators in combination with two dispersion sections to generate a high-harmonic density modulation in a relativistic beam. This seeding technique holds promise of a one-stage soft x-ray FEL that radiates not only transversely but also longitudinally coherent pulses. Currently, an experimental verification of the concept is being conducted at the SLAC National Accelerator Laboratory aimed at the demonstration of the EEHG.

  13. Technologies for Networked Enabled Operations

    NASA Technical Reports Server (NTRS)

    Glass, B.; Levine, J.

    2005-01-01

    Current point-to-point data links will not scale to support future integration of surveillance, security, and globally-distributed air traffic data, and already hinders efficiency and capacity. While the FAA and industry focus on a transition to initial system-wide information management (SWIM) capabilities, this paper describes a set of initial studies of NAS network-enabled operations technology gaps targeted for maturity in later SWIM spirals (201 5-2020 timeframe).

  14. Nanofluidics: enabling processes for biotech

    NASA Astrophysics Data System (ADS)

    Ulmanella, Umberto; Ho, Chih-Ming

    2001-10-01

    The advance of micro and nanodevice manufacturing technology enables us to carry out biological and chemical processes in a more efficient manner. In fact, fluidic processes connect the macro and the micro/nano worlds. For devices approaching the size of the fluid molecules, many physical phenomena occur that are not observed in macro flows. In this brief review, we discuss a few selected topics which of are interest for basic research and are important for applications in biotechnology.

  15. New Generation Sensor Web Enablement

    PubMed Central

    Bröring, Arne; Echterhoff, Johannes; Jirka, Simon; Simonis, Ingo; Everding, Thomas; Stasch, Christoph; Liang, Steve; Lemmens, Rob

    2011-01-01

    Many sensor networks have been deployed to monitor Earth’s environment, and more will follow in the future. Environmental sensors have improved continuously by becoming smaller, cheaper, and more intelligent. Due to the large number of sensor manufacturers and differing accompanying protocols, integrating diverse sensors into observation systems is not straightforward. A coherent infrastructure is needed to treat sensors in an interoperable, platform-independent and uniform way. The concept of the Sensor Web reflects such a kind of infrastructure for sharing, finding, and accessing sensors and their data across different applications. It hides the heterogeneous sensor hardware and communication protocols from the applications built on top of it. The Sensor Web Enablement initiative of the Open Geospatial Consortium standardizes web service interfaces and data encodings which can be used as building blocks for a Sensor Web. This article illustrates and analyzes the recent developments of the new generation of the Sensor Web Enablement specification framework. Further, we relate the Sensor Web to other emerging concepts such as the Web of Things and point out challenges and resulting future work topics for research on Sensor Web Enablement. PMID:22163760

  16. 'Ethos' Enabling Organisational Knowledge Creation

    NASA Astrophysics Data System (ADS)

    Matsudaira, Yoshito

    This paper examines knowledge creation in relation to improvements on the production line in the manufacturing department of Nissan Motor Company and aims to clarify embodied knowledge observed in the actions of organisational members who enable knowledge creation will be clarified. For that purpose, this study adopts an approach that adds a first, second, and third-person's viewpoint to the theory of knowledge creation. Embodied knowledge, observed in the actions of organisational members who enable knowledge creation, is the continued practice of 'ethos' (in Greek) founded in Nissan Production Way as an ethical basis. Ethos is knowledge (intangible) assets for knowledge creating companies. Substantiated analysis classifies ethos into three categories: the individual, team and organisation. This indicates the precise actions of the organisational members in each category during the knowledge creation process. This research will be successful in its role of showing the indispensability of ethos - the new concept of knowledge assets, which enables knowledge creation -for future knowledge-based management in the knowledge society.

  17. Enablement as a Positive Force in Counselling.

    ERIC Educational Resources Information Center

    Stamler, Lynnette Leeseberg

    1996-01-01

    Explores the concept of enablement and provides a framework for the counselor and counselor educator to use in facilitating client- or student-directed goals. Examines use of enablement, enablement versus co-dependency, the use of the enablement framework in the counseling role, and clinical examples of the enablement framework. (RJM)

  18. Noise-enabled optical ratchets

    PubMed Central

    León-Montiel, Roberto de J.; Quinto-Su, Pedro A.

    2017-01-01

    In this contribution, we report on the implementation of a novel noise-enabled optical ratchet system. We demonstrate that, unlike commonly-used ratchet schemes—where complex asymmetric optical potentials are needed—efficient transport of microparticles across a one-dimensional optical lattice can be produced by introducing controllable noise in the system. This work might open interesting routes towards the development of new technologies aimed at enhancing the efficiency of transport occurring at the micro- and nanoscale, from novel particle-sorting tools to efficient molecular motors. PMID:28287152

  19. Optimized microsystems-enabled photovoltaics

    DOEpatents

    Cruz-Campa, Jose Luis; Nielson, Gregory N.; Young, Ralph W.; Resnick, Paul J.; Okandan, Murat; Gupta, Vipin P.

    2015-09-22

    Technologies pertaining to designing microsystems-enabled photovoltaic (MEPV) cells are described herein. A first restriction for a first parameter of an MEPV cell is received. Subsequently, a selection of a second parameter of the MEPV cell is received. Values for a plurality of parameters of the MEPV cell are computed such that the MEPV cell is optimized with respect to the second parameter, wherein the values for the plurality of parameters are computed based at least in part upon the restriction for the first parameter.

  20. Noise-enabled optical ratchets

    NASA Astrophysics Data System (ADS)

    León-Montiel, Roberto De J.; Quinto-Su, Pedro A.

    2017-03-01

    In this contribution, we report on the implementation of a novel noise-enabled optical ratchet system. We demonstrate that, unlike commonly-used ratchet schemes—where complex asymmetric optical potentials are needed—efficient transport of microparticles across a one-dimensional optical lattice can be produced by introducing controllable noise in the system. This work might open interesting routes towards the development of new technologies aimed at enhancing the efficiency of transport occurring at the micro- and nanoscale, from novel particle-sorting tools to efficient molecular motors.

  1. Autonomy enables new science missions

    NASA Astrophysics Data System (ADS)

    Doyle, Richard J.; Gor, Victoria; Man, Guy K.; Stolorz, Paul E.; Chapman, Clark; Merline, William J.; Stern, Alan

    1997-01-01

    The challenge of space flight in NASA's future is to enable smaller, more frequent and intensive space exploration at much lower total cost without substantially decreasing mission reliability, capability, or the scientific return on investment. The most effective way to achieve this goal is to build intelligent capabilities into the spacecraft themselves. Our technological vision for meeting the challenge of returning quality science through limited communication bandwidth will actually put scientists in a more direct link with the spacecraft than they have enjoyed to date. Technologies such as pattern recognition and machine learning can place a part of the scientist's awareness onboard the spacecraft to prioritize downlink or to autonomously trigger time-critical follow-up observations-particularly important in flyby missions-without ground interaction. Onboard knowledge discovery methods can be used to include candidate discoveries in each downlink for scientists' scrutiny. Such capabilities will allow scientists to quickly reprioritize missions in a much more intimate and efficient manner than is possible today. Ultimately, new classes of exploration missions will be enabled.

  2. Nanomaterial-Enabled Neural Stimulation

    PubMed Central

    Wang, Yongchen; Guo, Liang

    2016-01-01

    Neural stimulation is a critical technique in treating neurological diseases and investigating brain functions. Traditional electrical stimulation uses electrodes to directly create intervening electric fields in the immediate vicinity of neural tissues. Second-generation stimulation techniques directly use light, magnetic fields or ultrasound in a non-contact manner. An emerging generation of non- or minimally invasive neural stimulation techniques is enabled by nanotechnology to achieve a high spatial resolution and cell-type specificity. In these techniques, a nanomaterial converts a remotely transmitted primary stimulus such as a light, magnetic or ultrasonic signal to a localized secondary stimulus such as an electric field or heat to stimulate neurons. The ease of surface modification and bio-conjugation of nanomaterials facilitates cell-type-specific targeting, designated placement and highly localized membrane activation. This review focuses on nanomaterial-enabled neural stimulation techniques primarily involving opto-electric, opto-thermal, magneto-electric, magneto-thermal and acousto-electric transduction mechanisms. Stimulation techniques based on other possible transduction schemes and general consideration for these emerging neurotechnologies are also discussed. PMID:27013938

  3. Enabling Exploration Through Docking Standards

    NASA Technical Reports Server (NTRS)

    Hatfield, Caris A.

    2012-01-01

    Human exploration missions beyond low earth orbit will likely require international cooperation in order to leverage limited resources. International standards can help enable cooperative missions by providing well understood, predefined interfaces allowing compatibility between unique spacecraft and systems. The International Space Station (ISS) partnership has developed a publicly available International Docking System Standard (IDSS) that provides a solution to one of these key interfaces by defining a common docking interface. The docking interface provides a way for even dissimilar spacecraft to dock for exchange of crew and cargo, as well as enabling the assembly of large space systems. This paper provides an overview of the key attributes of the IDSS, an overview of the NASA Docking System (NDS), and the plans for updating the ISS with IDSS compatible interfaces. The NDS provides a state of the art, low impact docking system that will initially be made available to commercial crew and cargo providers. The ISS will be used to demonstrate the operational utility of the IDSS interface as a foundational technology for cooperative exploration.

  4. Directory Enabled Policy Based Networking

    SciTech Connect

    KELIIAA, CURTIS M.

    2001-10-01

    This report presents a discussion of directory-enabled policy-based networking with an emphasis on its role as the foundation for securely scalable enterprise networks. A directory service provides the object-oriented logical environment for interactive cyber-policy implementation. Cyber-policy implementation includes security, network management, operational process and quality of service policies. The leading network-technology vendors have invested in these technologies for secure universal connectivity that transverses Internet, extranet and intranet boundaries. Industry standards are established that provide the fundamental guidelines for directory deployment scalable to global networks. The integration of policy-based networking with directory-service technologies provides for intelligent management of the enterprise network environment as an end-to-end system of related clients, services and resources. This architecture allows logical policies to protect data, manage security and provision critical network services permitting a proactive defense-in-depth cyber-security posture. Enterprise networking imposes the consideration of supporting multiple computing platforms, sites and business-operation models. An industry-standards based approach combined with principled systems engineering in the deployment of these technologies allows these issues to be successfully addressed. This discussion is focused on a directory-based policy architecture for the heterogeneous enterprise network-computing environment and does not propose specific vendor solutions. This document is written to present practical design methodology and provide an understanding of the risks, complexities and most important, the benefits of directory-enabled policy-based networking.

  5. Web-Enabled Design Review and Lessons Learned

    DTIC Science & Technology

    1998-04-01

    Additional information regarding cookie variables may be found through any WWW search engine . Computer Hardware Resource Utilization The extent to...i:. US Army Corps of Engineers Construction Engineering Research Laboratories USACERL Technical Report 98/31 April 1998 Web-Enabled Design...Information source, design data or procedure, management procedure, source of ideas, etc.) 3. Has the information in this report led to any quantitative

  6. Spatially Enabling the Health Sector

    PubMed Central

    Weeramanthri, Tarun Stephen; Woodgate, Peter

    2016-01-01

    Spatial information describes the physical location of either people or objects, and the measured relationships between them. In this article, we offer the view that greater utilization of spatial information and its related technology, as part of a broader redesign of the architecture of health information at local and national levels, could assist and speed up the process of health reform, which is taking place across the globe in richer and poorer countries alike. In making this point, we describe the impetus for health sector reform, recent developments in spatial information and analytics, and current Australasian spatial health research. We highlight examples of uptake of spatial information by the health sector, as well as missed opportunities. Our recommendations to spatially enable the health sector are applicable to high- and low-resource settings. PMID:27867933

  7. Simulation Enabled Safeguards Assessment Methodology

    SciTech Connect

    Robert Bean; Trond Bjornard; Thomas Larson

    2007-09-01

    It is expected that nuclear energy will be a significant component of future supplies. New facilities, operating under a strengthened international nonproliferation regime will be needed. There is good reason to believe virtual engineering applied to the facility design, as well as to the safeguards system design will reduce total project cost and improve efficiency in the design cycle. Simulation Enabled Safeguards Assessment MEthodology (SESAME) has been developed as a software package to provide this capability for nuclear reprocessing facilities. The software architecture is specifically designed for distributed computing, collaborative design efforts, and modular construction to allow step improvements in functionality. Drag and drop wireframe construction allows the user to select the desired components from a component warehouse, render the system for 3D visualization, and, linked to a set of physics libraries and/or computational codes, conduct process evaluations of the system they have designed.

  8. Context-Enabled Business Intelligence

    SciTech Connect

    Troy Hiltbrand

    2012-04-01

    To truly understand context and apply it in business intelligence, it is vital to understand what context is and how it can be applied in addressing organizational needs. Context describes the facets of the environment that impact the way that end users interact with the system. Context includes aspects of location, chronology, access method, demographics, social influence/ relationships, end-user attitude/ emotional state, behavior/ past behavior, and presence. To be successful in making Business Intelligence content enabled, it is important to be able to capture the context of use user. With advances in technology, there are a number of ways in which this user based information can be gathered and exposed to enhance the overall end user experience.

  9. Enabling the Kepler Exoplanet Census

    NASA Astrophysics Data System (ADS)

    Morton, Tim

    2013-01-01

    The Kepler mission, with its unrivaled photometric precision and nearly continuous monitoring, provides an unprecedented opportunity for an exoplanet census. However, while it has identified thousands of transiting planet candidates, only relatively few have yet been dynamically confirmed as bona fide planets, with only a handful more even conceivably amenable to future dynamical confirmation. As a result, the ability to draw detailed conclusions about the diversity of exoplanet systems from Kepler detections relies critically on assigning false positive probabilities to thousands of unconfirmed candidates. I have developed a procedure to calculate these probabilities using only the Kepler photometry, optionally including available follow-up observations. Using this, I can often statistically validate a candidate signal using two single-epoch observations: a stellar spectrum and a high-resolution image. Accordingly, I have applied this procedure to the publicly released Kepler Objects of Interest (KOIs) using results from several large follow-up campaigns: spectroscopic surveys of >100 KOIs with TripleSpec at the Palomar 200-in and >700 KOIs with Keck/HIRES, and imaging surveys of >60 KOIs with Keck/NIRC2 and nearly 1000 KOIs with the Palomar Robo-AO system. This has enabled me to identify hundreds of Kepler candidates as newly secure planets, identify some as likely false positives, and prescribe the most useful follow-up observations for the rest of the KOIs. These results will enable Kepler to fulfill its promise as a mission to study the statistics of exoplanet systems. In addition, this new approach to transit survey follow-up---detailed probabilistic assessment of large numbers of candidates in order to inform the application of relatively scarce follow-up resources---will remain fruitful as transit surveys continue to produce many more candidates than can be followed up with traditional strategies.

  10. Imaging enabled platforms for development of therapeutics

    NASA Astrophysics Data System (ADS)

    Celli, Jonathan; Rizvi, Imran; Blanden, Adam R.; Evans, Conor L.; Abu-Yousif, Adnan O.; Spring, Bryan Q.; Muzikansky, Alona; Pogue, Brian W.; Finkelstein, Dianne M.; Hasan, Tayyaba

    2011-03-01

    Advances in imaging and spectroscopic technologies have enabled the optimization of many therapeutic modalities in cancer and noncancer pathologies either by earlier disease detection or by allowing therapy monitoring. Amongst the therapeutic options benefiting from developments in imaging technologies, photodynamic therapy (PDT) is exceptional. PDT is a photochemistry-based therapeutic approach where a light-sensitive molecule (photosensitizer) is activated with light of appropriate energy (wavelength) to produce reactive molecular species such as free radicals and singlet oxygen. These molecular entities then react with biological targets such as DNA, membranes and other cellular components to impair their function and lead to eventual cell and tissue death. Development of PDT-based imaging also provides a platform for rapid screening of new therapeutics in novel in vitro models prior to expensive and labor-intensive animal studies. In this study we demonstrate how an imaging platform can be used for strategizing a novel combination treatment strategy for multifocal ovarian cancer. Using an in vitro 3D model for micrometastatic ovarian cancer in conjunction with quantitative imaging we examine dose and scheduling strategies for PDT in combination with carboplatin, a chemotherapeutic agent presently in clinical use for management of this deadly form of cancer.

  11. Enabling Participation In Exoplanet Science

    NASA Astrophysics Data System (ADS)

    Taylor, Stuart F.

    2015-08-01

    Determining the distribution of exoplanets has required the contributions of a community of astronomers, who all require the support of colleagues to finish their projects in a manner to enable them to enter new collaborations to continue to contribute to understanding exoplanet science.The contributions of each member of the astronomy community are to be encouraged and must never be intentionally obstructed.We present a member’s long pursuit to be a contributing part of the exoplanet community through doing transit photometry as a means of commissioning the telescopes for a new observatory, followed by pursuit of interpreting the distributions in exoplanet parameter data.We present how the photometry projects have been presented as successful by the others who have claimed to have completed them, but how by requiring its employees to present results while omitting one member has been obstructive against members working together and has prevented the results from being published in what can genuinely be called a peer-reviewed fashion.We present how by tolerating one group to obstruct one member from finishing participation and then falsely denying credit is counterproductive to doing science.We show how expecting one member to attempt to go around an ostracizing group by starting something different is destructive to the entire profession. We repeat previously published appeals to help ostracized members to “go around the observatory” by calling for discussion on how the community must act to reverse cases of shunning, bullying, and other abuses. Without better recourse and support from the community, actions that do not meet standard good collegial behavior end up forcing good members from the community. The most important actions are to enable an ostracized member to have recourse to participating in group papers by either working through other authors or through the journal. All journals and authors must expect that no co-author is keeping out a major

  12. Enabling technology for human collaboration.

    SciTech Connect

    Murphy, Tim Andrew; Jones, Wendell Bruce; Warner, David Jay; Doser, Adele Beatrice; Johnson, Curtis Martin; Merkle, Peter Benedict

    2003-11-01

    This report summarizes the results of a five-month LDRD late start project which explored the potential of enabling technology to improve the performance of small groups. The purpose was to investigate and develop new methods to assist groups working in high consequence, high stress, ambiguous and time critical situations, especially those for which it is impractical to adequately train or prepare. A testbed was constructed for exploratory analysis of a small group engaged in tasks with high cognitive and communication performance requirements. The system consisted of five computer stations, four with special devices equipped to collect physiologic, somatic, audio and video data. Test subjects were recruited and engaged in a cooperative video game. Each team member was provided with a sensor array for physiologic and somatic data collection while playing the video game. We explored the potential for real-time signal analysis to provide information that enables emergent and desirable group behavior and improved task performance. The data collected in this study included audio, video, game scores, physiological, somatic, keystroke, and mouse movement data. The use of self-organizing maps (SOMs) was explored to search for emergent trends in the physiological data as it correlated with the video, audio and game scores. This exploration resulted in the development of two approaches for analysis, to be used concurrently, an individual SOM and a group SOM. The individual SOM was trained using the unique data of each person, and was used to monitor the effectiveness and stress level of each member of the group. The group SOM was trained using the data of the entire group, and was used to monitor the group effectiveness and dynamics. Results suggested that both types of SOMs were required to adequately track evolutions and shifts in group effectiveness. Four subjects were used in the data collection and development of these tools. This report documents a proof of concept

  13. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  14. Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration.

    PubMed

    Chen, Yanling; Lu, Bingwen; Yang, Qingkai; Fearns, Colleen; Yates, John R; Lee, Jiing-Dwan

    2009-04-15

    Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.

  15. Classic Ras Proteins Promote Proliferation and Survival Via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells

    PubMed Central

    Brossier, Nicole M.; Prechtl, Amanda M.; Longo, Jody Fromm; Barnes, Stephen; Wilson, Landon S.; Byer, Stephanie J.; Brosius, Stephanie N.; Carroll, Steven L.

    2015-01-01

    Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras and K-Ras are coexpressed with their activators, (guanine nucleotide exchange factors), in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades. PMID:25946318

  16. Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2α Kinase Levels in NRAS(Q61) Mutant Cells.

    PubMed

    Posch, Christian; Sanlorenzo, Martina; Vujic, Igor; Oses-Prieto, Juan A; Cholewa, Brian D; Kim, Sarasa T; Ma, Jeffrey; Lai, Kevin; Zekhtser, Mitchell; Esteve-Puig, Rosaura; Green, Gary; Chand, Shreya; Burlingame, Alma L; Panzer-Grümayer, Renate; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-10-01

    In melanoma, mutant and thereby constantly active neuroblastoma rat sarcoma (NRAS) affects 15-20% of tumors, contributing to tumor initiation, growth, invasion, and metastasis. Recent therapeutic approaches aim to mimic RAS extinction by interfering with critical signaling pathways downstream of the mutant protein. This study investigates the phosphoproteome of primary human melanocytes bearing mutations in the two hot spots of NRAS, NRAS(G12) and NRAS(Q61). Stable isotope labeling by amino acids in cell culture followed by mass spectrometry identified 14,155 spectra of 3,371 unique phosphopeptides mapping to 1,159 proteins (false discovery rate < 2%). Data revealed pronounced PI3K/AKT signaling in NRAS(G12V) mutant cells and pronounced mitogen-activated protein kinase (MAPK) signaling in NRAS(Q61L) variants. Computer-based prediction models for kinases involved, revealed that CK2α is significantly overrepresented in primary human melanocytes bearing NRAS(Q61L) mutations. Similar differences were found in human NRAS(Q61) mutant melanoma cell lines that were also more sensitive to pharmacologic CK2α inhibition compared with NRAS(G12) mutant cells. Furthermore, CK2α levels were pronounced in patient samples of NRAS(Q61) mutant melanoma at the mRNA and protein level. The preclinical findings of this study reveal that codon 12 and 61 mutant NRAS cells have distinct signaling characteristics that could allow for the development of more effective, mutation-specific treatment modalities.

  17. Quantitative mass spectrometry: an overview

    NASA Astrophysics Data System (ADS)

    Urban, Pawel L.

    2016-10-01

    Mass spectrometry (MS) is a mainstream chemical analysis technique in the twenty-first century. It has contributed to numerous discoveries in chemistry, physics and biochemistry. Hundreds of research laboratories scattered all over the world use MS every day to investigate fundamental phenomena on the molecular level. MS is also widely used by industry-especially in drug discovery, quality control and food safety protocols. In some cases, mass spectrometers are indispensable and irreplaceable by any other metrological tools. The uniqueness of MS is due to the fact that it enables direct identification of molecules based on the mass-to-charge ratios as well as fragmentation patterns. Thus, for several decades now, MS has been used in qualitative chemical analysis. To address the pressing need for quantitative molecular measurements, a number of laboratories focused on technological and methodological improvements that could render MS a fully quantitative metrological platform. In this theme issue, the experts working for some of those laboratories share their knowledge and enthusiasm about quantitative MS. I hope this theme issue will benefit readers, and foster fundamental and applied research based on quantitative MS measurements. This article is part of the themed issue 'Quantitative mass spectrometry'.

  18. Quantitative mass spectrometry: an overview

    PubMed Central

    2016-01-01

    Mass spectrometry (MS) is a mainstream chemical analysis technique in the twenty-first century. It has contributed to numerous discoveries in chemistry, physics and biochemistry. Hundreds of research laboratories scattered all over the world use MS every day to investigate fundamental phenomena on the molecular level. MS is also widely used by industry—especially in drug discovery, quality control and food safety protocols. In some cases, mass spectrometers are indispensable and irreplaceable by any other metrological tools. The uniqueness of MS is due to the fact that it enables direct identification of molecules based on the mass-to-charge ratios as well as fragmentation patterns. Thus, for several decades now, MS has been used in qualitative chemical analysis. To address the pressing need for quantitative molecular measurements, a number of laboratories focused on technological and methodological improvements that could render MS a fully quantitative metrological platform. In this theme issue, the experts working for some of those laboratories share their knowledge and enthusiasm about quantitative MS. I hope this theme issue will benefit readers, and foster fundamental and applied research based on quantitative MS measurements. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644965

  19. Quantitative NIR chemical imaging in heritage science.

    PubMed

    Cséfalvayová, Linda; Strlič, Matija; Karjalainen, Harri

    2011-07-01

    Until recently, applications of spectral imaging in heritage science mostly focused on qualitative examination of artworks. This is partly due to the complexity of artworks and partly due to the lack of appropriate standard materials. With the recent advance of NIR imaging spectrometers, the interval 1000-2500 nm became available for exploration, enabling us to extract quantitative chemical information from artworks. In this contribution, the development of 2D NIR quantitative chemical maps of heritage objects is discussed along with presentation of the first quantitative image. Further case studies include semiquantitative mapping of plasticiser distribution in a plastic object and identification of historic plastic materials. In the NIR imaging studies discussed, sets of 256 spatially registered images were collected at different wavelengths in the NIR region of 1000-2500 nm. The data was analyzed as a spectral cube, both as a stack of wavelength-resolved images and as a series of spectra, one per each sample pixel, using multivariate analysis. This approach is only possible using well-characterized reference sample collections, as quantitative imaging applications need to be developed, thus enabling spatial maps of damaged and degraded areas to be visualized to a level of chemical detail previously not possible. Such quantitative chemical mapping of vulnerable areas of heritage objects is invaluable, as it enables damage to historic objects to be quantitatively visualized.

  20. CUDA Enabled Graph Subset Examiner

    SciTech Connect

    Johnston, Jeremy T.

    2016-12-22

    Finding Godsil-McKay switching sets in graphs is one way to demonstrate that a specific graph is not determined by its spectrum--the eigenvalues of its adjacency matrix. An important area of active research in pure mathematics is determining which graphs are determined by their spectra, i.e. when the spectrum of the adjacency matrix uniquely determines the underlying graph. We are interested in exploring the spectra of graphs in the Johnson scheme and specifically seek to determine which of these graphs are determined by their spectra. Given a graph G, a Godsil-McKay switching set is an induced subgraph H on 2k vertices with the following properties: I) H is regular, ii) every vertex in G/H is adjacent to either 0, k, or 2k vertices of H, and iii) at least one vertex in G/H is adjacent to k vertices in H. The software package examines each subset of a user specified size to determine whether or not it satisfies those 3 conditions. The software makes use of the massive parallel processing power of CUDA enabled GPUs. It also exploits the vertex transitivity of graphs in the Johnson scheme by reasoning that if G has a Godsil-McKay switching set, then it has a switching set which includes vertex 1. While the code (in its current state) is tuned to this specific problem, the method of examining each induced subgraph of G can be easily re-written to check for any user specified conditions on the subgraphs and can therefore be used much more broadly.

  1. Solar Glitter -- Microsystems Enabled Photovoltaics

    NASA Astrophysics Data System (ADS)

    Nielson, Gregory N.

    2012-02-01

    Many products have significantly benefitted from, or been enabled by, the ability to manufacture structures at an ever decreasing length scale. Obvious examples of this include integrated circuits, flat panel displays, micro-scale sensors, and LED lighting. These industries have benefited from length scale effects in terms of improved performance, reduced cost, or new functionality (or a combination of these). In a similar manner, we are working to take advantage of length scale effects that exist within solar photovoltaic (PV) systems. While this is a significant step away from traditional approaches to solar power systems, the benefits in terms of new functionality, improved performance, and reduced cost for solar power are compelling. We are exploring scale effects that result from the size of the solar cells within the system. We have developed unique cells of both crystalline silicon and III-V materials that are very thin (5-20 microns thick) and have very small lateral dimensions (on the order of hundreds of microns across). These cells minimize the amount of expensive semiconductor material required for the system, allow improved cell performance, and provide an expanded design space for both module and system concepts allowing optimized power output and reduced module and balance of system costs. Furthermore, the small size of the cells allows for unique high-efficiency, high-flexibility PV panels and new building-integrated PV options that are currently unavailable. These benefits provide a pathway for PV power to become cost competitive with grid power and allow unique power solutions independent of grid power.

  2. Enabling individualized therapy through nanotechnology

    PubMed Central

    Sakamoto, Jason H.; van de Ven, Anne L.; Godin, Biana; Blanco, Elvin; Serda, Rita E.; Grattoni, Alessandro; Ziemys, Arturas; Bouamrani, Ali; Hu, Tony; Ranganathan, Shivakumar I.; De Rosa, Enrica; Martinez, Jonathan O.; Smid, Christine A.; Buchanan, Rachel M.; Lee, Sei-Young; Srinivasan, Srimeenakshi; Landry, Matthew; Meyn, Anne; Tasciotti, Ennio; Liu, Xuewu; Decuzzi, Paolo; Ferrari, Mauro

    2010-01-01

    Individualized medicine is the healthcare strategy that rebukes the idiomatic dogma of ‘losing sight of the forest for the trees’. We are entering a new era of healthcare where it is no longer acceptable to develop and market a drug that is effective for only 80% of the patient population. The emergence of “-omic” technologies (e.g. genomics, transcriptomics, proteomics, metabolomics) and advances in systems biology are magnifying the deficiencies of standardized therapy, which often provide little treatment latitude for accommodating patient physiologic idiosyncrasies. A personalized approach to medicine is not a novel concept. Ever since the scientific community began unraveling the mysteries of the genome, the promise of discarding generic treatment regimens in favor of patient-specific therapies became more feasible and realistic. One of the major scientific impediments of this movement towards personalized medicine has been the need for technological enablement. Nanotechnology is projected to play a critical role in patient-specific therapy; however, this transition will depend heavily upon the evolutionary development of a systems biology approach to clinical medicine based upon “-omic” technology analysis and integration. This manuscript provides a forward looking assessment of the promise of nanomedicine as it pertains to individualized medicine and establishes a technology “snapshot” of the current state of nano-based products over a vast array of clinical indications and range of patient specificity. Other issues such as market driven hurdles and regulatory compliance reform are anticipated to “self-correct” in accordance to scientific advancement and healthcare demand. These peripheral, non-scientific concerns are not addressed at length in this manuscript; however they do exist, and their impact to the paradigm shifting healthcare transformation towards individualized medicine will be critical for its success. PMID:20045055

  3. Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex

    PubMed Central

    McClatchy, Daniel B.; Savas, Jeffrey N.; Martínez-Bartolomé, Salvador; Park, Sung Kyu; Maher, Pamela; Powell, Susan B.; Yates, John R.

    2015-01-01

    Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP down-regulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this dataset identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating. PMID:25869802

  4. Global quantitative analysis of phosphorylation underlying phencyclidine signaling and sensorimotor gating in the prefrontal cortex.

    PubMed

    McClatchy, D B; Savas, J N; Martínez-Bartolomé, S; Park, S K; Maher, P; Powell, S B; Yates, J R

    2016-02-01

    Prepulse inhibition (PPI) is an example of sensorimotor gating and deficits in PPI have been demonstrated in schizophrenia patients. Phencyclidine (PCP) suppression of PPI in animals has been studied to elucidate the pathological elements of schizophrenia. However, the molecular mechanisms underlying PCP treatment or PPI in the brain are still poorly understood. In this study, quantitative phosphoproteomic analysis was performed on the prefrontal cortex from rats that were subjected to PPI after being systemically injected with PCP or saline. PCP downregulated phosphorylation events were significantly enriched in proteins associated with long-term potentiation (LTP). Importantly, this data set identifies functionally novel phosphorylation sites on known LTP-associated signaling molecules. In addition, mutagenesis of a significantly altered phosphorylation site on xCT (SLC7A11), the light chain of system xc-, the cystine/glutamate antiporter, suggests that PCP also regulates the activity of this protein. Finally, new insights were also derived on PPI signaling independent of PCP treatment. This is the first quantitative phosphorylation proteomic analysis providing new molecular insights into sensorimotor gating.

  5. Quantitative multiplexed quantum dot immunohistochemistry

    SciTech Connect

    Sweeney, E.; Ward, T.H.; Gray, N.; Womack, C.; Jayson, G.; Hughes, A.; Dive, C.; Byers, R.

    2008-09-19

    Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8 h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

  6. EBprot: Statistical analysis of labeling-based quantitative proteomics data.

    PubMed

    Koh, Hiromi W L; Swa, Hannah L F; Fermin, Damian; Ler, Siok Ghee; Gunaratne, Jayantha; Choi, Hyungwon

    2015-08-01

    Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/).

  7. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana.

    PubMed

    Wang, Zhi-Kang; Wang, Jie; Liu, Jing; Ying, Sheng-Hua; Peng, Xiao-Jun; Feng, Ming-Guang

    2016-01-01

    Cdc14 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in Beauveria bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdc14 in the signaling network of B. bassiana by analyzing the Cdc14-specific proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions, 154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H2O2 (3 mM) and Congo red (0.15 mg/ml) resulted in the upregulation / downregulation of 23/63, 41/39 and 79/79 proteins and of 127/112, 52/47 and 105/226 phosphorylation sites in 85/92, 45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdc14 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdc14 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways. Fourteen conserved and novel motifs were identified in the fungal phosphorylation events.

  8. Proteomic and Phosphoproteomic Insights into a Signaling Hub Role for Cdc14 in Asexual Development and Multiple Stress Responses in Beauveria bassiana

    PubMed Central

    Wang, Zhi-Kang; Wang, Jie; Liu, Jing; Ying, Sheng-Hua; Peng, Xiao-Jun; Feng, Ming-Guang

    2016-01-01

    Cdc14 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phosphothreonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in Beauveria bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdc14 in the signaling network of B. bassiana by analyzing the Cdc14-specific proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions, 154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H2O2 (3 mM) and Congo red (0.15 mg/ml) resulted in the upregulation / downregulation of 23/63, 41/39 and 79/79 proteins and of 127/112, 52/47 and 105/226 phosphorylation sites in 85/92, 45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdc14 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdc14 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways. Fourteen conserved and novel motifs were identified in the fungal phosphorylation events. PMID:27055109

  9. Phosphoproteomic identification of targets of the Arabidopsis sucrose nonfermenting-like kinase SnRK2.8 reveals a connection to metabolic processes

    PubMed Central

    Shin, Ryoung; Alvarez, Sophie; Burch, Adrien Y.; Jez, Joseph M.; Schachtman, Daniel P.

    2007-01-01

    SnRK2.8 is a member of the sucrose nonfermenting-related kinase family that is down-regulated when plants are deprived of nutrients and growth is reduced. When this kinase is over expressed in Arabidopsis, the plants grow larger. To understand how this kinase modulates growth, we identified some of the proteins that are phosphorylated by this kinase. A new phosphoproteomic method was used in which total protein from plants overexpressing the kinase was compared with total protein from plants in which the kinase was inactivated. Protein profiles were compared on two-dimensional gels following staining by a dye that recognizes phosphorylated amino acids. Candidate target proteins were confirmed with in vitro phosphorylation assays, using the kinase and target proteins that were purified from Escherichia coli. Seven target proteins were confirmed as being phosphorylated by SnRK2.8. Certain targets, such as 14-3-3 proteins, regulate as yet unidentified proteins, whereas other targets, such as glyoxalase I and ribose 5-phosphate isomerase, detoxify byproducts from glycolysis and catalyze one of the final steps in carbon fixation, respectively. Also, adenosine kinase and 60S ribosomal protein were confirmed as targets of SnRK2.8. Using mass spectrometry, we identified phosphorylated residues in the SnRK2.8, the 14-3-3κ, and the 14-3-3χ. These data show that the expression of SnRK2.8 is correlated with plant growth, which may in part be due to the phosphorylation of enzymes involved in metabolic processes. PMID:17404219

  10. An Internet enabled impact limiter material database

    SciTech Connect

    Wix, S.; Kanipe, F.; McMurtry, W.

    1998-09-01

    This paper presents a detailed explanation of the construction of an interest enabled database, also known as a database driven web site. The data contained in the internet enabled database are impact limiter material and seal properties. The technique used in constructing the internet enabled database presented in this paper are applicable when information that is changing in content needs to be disseminated to a wide audience.

  11. Rigour in quantitative research.

    PubMed

    Claydon, Leica Sarah

    2015-07-22

    This article which forms part of the research series addresses scientific rigour in quantitative research. It explores the basis and use of quantitative research and the nature of scientific rigour. It examines how the reader may determine whether quantitative research results are accurate, the questions that should be asked to determine accuracy and the checklists that may be used in this process. Quantitative research has advantages in nursing, since it can provide numerical data to help answer questions encountered in everyday practice.

  12. Commentary: Academic Enablers and School Learning.

    ERIC Educational Resources Information Center

    Keith, Timothy Z.

    2002-01-01

    This commentary presents academic enablers within the broader, overlapping context of school learning theory, including the theories of Carroll, Harnishfeger and Wiley, Walberg, and others. Multivariate models are needed to understand the influences of academic enabler and school learning variables on learning, as well as the influences of these…

  13. Evaluating the promiscuous nature of tyrosine kinase inhibitors assessed in A431 epidermoid carcinoma cells by both chemical- and phosphoproteomics.

    PubMed

    Giansanti, Piero; Preisinger, Christian; Huber, Kilian V M; Gridling, Manuela; Superti-Furga, Giulio; Bennett, Keiryn L; Heck, Albert J R

    2014-07-18

    Deregulation of protein tyrosine kinase signaling has been linked to many diseases, most notably cancer. As a consequence, small molecule inhibitors of protein tyrosine kinases may provide powerful strategies for treatment. Following the successful introduction of imatinib in the treatment of chronic myelogenous leukemia, such drugs are also now evaluated for other types of cancer. However, many developed kinase inhibitors are not very target-specific and therefore may induce side effects. The importance of such side effects is certainly cell-proteome dependent. Understanding the all-inclusive action of a tyrosine kinase inhibitor on each individual cell-type entails the identification of potential targets, combined with monitoring the downstream effects revealing the signaling networks involved. Here, we explored a multilevel quantitative mass spectrometry-based proteomic strategy to identify the direct targets and downstream signaling effect of four tyrosine kinase inhibitors (imatinib, dasatinib, bosutinib, and nilotinib) in epidermoid carcinoma cells, as a model system for skin-cancer. More than 25 tyrosine kinases showed affinity to the drugs, with imatinib and nilotinib displaying a high specificity, especially when compared to dasatinib and bosutinib. Consequently, the latter two drugs showed a larger effect on downstream phosphotyrosine signaling. Many of the proteins affected are key regulators in cell adhesion and invasion. Our data represents a multiplexed view on the promiscuous action of certain tyrosine kinase inhibitors that needs to be taking into consideration prior to the application of these drugs in the treatment of different forms of cancer.

  14. Integrative Quantitative Proteomics Unveils Proteostasis Imbalance in Human Hepatocellular Carcinoma Developed on Nonfibrotic Livers*

    PubMed Central

    Negroni, Luc; Taouji, Said; Arma, Daniela; Pallares-Lupon, Nestor; Leong, Kristen; Beausang, Lee Anne; Latterich, Martin; Bossé, Roger; Balabaud, Charles; Schmitter, Jean-Marie; Bioulac-Sage, Paulette; Zucman-Rossi, Jessica; Rosenbaum, Jean; Chevet, Eric

    2014-01-01

    Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen®, and AMMP®) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253. PMID:25225353

  15. Non Linear Programming (NLP) Formulation for Quantitative Modeling of Protein Signal Transduction Pathways

    PubMed Central

    Morris, Melody K.; Saez-Rodriguez, Julio; Lauffenburger, Douglas A.; Alexopoulos, Leonidas G.

    2012-01-01

    Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms. PMID:23226239

  16. Non Linear Programming (NLP) formulation for quantitative modeling of protein signal transduction pathways.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Morris, Melody K; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Alexopoulos, Leonidas G

    2012-01-01

    Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms.

  17. Integrative quantitative proteomics unveils proteostasis imbalance in human hepatocellular carcinoma developed on nonfibrotic livers.

    PubMed

    Negroni, Luc; Taouji, Said; Arma, Daniela; Pallares-Lupon, Nestor; Leong, Kristen; Beausang, Lee Anne; Latterich, Martin; Bossé, Roger; Balabaud, Charles; Schmitter, Jean-Marie; Bioulac-Sage, Paulette; Zucman-Rossi, Jessica; Rosenbaum, Jean; Chevet, Eric

    2014-12-01

    Proteomics-based clinical studies represent promising resources for the discovery of novel biomarkers or for unraveling molecular mechanisms underlying particular diseases. Here, we present a discovery study of hepatocellular carcinoma developed on nonfibrotic liver (nfHCC) that combines complementary quantitative iTRAQ-based proteomics and phosphoproteomics approaches. Using both approaches, we compared a set of 24 samples (18 nfHCC versus six nontumor liver tissue). We identified 43 proteins (67 peptides) differentially expressed and 32 peptides differentially phosphorylated between the experimental groups. The functional analysis of the two data sets pointed toward the deregulation of a protein homeostasis (proteostasis) network including the up-regulation of the Endoplasmic Reticulum (ER) resident HSPA5, HSP90B1, PDIA6, and P4HB and of the cytosolic HSPA1B, HSP90AA1, HSPA9, UBC, CNDP2, TXN, and VCP as well as the increased phosphorylation of the ER resident calnexin at Ser583. Antibody-based validation approaches (immunohistochemistry, immunoblot, Alphascreen(®), and AMMP(®)) on independent nfHCC tumor sets (up to 77 samples) confirmed these observations, thereby indicating a common mechanism occurring in nfHCC tumors. Based on these results we propose that adaptation to proteostasis imbalance in nfHCC tumors might confer selective advantages to those tumors. As such, this model could provide an additional therapeutic opportunity for those tumors arising on normal liver by targeting the tumor proteostasis network. Data are available via ProteomeXchange with identifier PXD001253.

  18. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

    PubMed Central

    Li, Renfeng; Pinto, Sneha M.; Shaw, Patrick G.; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S.; Pandey, Akhilesh; Hayward, S. Diane

    2015-01-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  19. Utility Energy Services Contracts: Enabling Documents

    SciTech Connect

    Thomas, Karen; Vasquez, Deb

    2017-01-01

    The Federal Energy Management Program's 'Utility Energy Service Contracts: Enabling Documents' provide legislative information and materials that clarify the authority for federal agencies to enter into utility energy service contracts, or UESCs.

  20. Utility Energy Services Contracts: Enabling Documents

    SciTech Connect

    2009-05-01

    Utility Energy Services Contracts: Enabling Documents provides materials that clarify the authority for Federal agencies to enter into utility energy services contracts (UESCs), as well as sample documents and resources to ease utility partnership contracting.

  1. Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells

    PubMed Central

    Xue, Min; Wei, Wei; Su, Yapeng; Johnson, Dazy; Heath, James R.

    2016-01-01

    We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip (SCBC) with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady state model that describes energy potential in single cells, and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors. PMID:26916347

  2. Peptide Immunoaffinity Enrichment and Targeted Mass Spectrometry Enables Multiplex, Quantitative Pharmacodynamic Studies of Phospho-Signaling*

    PubMed Central

    Whiteaker, Jeffrey R.; Zhao, Lei; Yan, Ping; Ivey, Richard G.; Voytovich, Uliana J.; Moore, Heather D.; Lin, Chenwei; Paulovich, Amanda G.

    2015-01-01

    In most cell signaling experiments, analytes are measured one Western blot lane at a time in a semiquantitative and often poorly specific manner, limiting our understanding of network biology and hindering the translation of novel therapeutics and diagnostics. We show the feasibility of using multiplex immuno-MRM for phospho-pharmacodynamic measurements, establishing the potential for rapid and precise quantification of cell signaling networks. A 69-plex immuno-MRM assay targeting the DNA damage response network was developed and characterized by response curves and determinations of intra- and inter-assay repeatability. The linear range was ≥3 orders of magnitude, the median limit of quantification was 2.0 fmol/mg, the median intra-assay variability was 10% CV, and the median interassay variability was 16% CV. The assay was applied in proof-of-concept studies to immortalized and primary human cells and surgically excised cancer tissues to quantify exposure–response relationships and the effects of a genomic variant (ATM kinase mutation) or pharmacologic (kinase) inhibitor. The study shows the utility of multiplex immuno-MRM for simultaneous quantification of phosphorylated and nonmodified peptides, showing feasibility for development of targeted assay panels to cell signaling networks. PMID:25987412

  3. Tagging CO2 to Enable Quantitative Inventories of Geological Carbon Storage

    SciTech Connect

    Lackner, Klaus; Matter, Juerg; Park, Ah-Hyung; Stute, Martin; Carson, Cantwell; Ji, Yinghuang

    2014-06-30

    In the wake of concerns about the long term integrity and containment of sub-surface CO2 sequestration reservoirs, many efforts have been made to improve the monitoring, verification, and accounting methods for geo-sequestered CO2. Our project aimed to demonstrate the feasibility of a system designed to tag CO2 with carbon isotope 14C immediately prior to sequestration to a level that is normal on the surface (one part per trillion). Because carbon found at depth is naturally free of 14C, this tag would easily differentiate pre-existing carbon from anthropogenic injected carbon and provide an excellent handle for monitoring its whereabouts in the subsurface. It also creates an excellent handle for adding up anthropogenic carbon inventories. Future inventories in effect count 14C atoms. Accordingly, we have developed a 14C tagging system suitable for use at the part-per-trillion level. This system consists of a gas-exchange apparatus to make disposable cartridges ready for controlled injection into a fast flowing stream of pressurized CO2. We built a high-pressure injection and tagging system, and a 14C detection system. The disposable cartridge and injection system have been successfully demonstrated in the lab with a high-pressure flow reactor, as well as in the field at the CarbFix CO2 sequestration site in Iceland. The laser-based 14C detection system originally conceived has been shown to possess inadequate sensitivity for ambient levels. Alternative methods for detecting 14C, such as saturated cavity absorption ringdown spectroscopy and scintillation counting, may still be suitable. KEYWORDS

  4. Genome-enabled prediction of quantitative traits in chickens using genomic annotation

    PubMed Central

    2014-01-01

    Background Genome-wide association studies have been deemed successful for identifying statistically associated genetic variants of large effects on complex traits. Past studies have found enrichment of trait-associated SNPs in functionally annotated regions, while depletion was reported for intergenic regions (IGR). However, no systematic examination of connections between genomic regions and predictive ability of complex phenotypes has been carried out. Results In this study, we partitioned SNPs based on their annotation to characterize genomic regions that deliver low and high predictive power for three broiler traits in chickens using a whole-genome approach. Additive genomic relationship kernels were constructed for each of the genic regions considered, and a kernel-based Bayesian ridge regression was employed as prediction machine. We found that the predictive performance for ultrasound area of breast meat from using genic regions marked by SNPs was consistently better than that from SNPs in IGR, while IGR tagged by SNPs were better than the genic regions for body weight and hen house egg production. We also noted that predictive ability delivered by the whole battery of markers was close to the best prediction achieved by one of the genomic regions. Conclusions Whole-genome regression methods use all available quality filtered SNPs into a model, contrary to accommodating only validated SNPs from exonic or coding regions. Our results suggest that, while differences among genomic regions in terms of predictive ability were observed, the whole-genome approach remains as a promising tool if interest is on prediction of complex traits. PMID:24502227

  5. ISS - Enabling Exploration Through Docking Standards

    NASA Technical Reports Server (NTRS)

    Hatfield, Caris A.

    2011-01-01

    NASA and the ISS partnership are jointly developing a key standard to enable future collaborative exploration. The IDSS is based on flight proven design while incorporating new low impact technology. Low impact technology accommodates a wide range of vehicle contact and capture conditions. This standard will get early demonstration on the ISS. Experience gained here will enable operational experience and the opportunity to refine the standard.

  6. GIS Enabled Modeling and Simulation (GEMS)

    DTIC Science & Technology

    2007-06-01

    polygons, visual databases include texture information to provide a visualization of ground and material types. These databases include 3D models ...US Army TEC GIS-Enabled Modeling and Simulation project, and was the lead developer on the building interior semantic information portion of the...GIS Enabled Modeling and Simulation (GEMS) Thomas Stanzione Kevin Johnson MAK Technologies 68 Moulton Street Cambridge, MA 02138 (617) 876

  7. Lipid Informed Quantitation and Identification

    SciTech Connect

    Kevin Crowell, PNNL

    2014-07-21

    LIQUID (Lipid Informed Quantitation and Identification) is a software program that has been developed to enable users to conduct both informed and high-throughput global liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics analysis. This newly designed desktop application can quickly identify and quantify lipids from LC-MS/MS datasets while providing a friendly graphical user interface for users to fully explore the data. Informed data analysis simply involves the user specifying an electrospray ionization mode, lipid common name (i.e. PE(16:0/18:2)), and associated charge carrier. A stemplot of the isotopic profile and a line plot of the extracted ion chromatogram are also provided to show the MS-level evidence of the identified lipid. In addition to plots, other information such as intensity, mass measurement error, and elution time are also provided. Typically, a global analysis for 15,000 lipid targets

  8. Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.

    PubMed

    Tsiatsiani, Liana; Giansanti, Piero; Scheltema, Richard A; van den Toorn, Henk; Overall, Christopher M; Altelaar, A F Maarten; Heck, Albert J R

    2017-02-03

    A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)nK/R) and LysargiNase (i.e., K/R(X)n) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.

  9. Quantitative analysis of blood vessel geometry

    NASA Astrophysics Data System (ADS)

    Fuhrman, Michael G.; Abdul-Karim, Othman; Shah, Sujal; Gilbert, Steven G.; Van Bibber, Richard

    2001-07-01

    Re-narrowing or restenosis of a human coronary artery occurs within six months in one third of balloon angioplasty procedures. Accurate and repeatable quantitative analysis of vessel shape is important to characterize the progression and type of restenosis, and to evaluate effects new therapies might have. A combination of complicated geometry and image variability, and the need for high resolution and large image size makes visual/manual analysis slow, difficult, and prone to error. The image processing and analysis described here was developed to automate feature extraction of the lumen, internal elastic lamina, neointima, external elastic lamina, and tunica adventitia and to enable an objective, quantitative definition of blood vessel geometry. The quantitative geometrical analysis enables the measurement of several features including perimeter, area, and other metrics of vessel damage. Automation of feature extraction creates a high throughput capability that enables analysis of serial sections for more accurate measurement of restenosis dimensions. Measurement results are input into a relational database where they can be statistically analyzed compared across studies. As part of the integrated process, results are also imprinted on the images themselves to facilitate auditing of the results. The analysis is fast, repeatable and accurate while allowing the pathologist to control the measurement process.

  10. New Labour and the enabling state.

    PubMed

    Taylor, Ian

    2000-11-01

    The notion of the 'enabling state' gained currency in the UK during the 1990s as an alternative to the 'providing' or the welfare state. It reflected the process of contracting out in the NHS and compulsory competitive tendering (CCT) in local government during the 1980s, but was also associated with developments during the 1990s in health, social care and education in particular. The creation of an internal market in the NHS and the associated purchaser-provider split appeared to transfer 'ownership' of services increasingly to the providers - hospitals, General Practitioners (GPs) and schools. The mixed economy of care that was stimulated by the 1990 NHS and Community Care Act appeared to offer local authorities the opportunity to enable non state providers to offer care services in the community. The new service charters were part of the enablement process because they offered users more opportunity to influence provision. This article examines how far service providers were enabled and assesses the extent to which new Labour's policies enhance or reject the 'enabling state' in favour of more direct provision.

  11. Structures and practices enabling staff nurses to control their practice.

    PubMed

    Kramer, Marlene; Schmalenberg, Claudia; Maguire, Patricia; Brewer, Barbara B; Burke, Rebecca; Chmielewski, Linda; Cox, Karen; Kishner, Janice; Krugman, Mary; Meeks-Sjostrom, Diana; Waldo, Mary

    2008-08-01

    This mixed-methods study uses interviews, participant observations, and the CWEQII empowerment tool to identify structures and attributes of structures that promote control over nursing practice (CNP). Nearly 3,000 staff nurses completed the Essentials of Magnetism (EOM), an instrument that measures CNP, one of the eight staff nurse-identified essential attributes of a productive work environment. Strategic sampling is used to identify 101 high CNP-scoring clinical units in 8 high-EOM scoring magnet hospitals. In addition to 446 staff nurses, managers, and physicians on these high-scoring units, chief nursing officers, chief operating officers, and representatives from other professional departments are interviewed; participant observations are made of all unit/departmental/hospital council and interdisciplinary meetings held during a 4 to 6 day site visit. Structures and components of viable shared governance structures that enabled CNP are identified through constant comparative analysis of interviews and observations, and through analysis of quantitative measures.

  12. Quantitative plant proteomics.

    PubMed

    Bindschedler, Laurence V; Cramer, Rainer

    2011-02-01

    Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.

  13. Enabling the Discovery of Gravitational Radiation

    NASA Astrophysics Data System (ADS)

    Isaacson, Richard

    2017-01-01

    The discovery of gravitational radiation was announced with the publication of the results of a physics experiment involving over a thousand participants. This was preceded by a century of theoretical work, involving a similarly large group of physicists, mathematicians, and computer scientists. This huge effort was enabled by a substantial commitment of resources, both public and private, to develop the different strands of this complex research enterprise, and to build a community of scientists to carry it out. In the excitement following the discovery, the role of key enablers of this success has not always been adequately recognized in popular accounts. In this talk, I will try to call attention to a few of the key ingredients that proved crucial to enabling the successful discovery of gravitational waves, and the opening of a new field of science.

  14. Origami-enabled deformable silicon solar cells

    SciTech Connect

    Tang, Rui; Huang, Hai; Liang, Hanshuang; Liang, Mengbing; Tu, Hongen; Xu, Yong; Song, Zeming; Jiang, Hanqing; Yu, Hongyu

    2014-02-24

    Deformable electronics have found various applications and elastomeric materials have been widely used to reach flexibility and stretchability. In this Letter, we report an alternative approach to enable deformability through origami. In this approach, the deformability is achieved through folding and unfolding at the creases while the functional devices do not experience strain. We have demonstrated an example of origami-enabled silicon solar cells and showed that this solar cell can reach up to 644% areal compactness while maintaining reasonable good performance upon cyclic folding/unfolding. This approach opens an alternative direction of producing flexible, stretchable, and deformable electronics.

  15. Energy Savings Performance Contract (ESPC) ENABLE Program

    SciTech Connect

    2012-06-01

    The Energy Savings Performance Contract (ESPC) ENABLE program, a new project funding approach, allows small Federal facilities to realize energy and water savings in six months or less. ESPC ENABLE provides a standardized and streamlined process to install targeted energy conservation measures (ECMs) such as lighting, water, and controls with measurement and verification (M&V) appropriate for the size and scope of the project. This allows Federal facilities smaller than 200,000 square feet to make progress towards important energy efficiency and water conservation requirements.

  16. Enabling human HUMS with data modeling

    NASA Astrophysics Data System (ADS)

    Jaenisch, Holger M.; Handley, James W.; Jaenisch, Kristina K.; Hicklen, Michael L.

    2006-05-01

    We simulate a notional Navy SEAL rebreather diver on an extended mission using Model Predictive Control (MPC) theory. A mathematical framework for enabling physiological HUMS (Health Usage Management Systems) is shown. A rebreather simulation is used to derive MPC baseline Data Models of diver status by converting the simulation first into differential equations and then into lookup tables (LUT). When abnormal readings are indicated, sensor data from the diver is published to the ad hoc network, enabling intermittent upload. Mission success confidence is updated and determined during the mission. A novel method of converting MPC Data Models into lookup tables worn by the diver is given.

  17. Upgraded NERVA systems: Enabler nuclear system

    NASA Technical Reports Server (NTRS)

    Farbman, Gerry

    1991-01-01

    The NERVA/Rover Enabler technology enables to go on a low risk, short-term program to meet the requirements of the Mars mission and maybe some lunar missions. The following subject areas are covered: NERVA technology - the foundation for tomorrow's space missions; NERVA/Rover reactor system test sequence; NERVA engine development program; nuclear thermal reactor capability based on many related Westinghouse technology programs; investment in Rover/Nerva technology; synergistic applications of NERVA technology; flow schematic of the NDR engine; the NERVA nuclear subsystem; and technology evolution.

  18. Networking Technologies Enable Advances in Earth Science

    NASA Technical Reports Server (NTRS)

    Johnson, Marjory; Freeman, Kenneth; Gilstrap, Raymond; Beck, Richard

    2004-01-01

    This paper describes an experiment to prototype a new way of conducting science by applying networking and distributed computing technologies to an Earth Science application. A combination of satellite, wireless, and terrestrial networking provided geologists at a remote field site with interactive access to supercomputer facilities at two NASA centers, thus enabling them to validate and calibrate remotely sensed geological data in near-real time. This represents a fundamental shift in the way that Earth scientists analyze remotely sensed data. In this paper we describe the experiment and the network infrastructure that enabled it, analyze the data flow during the experiment, and discuss the scientific impact of the results.

  19. Network Enabled Operations: A Canadian Perspective

    DTIC Science & Technology

    2005-05-13

    Decisive: US Naval Institute Proceedings. ** VERDON , J. (2004) Transformation in the CF - People Implications of Effects-Based and Network-Enabled...Operations: A Canadian Perspective (U) 4. AUTHORS (First name, middle initial and last name. If military, show rank, e.g. Maj. John E. Doe.) Michael H

  20. Nanotechnologv Enabled Biological and Chemical Sensors

    NASA Technical Reports Server (NTRS)

    Koehne, Jessica; Meyyappan, M.

    2011-01-01

    Nanotechnology is an enabling technology that will impact almost all economic sectors: one of the most important and with great potential is the health/medical sector. - Nanomaterials for drug delivery - Early warning sensors - Implantable devices - Artificial parts with improved characteristics Carbon nanotubes and nanofibers show promise for use in sensor development, electrodes and other biomedical applications.

  1. School Bureaucracies That Work: Enabling, Not Coercive.

    ERIC Educational Resources Information Center

    Hoy, Wayne K.; Sweetland, Scott R.

    2000-01-01

    Attempts to reconcile two theoretically opposing perspectives of bureaucracy (as either alienating or facilitative) by creating and testing a new construct called "enabling bureaucracy." Empirical results are encouraging. Schools can be designed with formalized procedures and hierarchical structures that help rather than hinder teaching and…

  2. ICT-Enabled Learning: The Student Perspective

    ERIC Educational Resources Information Center

    Scott, Geoff; Grebennikov, Leonid; Gozzard, Terry

    2009-01-01

    This research seeks to contribute to current discussions in Australian higher education on how best to deploy ICT-enabled learning. Its particular focus is on examining the qualitative data from students on their experience of using Information and Communication Technologies (ICT) at one college in an Australian university. In total, about 71,240…

  3. Enabling a Comprehensive Teaching Strategy: Video Lectures

    ERIC Educational Resources Information Center

    Brecht, H. David; Ogilby, Suzanne M.

    2008-01-01

    This study empirically tests the feasibility and effectiveness of video lectures as a form of video instruction that enables a comprehensive teaching strategy used throughout a traditional classroom course. It examines student use patterns and the videos' effects on student learning, using qualitative and nonparametric statistical analyses of…

  4. Action Learning: Avoiding Conflict or Enabling Action

    ERIC Educational Resources Information Center

    Corley, Aileen; Thorne, Ann

    2006-01-01

    Action learning is based on the premise that action and learning are inextricably entwined and it is this potential, to enable action, which has contributed to the growth of action learning within education and management development programmes. However has this growth in action learning lead to an evolution or a dilution of Revan's classical…

  5. Safely Enabling Low-Altitude Airspace Operations

    NASA Technical Reports Server (NTRS)

    Kopardekar, Parimal

    2015-01-01

    Near-term Goal: Enable initial low-altitude airspace and UAS operations with demonstrated safety as early as possible, within 5 years. Long-term Goal: Accommodate increased UAS operations with highest safety, efficiency, and capacity as much autonomously as possible (10-15 years).

  6. Technology-Enabled Crime, Policing and Security

    ERIC Educational Resources Information Center

    McQuade, Sam

    2006-01-01

    Crime, policing and security are enabled by and co-evolve with technologies that make them possible. As criminals compete with security and policing officials for technological advantage perpetually complex crime, policing and security results in relatively confusing and therefore unmanageable threats to society. New, adaptive and ordinary crimes…

  7. Recapturing Quantitative Biology.

    ERIC Educational Resources Information Center

    Pernezny, Ken; And Others

    1996-01-01

    Presents a classroom activity on estimating animal populations. Uses shoe boxes and candies to emphasize the importance of mathematics in biology while introducing the methods of quantitative ecology. (JRH)

  8. Quantitative receptor autoradiography

    SciTech Connect

    Boast, C.A.; Snowhill, E.W.; Altar, C.A.

    1986-01-01

    Quantitative receptor autoradiography addresses the topic of technical and scientific advances in the sphere of quantitative autoradiography. The volume opens with a overview of the field from a historical and critical perspective. Following is a detailed discussion of in vitro data obtained from a variety of neurotransmitter systems. The next section explores applications of autoradiography, and the final two chapters consider experimental models. Methodological considerations are emphasized, including the use of computers for image analysis.

  9. Multivariate Quantitative Chemical Analysis

    NASA Technical Reports Server (NTRS)

    Kinchen, David G.; Capezza, Mary

    1995-01-01

    Technique of multivariate quantitative chemical analysis devised for use in determining relative proportions of two components mixed and sprayed together onto object to form thermally insulating foam. Potentially adaptable to other materials, especially in process-monitoring applications in which necessary to know and control critical properties of products via quantitative chemical analyses of products. In addition to chemical composition, also used to determine such physical properties as densities and strengths.

  10. Enabling room temperature sodium metal batteries

    SciTech Connect

    Cao, Ruiguo; Mishra, Kuber; Li, Xiaolin; Qian, Jiangfeng; Engelhard, Mark H.; Bowden, Mark E.; Han, Kee Sung; Mueller, Karl T.; Henderson, Wesley A.; Zhang, Ji-Guang

    2016-12-01

    Rechargeable batteries based upon sodium (Na+) cations are at the core of many new battery chemistries beyond Li-ion batteries. Rather than using carbon or alloy-based anodes, the direct utilization of solid sodium metal as an anode would be highly advantageous, but its use has been highly problematic due to its high reactivity. In this work, however, it is demonstrated that, by tailoring the electrolyte formulation, solid Na metal can be electrochemically plated/stripped at ambient temperature with high efficiency (> 99%) on both copper and inexpensive aluminum current collectors thereby enabling a shift in focus to new battery chemical couples based upon Na metal operating at ambient temperature. These highly concentrated electrolytes has enabled stable cycling of Na metal batteries based on a Na metal anode and Na3V2(PO4)3 cathode at high rates with very high efficiency.

  11. Implementation of Quantitative and Systems Pharmacology in Large Pharma

    PubMed Central

    Visser, S A G; de Alwis, D P; Kerbusch, T; Stone, J A; Allerheiligen, S R B

    2014-01-01

    Quantitative and systems pharmacology concepts and tools are the foundation of the model-informed drug development paradigm at Merck for integrating knowledge, enabling decisions, and enhancing submissions. Rigorous prioritization of modeling and simulation activities has enabled key drug development decisions and led to a high return on investment through significant cost avoidance. Critical factors for the successful implementation, examples on impact on decision making with associated return of investment, and drivers for continued success are discussed. PMID:25338195

  12. Enabling international adoption of LOINC through translation.

    PubMed

    Vreeman, Daniel J; Chiaravalloti, Maria Teresa; Hook, John; McDonald, Clement J

    2012-08-01

    Interoperable health information exchange depends on adoption of terminology standards, but international use of such standards can be challenging because of language differences between local concept names and the standard terminology. To address this important barrier, we describe the evolution of an efficient process for constructing translations of LOINC terms names, the foreign language functions in RELMA, and the current state of translations in LOINC. We also present the development of the Italian translation to illustrate how translation is enabling adoption in international contexts. We built a tool that finds the unique list of LOINC Parts that make up a given set of LOINC terms. This list enables translation of smaller pieces like the core component "hepatitis c virus" separately from all the suffixes that could appear with it, such "Ab.IgG", "DNA", and "RNA". We built another tool that generates a translation of a full LOINC name from all of these atomic pieces. As of version 2.36 (June 2011), LOINC terms have been translated into nine languages from 15 linguistic variants other than its native English. The five largest linguistic variants have all used the Part-based translation mechanism. However, even with efficient tools and processes, translation of standard terminology is a complex undertaking. Two of the prominent linguistic challenges that translators have faced include: the approach to handling acronyms and abbreviations, and the differences in linguistic syntax (e.g. word order) between languages. LOINC's open and customizable approach has enabled many different groups to create translations that met their needs and matched their resources. Distributing the standard and its many language translations at no cost worldwide accelerates LOINC adoption globally, and is an important enabler of interoperable health information exchange.

  13. Enabling Tactical Edge Mashups with Live Objects

    DTIC Science & Technology

    2010-06-01

    and visual Situation Reports (SITREPs) while airborne, via his Live Objects-enabled IP radio, to better vector the bird to the site of the downed...including the time for reviewing instructions, searching existing data sources , gathering and maintaining the data needed, and completing and...Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a

  14. NASA Missions Enabled by Space Nuclear Systems

    NASA Technical Reports Server (NTRS)

    Scott, John H.; Schmidt, George R.

    2009-01-01

    This viewgraph presentation reviews NASA Space Missions that are enabled by Space Nuclear Systems. The topics include: 1) Space Nuclear System Applications; 2) Trade Space for Electric Power Systems; 3) Power Generation Specific Energy Trade Space; 4) Radioisotope Power Generation; 5) Radioisotope Missions; 6) Fission Power Generation; 7) Solar Powered Lunar Outpost; 8) Fission Powered Lunar Outpost; 9) Fission Electric Power Generation; and 10) Fission Nuclear Thermal Propulsion.

  15. Enabling technologies for petascale electromagnetic accelerator simulation

    NASA Astrophysics Data System (ADS)

    Lee, Lie-Quan; Akcelik, Volkan; Chen, Sheng; Ge, Lixin; Prudencio, Ernesto; Schussman, Greg; Uplenchwar, Ravi; Ng, Cho; Ko, Kwok; Luo, Xiaojun; Shephard, Mark

    2007-07-01

    The SciDAC2 accelerator project at SLAC aims to simulate an entire three-cryomodule radio frequency (RF) unit of the International Linear Collider (ILC) main Linac. Petascale computing resources supported by advances in Applied Mathematics (AM) and Computer Science (CS) and INCITE Program are essential to enable such very large-scale electromagnetic accelerator simulations required by the ILC Global Design Effort. This poster presents the recent advances and achievements in the areas of CS/AM through collaborations.

  16. Open Architecture as an Enabler for FORCEnet

    DTIC Science & Technology

    2006-09-01

    Functional Area Manager FCP Fire Control Picture FCS Fire Control Solution FCQ Fire Control Quality FFBD Functional Flow Block Diagram Fn/OA FORCEnet...a) Firing Unit (FRU) receive Fire Control Quality ( FCQ ) data on threat from Remote Unit(s) (RUs). b) Validate FCQ data, enable FRU to act. c...Maintain Common Operational Picture (COP) of local tracks. d) Develop Fire Control Solution (FCS) based on FCQ data. e) Correlate FRU FCS with RU

  17. Enabling technologies: fermentation and downstream processing.

    PubMed

    Weuster-Botz, Dirk; Hekmat, Dariusch; Puskeiler, Robert; Franco-Lara, Ezequiel

    2007-01-01

    Efficient parallel tools for bioprocess design, consequent application of the concepts for metabolic process analysis as well as innovative downstream processing techniques are enabling technologies for new industrial bioprocesses from an engineering point of view. Basic principles, state-of-the-art techniques and cutting-edge technologies are briefly reviewed. Emphasis is on parallel bioreactors for bioprocess design, biochemical systems characterization and metabolic control analysis, as well as on preparative chromatography, affinity filtration and protein crystallization on a process scale.

  18. Enabling international adoption of LOINC through translation

    PubMed Central

    Vreeman, Daniel J.; Chiaravalloti, Maria Teresa; Hook, John; McDonald, Clement J.

    2012-01-01

    Interoperable health information exchange depends on adoption of terminology standards, but international use of such standards can be challenging because of language differences between local concept names and the standard terminology. To address this important barrier, we describe the evolution of an efficient process for constructing translations of LOINC terms names, the foreign language functions in RELMA, and the current state of translations in LOINC. We also present the development of the Italian translation to illustrate how translation is enabling adoption in international contexts. We built a tool that finds the unique list of LOINC Parts that make up a given set of LOINC terms. This list enables translation of smaller pieces like the core component “hepatitis c virus” separately from all the suffixes that could appear with it, such “Ab.IgG”, “DNA”, and “RNA”. We built another tool that generates a translation of a full LOINC name from all of these atomic pieces. As of version 2.36 (June 2011), LOINC terms have been translated into 9 languages from 15 linguistic variants other than its native English. The five largest linguistic variants have all used the Part-based translation mechanism. However, even with efficient tools and processes, translation of standard terminology is a complex undertaking. Two of the prominent linguistic challenges that translators have faced include: the approach to handling acronyms and abbreviations, and the differences in linguistic syntax (e.g. word order) between languages. LOINC’s open and customizable approach has enabled many different groups to create translations that met their needs and matched their resources. Distributing the standard and its many language translations at no cost worldwide accelerates LOINC adoption globally, and is an important enabler of interoperable health information exchange PMID:22285984

  19. Enabling Technologies for Petascale Electromagnetic Accelerator Simulation

    SciTech Connect

    Lee, Lie-Quan; Akcelik, Volkan; Chen, Sheng; Ge, Li-Xin; Prudencio, Ernesto; Schussman, Greg; Uplenchwar, Ravi; Ng, Cho; Ko, Kwok; Luo, Xiaojun; Shephard, Mark; /Rensselaer Poly.

    2007-11-09

    The SciDAC2 accelerator project at SLAC aims to simulate an entire three-cryomodule radio frequency (RF) unit of the International Linear Collider (ILC) main Linac. Petascale computing resources supported by advances in Applied Mathematics (AM) and Computer Science (CS) and INCITE Program are essential to enable such very large-scale electromagnetic accelerator simulations required by the ILC Global Design Effort. This poster presents the recent advances and achievements in the areas of CS/AM through collaborations.

  20. Enabling Research Tools for Sustained Climate Assessment

    NASA Technical Reports Server (NTRS)

    Leidner, Allison K.; Bosilovich, Michael G.; Jasinski, Michael F.; Nemani, Ramakrishna R.; Waliser, Duane Edward; Lee, Tsengdar J.

    2016-01-01

    The U.S. Global Change Research Program Sustained Assessment process benefits from long-term investments in Earth science research that enable the scientific community to conduct assessment-relevant science. To this end, NASA initiated several research programs over the past five years to support the Earth observation community in developing indicators, datasets, research products, and tools to support ongoing and future National Climate Assessments. These activities complement NASA's ongoing Earth science research programs. One aspect of the assessment portfolio funds four "enabling tools" projects at NASA research centers. Each tool leverages existing capacity within the center, but has developed tailored applications and products for National Climate Assessments. The four projects build on the capabilities of a global atmospheric reanalysis (MERRA-2), a continental U.S. land surface reanalysis (NCA-LDAS), the NASA Earth Exchange (NEX), and a Regional Climate Model Evaluation System (RCMES). Here, we provide a brief overview of each enabling tool, highlighting the ways in which it has advanced assessment science to date. We also discuss how the assessment community can access and utilize these tools for National Climate Assessments and other sustained assessment activities.

  1. DISK FORMATION ENABLED BY ENHANCED RESISTIVITY

    SciTech Connect

    Krasnopolsky, Ruben; Shang Hsien; Li Zhiyun

    2010-06-20

    Disk formation in magnetized cloud cores is hindered by magnetic braking. Previous work has shown that for realistic levels of core magnetization, the magnetic field suppresses the formation of rotationally supported disks during the protostellar mass accretion phase of low-mass star formation both in the ideal MHD limit and in the presence of ambipolar diffusion for typical rates of cosmic-ray ionization. Additional effects, such as ohmic dissipation, the Hall effect, and protostellar outflow, are needed to weaken the magnetic braking and enable the formation of persistent, rotationally supported, protostellar disks. In this paper, we first demonstrate that the classic microscopic resistivity is not large enough to enable disk formation by itself. We then experiment with a set of enhanced values for the resistivity in the range {eta} = 10{sup 17}-10{sup 22} cm{sup 2} s{sup -1}. We find that a value of order 10{sup 19} cm{sup 2} s{sup -1} is needed to enable the formation of a 10{sup 2} AU scale Keplerian disk; the value depends somewhat on the degree of core magnetization. The required resistivity is a few orders of magnitude larger than the classic microscopic values. Whether it can be achieved naturally during protostellar collapse remains to be determined.

  2. Enabling conditions and children's understanding of pretense.

    PubMed

    Sobel, David M

    2009-11-01

    Two experiments examined whether preschoolers' difficulties on tasks that required relating pretending and knowledge (e.g., Lillard, A. S. (1993a). Young children's conceptualization of pretense: Action or mental representational state? Child Development, 64, 372-386) were due to children's inability to appreciate the causal mechanism behind enabling conditions. In Experiment 1, 4-year-olds were told about a character who knew about one kind of animal and did not know about another. The character acted in a manner consistent with both animals. Children were asked whether the character was pretending to be the animal of which he was ignorant. The character's knowledge was either represented in a generic manner (as a picture) or in a manner that suggested a particular enabling condition relation that children found accessible (as a battery, which most 4-year-olds recognize is critical for making toys work). Children were more successful at relating knowledge and pretending in the battery condition. This improvement in performance extended to another task in which children had to identify the enabling condition relation between knowledge and identification, in which there were reduced demands on the inhibitory mechanisms necessary for success. Experiment 2 found that the results in Experiment 1 were not due to demands of the procedure used in Experiment 1. These results are discussed in the context of recent theories of theory of mind that focus on the importance of causal relations among mental states.

  3. Web-enabling technologies for the factory floor: a web-enabling strategy for emanufacturing

    NASA Astrophysics Data System (ADS)

    Velez, Ricardo; Lastra, Jose L. M.; Tuokko, Reijo O.

    2001-10-01

    This paper is intended to address the different technologies available for Web-enabling of the factory floor. It will give an overview of the importance of Web-enabling of the factory floor, in the application of the concepts of flexible and intelligent manufacturing, in conjunction with e-commerce. As a last section, it will try to define a Web-enabling strategy for the application in eManufacturing. This is made under the scope of the electronics manufacturing industry, so every application, technology or related matter is presented under such scope.

  4. Quantitative myocardial perfusion SPECT.

    PubMed

    Tsui, B M; Frey, E C; LaCroix, K J; Lalush, D S; McCartney, W H; King, M A; Gullberg, G T

    1998-01-01

    In recent years, there has been much interest in the clinical application of attenuation compensation to myocardial perfusion single photon emission computed tomography (SPECT) with the promise that accurate quantitative images can be obtained to improve clinical diagnoses. The different attenuation compensation methods that are available create confusion and some misconceptions. Also, attenuation-compensated images reveal other image-degrading effects including collimator-detector blurring and scatter that are not apparent in uncompensated images. This article presents basic concepts of the major factors that degrade the quality and quantitative accuracy of myocardial perfusion SPECT images, and includes a discussion of the various image reconstruction and compensation methods and misconceptions and pitfalls in implementation. The differences between the various compensation methods and their performance are demonstrated. Particular emphasis is directed to an approach that promises to provide quantitative myocardial perfusion SPECT images by accurately compensating for the 3-dimensional (3-D) attenuation, collimator-detector response, and scatter effects. With advances in the computer hardware and optimized implementation techniques, quantitatively accurate and high-quality myocardial perfusion SPECT images can be obtained in clinically acceptable processing time. Examples from simulation, phantom, and patient studies are used to demonstrate the various aspects of the investigation. We conclude that quantitative myocardial perfusion SPECT, which holds great promise to improve clinical diagnosis, is an achievable goal in the near future.

  5. Small-RPS Enabled Mars Rover Concept

    NASA Astrophysics Data System (ADS)

    Balint, Tibor S.

    2005-02-01

    Both the MER and the Mars Pathfinder rovers operated on Mars in an energy-limited mode, since the solar panels generated power during daylight hours only. At other times the rovers relied on power stored in batteries. In comparison, Radioisotope Power Systems (RPS) offer a power-enabled paradigm, where power can be generated for long mission durations (measured in years), independently from the Sun, and on a continuous basis. A study was performed at JPL to assess the feasibility of a small-RPS enabled MER-class rover concept and any associated advantages of its mission on Mars, The rover concept relied on design heritage from MER with two significant changes. First, the solar panels were replaced with two single GPHS module based small-RPSs. Second, the Mossbauer spectroscope was substituted with a laser Raman spectroscope, in order to move towards MEPAG defined astrobiology driven science goals. The highest power requirements were contributed to mobility and telecommunication type operating modes, hence influencing power system sizing. The resulting hybrid power system included two small-RPSs and two batteries. Each small-RPS was assumed to generate 50 We of power or 620 Wh/sol of energy (BOL), comparable to that of MER. The two 8 Ah batteries were considered available during peak power usage. Mission architecture, power trades, science instruments, data, communication, thermal and radiation environments, mobility, and mass issues were also addressed. The study demonstrated that a new set of RPS-enabled rover missions could be envisioned for Mars exploration within the next decade, targeting astrobiology oriented science objectives, while powered by 2 to 4 GPHS modules.

  6. A wireless sensor enabled by wireless power.

    PubMed

    Lee, Da-Sheng; Liu, Yu-Hong; Lin, Chii-Ruey

    2012-11-22

    Through harvesting energy by wireless charging and delivering data by wireless communication, this study proposes the concept of a wireless sensor enabled by wireless power (WPWS) and reports the fabrication of a prototype for functional tests. One WPWS node consists of wireless power module and sensor module with different chip-type sensors. Its main feature is the dual antenna structure. Following RFID system architecture, a power harvesting antenna was designed to gather power from a standard reader working in the 915 MHz band. Referring to the Modbus protocol, the other wireless communication antenna was integrated on a node to send sensor data in parallel. The dual antenna structure integrates both the advantages of an RFID system and a wireless sensor. Using a standard UHF RFID reader, WPWS can be enabled in a distributed area with a diameter up to 4 m. Working status is similar to that of a passive tag, except that a tag can only be queried statically, while the WPWS can send dynamic data from the sensors. The function is the same as a wireless sensor node. Different WPWSs equipped with temperature and humidity, optical and airflow velocity sensors are tested in this study. All sensors can send back detection data within 8 s. The accuracy is within 8% deviation compared with laboratory equipment. A wireless sensor network enabled by wireless power should be a totally wireless sensor network using WPWS. However, distributed WPWSs only can form a star topology, the simplest topology for constructing a sensor network. Because of shielding effects, it is difficult to apply other complex topologies. Despite this limitation, WPWS still can be used to extend sensor network applications in hazardous environments. Further research is needed to improve WPWS to realize a totally wireless sensor network.

  7. Astronomy Enabled by Ares V -- A Workshop

    NASA Astrophysics Data System (ADS)

    Lester, Daniel F.; Langhoff, S.; Worden, S. P.; Thronson, H.; Correll, R.

    2009-01-01

    On April 26th and 27th, 2008, NASA Ames Research Center hosted a two-day weekend workshop entitled "Astronomy Enabled by Ares V.” The primary goal of the workshop was to begin the process of bringing the Ares V designers together with senior representatives of the astronomical community to discuss the feasibility of using the Ares V heavy-lift launch vehicle to enable both new astronomical telescope architectures and new science. When developed in the latter part of the upcoming decade Ares V will be by far the most capable launch vehicle, with mass and volume launch capability many times that now available. The vehicle is understood to be the main workhorse in carrying humans and cargo to the Moon and beyond and, as such, is a key lynchpin for NASA's new space transportation architecture. Participants included experts from academia, industry, and NASA, including representatives of the Constellation architecture. Participants considered, in the context of identified astronomy needs: (1) Are there telescope concepts or missions capable of breakthrough science that are either enabled or significantly enhanced by the capabilities of an Ares V? (2) What demands do large telescopes place on the payload environment of the Ares V, such as mass, volume, fairing shape, cleanliness, acoustics, etc.? (3) What technology and environmental issues need to be addressed to facilitate launching observatories on an Ares V? (4) Is there a trade-off between mass and complexity that could reduce launch risk and, thereby, the cost of building large telescopes? We report on the results of this workshop, which included discussion on the operations model for such large-investment astronomical facilities. Such an operations model might well involve human and or robotic maintenance and servicing, in order to fully capitalize on the science potential of such facilities.

  8. A Wireless Sensor Enabled by Wireless Power

    PubMed Central

    Lee, Da-Sheng; Liu, Yu-Hong; Lin, Chii-Ruey

    2012-01-01

    Through harvesting energy by wireless charging and delivering data by wireless communication, this study proposes the concept of a wireless sensor enabled by wireless power (WPWS) and reports the fabrication of a prototype for functional tests. One WPWS node consists of wireless power module and sensor module with different chip-type sensors. Its main feature is the dual antenna structure. Following RFID system architecture, a power harvesting antenna was designed to gather power from a standard reader working in the 915 MHz band. Referring to the Modbus protocol, the other wireless communication antenna was integrated on a node to send sensor data in parallel. The dual antenna structure integrates both the advantages of an RFID system and a wireless sensor. Using a standard UHF RFID reader, WPWS can be enabled in a distributed area with a diameter up to 4 m. Working status is similar to that of a passive tag, except that a tag can only be queried statically, while the WPWS can send dynamic data from the sensors. The function is the same as a wireless sensor node. Different WPWSs equipped with temperature and humidity, optical and airflow velocity sensors are tested in this study. All sensors can send back detection data within 8 s. The accuracy is within 8% deviation compared with laboratory equipment. A wireless sensor network enabled by wireless power should be a totally wireless sensor network using WPWS. However, distributed WPWSs only can form a star topology, the simplest topology for constructing a sensor network. Because of shielding effects, it is difficult to apply other complex topologies. Despite this limitation, WPWS still can be used to extend sensor network applications in hazardous environments. Further research is needed to improve WPWS to realize a totally wireless sensor network. PMID:23443370

  9. ENABLER Nuclear Propulsion System Conceptual Design

    NASA Astrophysics Data System (ADS)

    Pauley, Keith A.; Woodham, Kurt; Ohi, Don; Haga, Heath; Henderson, Bo

    2004-02-01

    The Titan Corporation conducted a systems engineering study to develop an overall architecture that meets both the articulated and unarticulated requirements on the Prometheus Program with the least development effort. Key elements of the Titan-designed ENABLER system include a thermal fission reactor, thermionic power converters, sodium heat pipes, ion thruster engines, and a radiation shield and deployable truss to protect the payload. The overall design is scaleable over a wide range of power requirements from 10s of kilowatts to 10s of megawatts.

  10. Enabling Strain Hardening Simulations with Dislocation Dynamics

    SciTech Connect

    Arsenlis, A; Cai, W

    2006-12-20

    Numerical algorithms for discrete dislocation dynamics simulations are investigated for the purpose of enabling strain hardening simulations of single crystals on massively parallel computers. The algorithms investigated include the /(N) calculation of forces, the equations of motion, time integration, adaptive mesh refinement, the treatment of dislocation core reactions, and the dynamic distribution of work on parallel computers. A simulation integrating all of these algorithmic elements using the Parallel Dislocation Simulator (ParaDiS) code is performed to understand their behavior in concert, and evaluate the overall numerical performance of dislocation dynamics simulations and their ability to accumulate percents of plastic strain.

  11. Enabling Rapid Naval Architecture Design Space Exploration

    NASA Technical Reports Server (NTRS)

    Mueller, Michael A.; Dufresne, Stephane; Balestrini-Robinson, Santiago; Mavris, Dimitri

    2011-01-01

    Well accepted conceptual ship design tools can be used to explore a design space, but more precise results can be found using detailed models in full-feature computer aided design programs. However, defining a detailed model can be a time intensive task and hence there is an incentive for time sensitive projects to use conceptual design tools to explore the design space. In this project, the combination of advanced aerospace systems design methods and an accepted conceptual design tool facilitates the creation of a tool that enables the user to not only visualize ship geometry but also determine design feasibility and estimate the performance of a design.

  12. Camera-enabled techniques for organic synthesis

    PubMed Central

    Ingham, Richard J; O’Brien, Matthew; Browne, Duncan L

    2013-01-01

    Summary A great deal of time is spent within synthetic chemistry laboratories on non-value-adding activities such as sample preparation and work-up operations, and labour intensive activities such as extended periods of continued data collection. Using digital cameras connected to computer vision algorithms, camera-enabled apparatus can perform some of these processes in an automated fashion, allowing skilled chemists to spend their time more productively. In this review we describe recent advances in this field of chemical synthesis and discuss how they will lead to advanced synthesis laboratories of the future. PMID:23766820

  13. Enabling plant synthetic biology through genome engineering.

    PubMed

    Baltes, Nicholas J; Voytas, Daniel F

    2015-02-01

    Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code - enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology.

  14. PHM Enabled Autonomous Propellant Loading Operations

    NASA Technical Reports Server (NTRS)

    Walker, Mark; Figueroa, Fernando

    2017-01-01

    The utility of Prognostics and Health Management (PHM) software capability applied to Autonomous Operations (AO) remains an active research area within aerospace applications. The ability to gain insight into which assets and subsystems are functioning properly, along with the derivation of confident predictions concerning future ability, reliability, and availability, are important enablers for making sound mission planning decisions. When coupled with software that fully supports mission planning and execution, an integrated solution can be developed that leverages state assessment and estimation for the purposes of delivering autonomous operations. The authors have been applying this integrated, model-based approach to the autonomous loading of cryogenic spacecraft propellants at Kennedy Space Center.

  15. Quantitative Hydrocarbon Surface Analysis

    NASA Technical Reports Server (NTRS)

    Douglas, Vonnie M.

    2000-01-01

    The elimination of ozone depleting substances, such as carbon tetrachloride, has resulted in the use of new analytical techniques for cleanliness verification and contamination sampling. The last remaining application at Rocketdyne which required a replacement technique was the quantitative analysis of hydrocarbons by infrared spectrometry. This application, which previously utilized carbon tetrachloride, was successfully modified using the SOC-400, a compact portable FTIR manufactured by Surface Optics Corporation. This instrument can quantitatively measure and identify hydrocarbons from solvent flush of hardware as well as directly analyze the surface of metallic components without the use of ozone depleting chemicals. Several sampling accessories are utilized to perform analysis for various applications.

  16. Quantitative photoacoustic tomography

    PubMed Central

    Yuan, Zhen; Jiang, Huabei

    2009-01-01

    In this paper, several algorithms that allow for quantitative photoacoustic reconstruction of tissue optical, acoustic and physiological properties are described in a finite-element method based framework. These quantitative reconstruction algorithms are compared, and the merits and limitations associated with these methods are discussed. In addition, a multispectral approach is presented for concurrent reconstructions of multiple parameters including deoxyhaemoglobin, oxyhaemoglobin and water concentrations as well as acoustic speed. Simulation and in vivo experiments are used to demonstrate the effectiveness of the reconstruction algorithms presented. PMID:19581254

  17. Quantitative Decision Support Requires Quantitative User Guidance

    NASA Astrophysics Data System (ADS)

    Smith, L. A.

    2009-12-01

    Is it conceivable that models run on 2007 computer hardware could provide robust and credible probabilistic information for decision support and user guidance at the ZIP code level for sub-daily meteorological events in 2060? In 2090? Retrospectively, how informative would output from today’s models have proven in 2003? or the 1930’s? Consultancies in the United Kingdom, including the Met Office, are offering services to “future-proof” their customers from climate change. How is a US or European based user or policy maker to determine the extent to which exciting new Bayesian methods are relevant here? or when a commercial supplier is vastly overselling the insights of today’s climate science? How are policy makers and academic economists to make the closely related decisions facing them? How can we communicate deep uncertainty in the future at small length-scales without undermining the firm foundation established by climate science regarding global trends? Three distinct aspects of the communication of the uses of climate model output targeting users and policy makers, as well as other specialist adaptation scientists, are discussed. First, a brief scientific evaluation of the length and time scales at which climate model output is likely to become uninformative is provided, including a note on the applicability the latest Bayesian methodology to current state-of-the-art general circulation models output. Second, a critical evaluation of the language often employed in communication of climate model output, a language which accurately states that models are “better”, have “improved” and now “include” and “simulate” relevant meteorological processed, without clearly identifying where the current information is thought to be uninformative and misleads, both for the current climate and as a function of the state of the (each) climate simulation. And thirdly, a general approach for evaluating the relevance of quantitative climate model output

  18. Enabling Meaningful Affiliation Searches in the ADS

    NASA Astrophysics Data System (ADS)

    Grant, C. S.; Thompson, D. M.; Chyla, R.; Holachek, A.; Accomazzi, A.; Henneken, E. A.; Kurtz, M. J.; Luker, J.; Murray, S. S.

    2015-04-01

    For many years, users have wanted to search affiliations in the ADS in order to build institutional databases and to help with author disambiguation. Although we currently provide this capability upon request, we have yet to incorporate it as part of the operational Abstract Service. This is because it cannot be used reliably, primarily because of the lack of uniform representation of the affiliation data. In an effort to make affiliation searches more meaningful, we have designed a two-tiered hierarchy of standard institutional names based on Ringgold identifiers, with the expectation that this will enable us to implement a search by institution, which will work for the vast majority of institutions. It is our intention to provide the capability of searching the ADS both by standard affiliation name and original affiliation string, as well as to enable autosuggest of affiliations as a means of helping to disambiguate author identification. Some institutions are likely to require manual work, and we encourage interested librarians to assist us in standardizing the representation of their institutions in the affiliation field.

  19. Nano-enabled SERS reporting photosensitizers.

    PubMed

    Farhadi, Arash; Roxin, Áron; Wilson, Brian C; Zheng, Gang

    2015-01-01

    To impart effective cellular damage via photodynamic therapy (PDT), it is vital to deliver the appropriate light dose and photosensitizer concentration, and to monitor the PDT dose delivered at the site of interest. In vivo monitoring of photosensitizers has in large part relied on their fluorescence emission. Palladium-containing photosensitizers have shown promising clinical results by demonstrating near full conversion of light to PDT activity at the cost of having undetectable fluorescence. We demonstrate that, through the coupling of plasmonic nanoparticles with palladium-photosensitizers, surface-enhanced Raman scattering (SERS) provides both reporting and monitoring capability to otherwise quiescent molecules. Nano-enabled SERS reporting of photosensitizers allows for the decoupling of the therapeutic and imaging mechanisms so that both phenomena can be optimized independently. Most importantly, the design enables the use of the same laser wavelength to stimulate both the PDT and imaging features, opening the potential for real-time dosimetry of photosensitizer concentration and PDT dose delivery by SERS monitoring.

  20. The Master Enabler: In Orbit Servicing

    NASA Technical Reports Server (NTRS)

    Reed, Benjamin B.; Kienlen, Michael; Naasz, Bo; Roberts, Brian; Deweese, Keith; Cassidy, Justin

    2015-01-01

    Some of the most noteworthy missions in space exploration have occurred in the last two decades and owe their success to on-orbit servicing. The tremendously successful Hubble Space Telescope repair and upgrade missions, as well as the completed assembly of the International Space Station (ISS) and its full utilization, lead us to the next chapter and set of challenges. These include fully exploiting the many space systems already launched, assembling large structures in situ thereby enabling new scientific discoveries, and providing systems that reliably and cost-effectively support the next steps in space exploration. In-orbit servicing is a tool--a tool that can serve as the master enabler to create space architectures that would otherwise be unattainable. This paper will survey how NASA's satellite-servicing technology development efforts are being applied to the planning and execution of two such ambitious missions, specifically asteroid capture and the in-space assembly of a very large life-finding telescope.

  1. In-Orbit Servicing: The Master Enabler

    NASA Technical Reports Server (NTRS)

    Reed, Benjamin B.; Kienlen, Michael; Naasz, Bo; Roberts, Brian; Deweese, Keith

    2015-01-01

    Some of the most noteworthy missions in space exploration have occurred in the last two decades and owe their success to on-orbit servicing. The tremendously successful Hubble Space Telescope repair and upgrade missions, as well as the completed assembly of the International Space Station (ISS) and its full utilization, lead us to the next chapter and set of challenges. These include fully exploiting the many space systems already launched, assembling large structures in situ thereby enabling new scientific discoveries, and providing systems that reliably and cost-effectively support the next steps in space exploration. In-orbit servicing is a tool--a tool that can serve as the master enabler to create space architectures that would otherwise be unattainable. This paper will survey how NASA's satellite-servicing technology development efforts are being applied to the planning and execution of two such ambitious missions, specifically asteroid capture and the in-space assembly of a very large life-finding telescope.

  2. The "Master Enabler" - In-Orbit Servicing

    NASA Technical Reports Server (NTRS)

    Reed, Benjamin; Kienlen, Michael; Naasz, Bo; Roberts, Brian; Deweese, Keith; Cassidy, Justin

    2015-01-01

    Some of the most noteworthy missions in space exploration have occurred in the last two decades and owe their success to on-orbit servicing. The tremendously successful Hubble Space Telescope repair and upgrade missions, as well as the completed assembly of the International Space Station (ISS) and its full utilization, lead us to the next chapter and set of challenges. These include fully exploiting the many space systems already launched, assembling large structures in situ thereby enabling new scientific discoveries, and providing systems that reliably and cost-effectively support the next steps in space exploration. In-orbit servicing is a tool-a tool that can serve as the master enabler to create space architectures that would otherwise be unattainable. This paper will survey how NASA's satellite-servicing technology development efforts are being applied to the planning and execution of two such ambitious missions, specifically asteroid capture and the in-space assembly of a very large life-finding telescope.

  3. MENTOR: an enabler for interoperable intelligent systems

    NASA Astrophysics Data System (ADS)

    Sarraipa, João; Jardim-Goncalves, Ricardo; Steiger-Garcao, Adolfo

    2010-07-01

    A community with knowledge organisation based on ontologies will enable an increase in the computational intelligence of its information systems. However, due to the worldwide diversity of communities, a high number of knowledge representation elements, which are not semantically coincident, have appeared representing the same segment of reality, becoming a barrier to business communications. Even if a domain community uses the same kind of technologies in its information systems, such as ontologies, it doesn't solve its semantics differences. In order to solve this interoperability problem, a solution is to use a reference ontology as an intermediary in the communications between the community enterprises and the outside, while allowing the enterprises to keep their own ontology and semantics unchanged internally. This work proposes MENTOR, a methodology to support the development of a common reference ontology for a group of organisations sharing the same business domain. This methodology is based on the mediator ontology (MO) concept, which assists the semantic transformations among each enterprise's ontology and the referential one. The MO enables each organisation to keep its own terminology, glossary and ontological structures, while providing seamless communication and interaction with the others.

  4. Quantitative Simulation Games

    NASA Astrophysics Data System (ADS)

    Černý, Pavol; Henzinger, Thomas A.; Radhakrishna, Arjun

    While a boolean notion of correctness is given by a preorder on systems and properties, a quantitative notion of correctness is defined by a distance function on systems and properties, where the distance between a system and a property provides a measure of "fit" or "desirability." In this article, we explore several ways how the simulation preorder can be generalized to a distance function. This is done by equipping the classical simulation game between a system and a property with quantitative objectives. In particular, for systems that satisfy a property, a quantitative simulation game can measure the "robustness" of the satisfaction, that is, how much the system can deviate from its nominal behavior while still satisfying the property. For systems that violate a property, a quantitative simulation game can measure the "seriousness" of the violation, that is, how much the property has to be modified so that it is satisfied by the system. These distances can be computed in polynomial time, since the computation reduces to the value problem in limit average games with constant weights. Finally, we demonstrate how the robustness distance can be used to measure how many transmission errors are tolerated by error correcting codes.

  5. QUANTITY: An Isobaric Tag for Quantitative Glycomics

    PubMed Central

    Yang, Shuang; Wang, Meiyao; Chen, Lijun; Yin, Bojiao; Song, Guoqiang; Turko, Illarion V.; Phinney, Karen W.; Betenbaugh, Michael J.; Zhang, Hui; Li, Shuwei

    2015-01-01

    Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics - analysis of glycans at global level - however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering. PMID:26616285

  6. Quantitative Assessment of Robot-Generated Maps

    NASA Astrophysics Data System (ADS)

    Scrapper, C.; Madhavan, R.; Lakaemper, R.; Censi, A.; Godil, A.; Wagan, A.; Jacoff, A.

    Mobile robotic mapping is now considered to be a sufficiently mature field with demonstrated successes in various domains. While much progress has been made in the development of computationally efficient and consistent mapping schemes, it is still murky, at best, on how these maps can be evaluated. We are motivated by the absence of an accepted standard for quantitatively measuring the performance of robotic mapping systems against user-defined requirements. It is our belief that the development of standardized methods for quantitatively evaluating existing robotic technologies will improve the utility of mobile robots in already established application areas, such as vacuum cleaning, robot surveillance, and bomb disposal. This approach will also enable the proliferation and acceptance of such technologies in emerging markets. This chapter summarizes our preliminary efforts by bringing together the research community towards addressing this important problem which has ramifications not only from researchers' perspective but also from consumers', robot manufacturers', and developers' viewpoints.

  7. Critical Quantitative Inquiry in Context

    ERIC Educational Resources Information Center

    Stage, Frances K.; Wells, Ryan S.

    2014-01-01

    This chapter briefly traces the development of the concept of critical quantitative inquiry, provides an expanded conceptualization of the tasks of critical quantitative research, offers theoretical explanation and justification for critical research using quantitative methods, and previews the work of quantitative criticalists presented in this…

  8. Overview of quantitative susceptibility mapping.

    PubMed

    Deistung, Andreas; Schweser, Ferdinand; Reichenbach, Jürgen R

    2017-04-01

    Magnetic susceptibility describes the magnetizability of a material to an applied magnetic field and represents an important parameter in the field of MRI. With the recently introduced method of quantitative susceptibility mapping (QSM) and its conceptual extension to susceptibility tensor imaging (STI), the non-invasive assessment of this important physical quantity has become possible with MRI. Both methods solve the ill-posed inverse problem to determine the magnetic susceptibility from local magnetic fields. Whilst QSM allows the extraction of the spatial distribution of the bulk magnetic susceptibility from a single measurement, STI enables the quantification of magnetic susceptibility anisotropy, but requires multiple measurements with different orientations of the object relative to the main static magnetic field. In this review, we briefly recapitulate the fundamental theoretical foundation of QSM and STI, as well as computational strategies for the characterization of magnetic susceptibility with MRI phase data. In the second part, we provide an overview of current methodological and clinical applications of QSM with a focus on brain imaging. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Enabling Computational Technologies for Terascale Scientific Simulations

    SciTech Connect

    Ashby, S.F.

    2000-08-24

    We develop scalable algorithms and object-oriented code frameworks for terascale scientific simulations on massively parallel processors (MPPs). Our research in multigrid-based linear solvers and adaptive mesh refinement enables Laboratory programs to use MPPs to explore important physical phenomena. For example, our research aids stockpile stewardship by making practical detailed 3D simulations of radiation transport. The need to solve large linear systems arises in many applications, including radiation transport, structural dynamics, combustion, and flow in porous media. These systems result from discretizations of partial differential equations on computational meshes. Our first research objective is to develop multigrid preconditioned iterative methods for such problems and to demonstrate their scalability on MPPs. Scalability describes how total computational work grows with problem size; it measures how effectively additional resources can help solve increasingly larger problems. Many factors contribute to scalability: computer architecture, parallel implementation, and choice of algorithm. Scalable algorithms have been shown to decrease simulation times by several orders of magnitude.

  10. Enabling opportunistic resources for CMS Computing Operations

    SciTech Connect

    Hufnagel, Dick

    2015-11-19

    With the increased pressure on computing brought by the higher energy and luminosity from the LHC in Run 2, CMS Computing Operations expects to require the ability to utilize “opportunistic” resources — resources not owned by, or a priori configured for CMS — to meet peak demands. In addition to our dedicated resources we look to add computing resources from non CMS grids, cloud resources, and national supercomputing centers. CMS uses the HTCondor/glideinWMS job submission infrastructure for all its batch processing, so such resources will need to be transparently integrated into its glideinWMS pool. Bosco and parrot wrappers are used to enable access and bring the CMS environment into these non CMS resources. Here we describe our strategy to supplement our native capabilities with opportunistic resources and our experience so far using them.

  11. Enabling a New Planning and Scheduling Paradigm

    NASA Technical Reports Server (NTRS)

    Jaap, John; Davis, Elizabeth

    2004-01-01

    The Flight Projects Directorate at NASA's Marshall Space Flight Center is developing a new planning and scheduling environment and a new scheduling algorithm to enable a paradigm shift in planning and scheduling concepts. Over the past 33 years Marshall has developed and evolved a paradigm for generating payload timelines for Skylab, Spacelab, various other Shuttle payloads, and the International Space Station. The current paradigm starts by collecting the requirements, called "tasks models," from the scientists and technologists for the tasks that they want to be done. Because of shortcomings in the current modeling schema, some requirements are entered as notes. Next a cadre with knowledge of vehicle and hardware modifies these models to encompass and be compatible with the hardware model; again, notes are added when the modeling schema does not provide a better way to represent the requirements. Finally, another cadre further modifies the models to be compatible with the scheduling engine. This last cadre also submits the models to the scheduling engine or builds the timeline manually to accommodate requirements that are expressed in notes. A future paradigm would provide a scheduling engine that accepts separate science models and hardware models. The modeling schema would have the capability to represent all the requirements without resorting to notes. Furthermore, the scheduling engine would not require that the models be modified to account for the capabilities (limitations) of the scheduling engine. The enabling technology under development at Marshall has three major components. (1) A new modeling schema allows expressing all the requirements of the tasks without resorting to notes or awkward contrivances. The chosen modeling schema is both maximally expressive and easy to use. It utilizes graphics methods to show hierarchies of task constraints and networks of temporal relationships. (2) A new scheduling algorithm automatically schedules the models

  12. Enabling Communication in Emergency Response Environments

    PubMed Central

    Aldunate, Roberto G.; Schmidt, Klaus Nicholas; Herrera, Oriel

    2012-01-01

    Effective communication among first responders during response to natural and human-made large-scale catastrophes has increased tremendously during the last decade. However, most efforts to achieve a higher degree of effectiveness in communication lack synergy between the environment and the technology involved to support first responders operations. This article presents a natural and intuitive interface to support Stigmergy; or communication through the environment, based on intuitively marking and retrieving information from the environment with a pointer. A prototype of the system was built and tested in the field, however the pointing activity revealed challenges regarding accuracy due to limitations of the sensors used. The results obtained from these field tests were the basis for this research effort and will have the potential to enable communication through the environment for first responders operating in highly dynamical and inhospitable disaster relief environments. PMID:22778647

  13. Focus on atomtronics-enabled quantum technologies

    NASA Astrophysics Data System (ADS)

    Amico, Luigi; Birkl, Gerhard; Boshier, Malcolm; Kwek, Leong-Chuan

    2017-02-01

    Atomtronics is an emerging field in quantum technology that promises to realize ‘atomic circuit’ architectures exploiting ultra-cold atoms manipulated in versatile micro-optical circuits generated by laser fields of different shapes and intensities or micro-magnetic circuits known as atom chips. Although devising new applications for computation and information transfer is a defining goal of the field, atomtronics wants to enlarge the scope of quantum simulators and to access new physical regimes with novel fundamental science. With this focus issue we want to survey the state of the art of atomtronics-enabled quantum technology. We collect articles on both conceptual and applicative aspects of the field for diverse exploitations, both to extend the scope of the existing atom-based quantum devices and to devise platforms for new routes to quantum technology.

  14. Health-Enabled Smart Sensor Fusion Technology

    NASA Technical Reports Server (NTRS)

    Wang, Ray

    2012-01-01

    A process was designed to fuse data from multiple sensors in order to make a more accurate estimation of the environment and overall health in an intelligent rocket test facility (IRTF), to provide reliable, high-confidence measurements for a variety of propulsion test articles. The object of the technology is to provide sensor fusion based on a distributed architecture. Specifically, the fusion technology is intended to succeed in providing health condition monitoring capability at the intelligent transceiver, such as RF signal strength, battery reading, computing resource monitoring, and sensor data reading. The technology also provides analytic and diagnostic intelligence at the intelligent transceiver, enhancing the IEEE 1451.x-based standard for sensor data management and distributions, as well as providing appropriate communications protocols to enable complex interactions to support timely and high-quality flow of information among the system elements.

  15. Enabling MEMS technologies for communications systems

    NASA Astrophysics Data System (ADS)

    Lubecke, Victor M.; Barber, Bradley P.; Arney, Susanne

    2001-11-01

    Modern communications demands have been steadily growing not only in size, but sophistication. Phone calls over copper wires have evolved into high definition video conferencing over optical fibers, and wireless internet browsing. The technology used to meet these demands is under constant pressure to provide increased capacity, speed, and efficiency, all with reduced size and cost. Various MEMS technologies have shown great promise for meeting these challenges by extending the performance of conventional circuitry and introducing radical new systems approaches. A variety of strategic MEMS structures including various cost-effective free-space optics and high-Q RF components are described, along with related practical implementation issues. These components are rapidly becoming essential for enabling the development of progressive new communications systems technologies including all-optical networks, and low cost multi-system wireless terminals and basestations.

  16. Provision of enabling technology in professional sports.

    PubMed

    McBride, D K

    2000-06-01

    Multiple-round golf tournaments are designed intentionally to separate individuals' scores as play proceeds. Variance analyses and consideration of individual differences (vs group mean effects) for a sample of professional events confirm that 3-, 4-, and 5-round tournaments show significantly increased variability (though stable means) from first to last rounds. It is argued here that the dispersion of scores increases as play proceeds because the more physically or mentally fit players emerge and continue to perform best. Furthermore, a marginal income analysis indicates that the average gain in earnings from a one-shot improvement in score is approximately $8,000. An interpretation based on fatigue, competition, and stress supports the Professional Golf Association's claim that provision of enabling devices, like a golf cart for disabled players, is also an enhancement and is thus unfair.

  17. Enabling patients to manage altered body image.

    PubMed

    Price, Bob

    2016-12-14

    The author presented a model in the 1990s to explain altered body image, which has been used to characterise the difficulties encountered by patients who experience body change as a result of illness, injury or disability. However, it remains a challenge for nurses to establish care plans that can assist patients to manage the psychological adjustments associated with disfigurement. This article presents some simple questions to help patients narrate their psychological experiences and needs, and proposes a model of psychological change, based on the work of Kübler-Ross, to enable nurses to anticipate patient requirements that might arise at different stages of the individual's recovery and rehabilitation. Body-image rehabilitation may be protracted. Therefore, it is essential for nurses to understand what the patient is thinking and feeling throughout the rehabilitation process and which stage of psychological change the patient is working through.

  18. Enabling communication concurrency through flexible MPI endpoints

    DOE PAGES

    Dinan, James; Grant, Ryan E.; Balaji, Pavan; ...

    2014-09-23

    MPI defines a one-to-one relationship between MPI processes and ranks. This model captures many use cases effectively; however, it also limits communication concurrency and interoperability between MPI and programming models that utilize threads. Our paper describes the MPI endpoints extension, which relaxes the longstanding one-to-one relationship between MPI processes and ranks. Using endpoints, an MPI implementation can map separate communication contexts to threads, allowing them to drive communication independently. Also, endpoints enable threads to be addressable in MPI operations, enhancing interoperability between MPI and other programming models. Furthermore, these characteristics are illustrated through several examples and an empirical study thatmore » contrasts current multithreaded communication performance with the need for high degrees of communication concurrency to achieve peak communication performance.« less

  19. Science Missions Enabled by the Ares V

    NASA Technical Reports Server (NTRS)

    Worden, Simon Peter; Weiler, Edward J.

    2008-01-01

    NASA's planned heavy-lift Ares V rocket is a centerpiece of U.S. Space Exploration Policy. With approximately 30% more capacity to Trans-Lunar Injection (TLI) than the Saturn V, Ares V could also enable additional science and exploration missions currently unachievable or extremely unworkable under current launch vehicle architectures. During the spring and summer of 2008, NASA held two workshops dedicated to the discussion of these new mission concepts for the Ares V rocket. The first workshop dealt with astronomy and astrophysics, and the second dealt primarily with planetary science and exploration, but did touch on Earth science and heliophysics. We present here the summary results and outcomes of these meetings, including a discussion of specific mission concepts and ideas, as well as suggestions on design for the Ares V fairing and flight configurations that improve science return.

  20. Enabling opportunistic resources for CMS Computing Operations

    NASA Astrophysics Data System (ADS)

    Hufnagel, D.; CMS Collaboration

    2015-12-01

    With the increased pressure on computing brought by the higher energy and luminosity from the LHC in Run 2, CMS Computing Operations expects to require the ability to utilize opportunistic resources resources not owned by, or a priori configured for CMS to meet peak demands. In addition to our dedicated resources we look to add computing resources from non CMS grids, cloud resources, and national supercomputing centers. CMS uses the HTCondor/glideinWMS job submission infrastructure for all its batch processing, so such resources will need to be transparently integrated into its glideinWMS pool. Bosco and parrot wrappers are used to enable access and bring the CMS environment into these non CMS resources. Here we describe our strategy to supplement our native capabilities with opportunistic resources and our experience so far using them.

  1. Enabling communication concurrency through flexible MPI endpoints

    SciTech Connect

    Dinan, James; Grant, Ryan E.; Balaji, Pavan; Goodell, David; Miller, Douglas; Snir, Marc; Thakur, Rajeev

    2014-09-23

    MPI defines a one-to-one relationship between MPI processes and ranks. This model captures many use cases effectively; however, it also limits communication concurrency and interoperability between MPI and programming models that utilize threads. Our paper describes the MPI endpoints extension, which relaxes the longstanding one-to-one relationship between MPI processes and ranks. Using endpoints, an MPI implementation can map separate communication contexts to threads, allowing them to drive communication independently. Also, endpoints enable threads to be addressable in MPI operations, enhancing interoperability between MPI and other programming models. Furthermore, these characteristics are illustrated through several examples and an empirical study that contrasts current multithreaded communication performance with the need for high degrees of communication concurrency to achieve peak communication performance.

  2. Microdevices enabled by rarefied flow phenomena

    NASA Astrophysics Data System (ADS)

    Alexeenko, Alina A.; Strongrich, A. D.; Cofer, A. G.; Pikus, A.; Sebastiao, I. B.; Tholeti, S. S.; Shivkumar, G.

    2016-11-01

    In this paper we review emerging applications of rarefied gas dynamics for microscale sensing, actuation, power generation and thermal management. The performance of conventional fluidic devices such as pumps, combustors and heat engines drops with the decrease of characteristic length scale due to greater viscous and heat transfer losses. However, the close coupling between non-equilibrium gas, liquid and solid-state transport and electromagnetic phenomena enables unconventional micro/nanodevices. We specifically consider three distinct examples of devices with non-equilibrium gas-phase transport based on i) very large thermal gradients; ii) increased capillary forces; iii) high electric fields - all of which are generated by scaling down device size by using nano/micromanufacturing techniques.

  3. Microsystem enabled photovoltaic modules and systems

    DOEpatents

    Nielson, Gregory N; Sweatt, William C; Okandan, Murat

    2015-05-12

    A microsystem enabled photovoltaic (MEPV) module including: an absorber layer; a fixed optic layer coupled to the absorber layer; a translatable optic layer; a translation stage coupled between the fixed and translatable optic layers; and a motion processor electrically coupled to the translation stage to controls motion of the translatable optic layer relative to the fixed optic layer. The absorber layer includes an array of photovoltaic (PV) elements. The fixed optic layer includes an array of quasi-collimating (QC) micro-optical elements designed and arranged to couple incident radiation from an intermediate image formed by the translatable optic layer into one of the PV elements such that it is quasi-collimated. The translatable optic layer includes an array of focusing micro-optical elements corresponding to the QC micro-optical element array. Each focusing micro-optical element is designed to produce a quasi-telecentric intermediate image from substantially collimated radiation incident within a predetermined field of view.

  4. Laboratory Astrophysics: Enabling Scientific Discovery and Understanding

    NASA Technical Reports Server (NTRS)

    Kirby, K.

    2006-01-01

    NASA's Science Strategic Roadmap for Universe Exploration lays out a series of science objectives on a grand scale and discusses the various missions, over a wide range of wavelengths, which will enable discovery. Astronomical spectroscopy is arguably the most powerful tool we have for exploring the Universe. Experimental and theoretical studies in Laboratory Astrophysics convert "hard-won data into scientific understanding". However, the development of instruments with increasingly high spectroscopic resolution demands atomic and molecular data of unprecedented accuracy and completeness. How to meet these needs, in a time of severe budgetary constraints, poses a significant challenge both to NASA, the astronomical observers and model-builders, and the laboratory astrophysics community. I will discuss these issues, together with some recent examples of productive astronomy/lab astro collaborations.

  5. Bluetooth-enabled teleradiology: applications and complications.

    PubMed

    Hura, Angela M

    2002-01-01

    Wireless personal area networks and local area networks are becoming increasingly more prevalent in the teleradiology and telemedicine industry. Although there has been much debate about the role that Bluetooth will play in the future of wireless technology, both promoters and doubters acknowledge that Bluetooth will have an impact on networking, even if only as a "niche" product. This article provides an overview of the Bluetooth standard and highlights current and future areas of inclusion for use in a teleradiology environment. The possibilities for Bluetooth in a teleradiology environment without wires are nearly boundless and an overview of current and proposed Bluetooth-enabled radiology equipment and vendors is provided. A comparison of Bluetooth and other wireless technologies is provided, including areas of similarity and potential conflict. Bluetooth and other wireless technologies can not only peacefully coexist but also complement each other and provide enhanced teleradiology services.

  6. Enabling opportunistic resources for CMS Computing Operations

    SciTech Connect

    Hufnagel, Dirk

    2015-12-23

    With the increased pressure on computing brought by the higher energy and luminosity from the LHC in Run 2, CMS Computing Operations expects to require the ability to utilize opportunistic resources resources not owned by, or a priori configured for CMS to meet peak demands. In addition to our dedicated resources we look to add computing resources from non CMS grids, cloud resources, and national supercomputing centers. CMS uses the HTCondor/glideinWMS job submission infrastructure for all its batch processing, so such resources will need to be transparently integrated into its glideinWMS pool. Bosco and parrot wrappers are used to enable access and bring the CMS environment into these non CMS resources. Finally, we describe our strategy to supplement our native capabilities with opportunistic resources and our experience so far using them.

  7. Grid Enabled Geospatial Catalogue Web Service

    NASA Technical Reports Server (NTRS)

    Chen, Ai-Jun; Di, Li-Ping; Wei, Ya-Xing; Liu, Yang; Bui, Yu-Qi; Hu, Chau-Min; Mehrotra, Piyush

    2004-01-01

    Geospatial Catalogue Web Service is a vital service for sharing and interoperating volumes of distributed heterogeneous geospatial resources, such as data, services, applications, and their replicas over the web. Based on the Grid technology and the Open Geospatial Consortium (0GC) s Catalogue Service - Web Information Model, this paper proposes a new information model for Geospatial Catalogue Web Service, named as GCWS which can securely provides Grid-based publishing, managing and querying geospatial data and services, and the transparent access to the replica data and related services under the Grid environment. This information model integrates the information model of the Grid Replica Location Service (RLS)/Monitoring & Discovery Service (MDS) with the information model of OGC Catalogue Service (CSW), and refers to the geospatial data metadata standards from IS0 19115, FGDC and NASA EOS Core System and service metadata standards from IS0 191 19 to extend itself for expressing geospatial resources. Using GCWS, any valid geospatial user, who belongs to an authorized Virtual Organization (VO), can securely publish and manage geospatial resources, especially query on-demand data in the virtual community and get back it through the data-related services which provide functions such as subsetting, reformatting, reprojection etc. This work facilitates the geospatial resources sharing and interoperating under the Grid environment, and implements geospatial resources Grid enabled and Grid technologies geospatial enabled. It 2!so makes researcher to focus on science, 2nd not cn issues with computing ability, data locztic, processir,g and management. GCWS also is a key component for workflow-based virtual geospatial data producing.

  8. Enabling Smart Air Conditioning by Sensor Development: A Review

    PubMed Central

    Cheng, Chin-Chi; Lee, Dasheng

    2016-01-01

    The study investigates the development of sensors, in particular the use of thermo-fluidic sensors and occupancy detectors, to achieve smart operation of air conditioning systems. Smart operation refers to the operation of air conditioners by the reinforcement of interaction to achieve both thermal comfort and energy efficiency. Sensors related to thermal comfort include those of temperature, humidity, and pressure and wind velocity anemometers. Improvements in their performance in the past years have been studied by a literature survey. Traditional occupancy detection using passive infra-red (PIR) sensors and novel methodologies using smartphones and wearable sensors are both discussed. Referring to the case studies summarized in this study, air conditioning energy savings are evaluated quantitatively. Results show that energy savings of air conditioners before 2000 was 11%, and 30% after 2000 by the integration of thermo-fluidic sensors and occupancy detectors. By utilizing wearable sensing to detect the human motions, metabolic rates and related information, the energy savings can reach up to 46.3% and keep the minimum change of predicted mean vote (∆PMV→0), which means there is no compromise in thermal comfort. This enables smart air conditioning to compensate for the large variations from person to person in terms of physiological and psychological satisfaction, and find an optimal temperature for everyone in a given space. However, this tendency should be evidenced by more experimental results in the future. PMID:27916906

  9. Stratified charge rotary engine critical technology enablement. Volume 2: Appendixes

    NASA Technical Reports Server (NTRS)

    Irion, C. E.; Mount, R. E.

    1992-01-01

    This second volume of appendixes is a companion to Volume 1 of this report which summarizes results of a critical technology enablement effort with the stratified charge rotary engine (SCRE) focusing on a power section of 0.67 liters (40 cu. in.) per rotor in single and two rotor versions. The work is a continuation of prior NASA Contracts NAS3-23056 and NAS3-24628. Technical objectives are multi-fuel capability, including civil and military jet fuel and DF-2, fuel efficiency of 0.355 Lbs/BHP-Hr. at best cruise condition above 50 percent power, altitude capability of up to 10Km (33,000 ft.) cruise, 2000 hour TBO and reduced coolant heat rejection. Critical technologies for SCRE's that have the potential for competitive performance and cost in a representative light-aircraft environment were examined. Objectives were: the development and utilization of advanced analytical tools, i.e. higher speed and enhanced three dimensional combustion modeling; identification of critical technologies; development of improved instrumentation; and to isolate and quantitatively identify the contribution to performance and efficiency of critical components or subsystems. A family of four-stage third-order explicit Runge-Kutta schemes is derived that required only two locations and has desirable stability characteristics. Error control is achieved by embedding a second-order scheme within the four-stage procedure. Certain schemes are identified that are as efficient and accurate as conventional embedded schemes of comparable order and require fewer storage locations.

  10. Enabling Smart Air Conditioning by Sensor Development: A Review.

    PubMed

    Cheng, Chin-Chi; Lee, Dasheng

    2016-11-30

    The study investigates the development of sensors, in particular the use of thermo-fluidic sensors and occupancy detectors, to achieve smart operation of air conditioning systems. Smart operation refers to the operation of air conditioners by the reinforcement of interaction to achieve both thermal comfort and energy efficiency. Sensors related to thermal comfort include those of temperature, humidity, and pressure and wind velocity anemometers. Improvements in their performance in the past years have been studied by a literature survey. Traditional occupancy detection using passive infra-red (PIR) sensors and novel methodologies using smartphones and wearable sensors are both discussed. Referring to the case studies summarized in this study, air conditioning energy savings are evaluated quantitatively. Results show that energy savings of air conditioners before 2000 was 11%, and 30% after 2000 by the integration of thermo-fluidic sensors and occupancy detectors. By utilizing wearable sensing to detect the human motions, metabolic rates and related information, the energy savings can reach up to 46.3% and keep the minimum change of predicted mean vote (∆PMV→0), which means there is no compromise in thermal comfort. This enables smart air conditioning to compensate for the large variations from person to person in terms of physiological and psychological satisfaction, and find an optimal temperature for everyone in a given space. However, this tendency should be evidenced by more experimental results in the future.

  11. Stratified Charge Rotary Engine Critical Technology Enablement, Volume 1

    NASA Technical Reports Server (NTRS)

    Irion, C. E.; Mount, R. E.

    1992-01-01

    This report summarizes results of a critical technology enablement effort with the stratified charge rotary engine (SCRE) focusing on a power section of 0.67 liters (40 cu. in.) per rotor in single and two rotor versions. The work is a continuation of prior NASA Contracts NAS3-23056 and NAS3-24628. Technical objectives are multi-fuel capability, including civil and military jet fuel and DF-2, fuel efficiency of 0.355 Lbs/BHP-Hr. at best cruise condition above 50 percent power, altitude capability of up to 10Km (33,000 ft.) cruise, 2000 hour TBO and reduced coolant heat rejection. Critical technologies for SCRE's that have the potential for competitive performance and cost in a representative light-aircraft environment were examined. Objectives were: the development and utilization of advanced analytical tools, i.e. higher speed and enhanced three dimensional combustion modeling; identification of critical technologies; development of improved instrumentation, and to isolate and quantitatively identify the contribution to performance and efficiency of critical components or subsystems.

  12. Energy & Climate: Getting Quantitative

    NASA Astrophysics Data System (ADS)

    Wolfson, Richard

    2011-11-01

    A noted environmentalist claims that buying an SUV instead of a regular car is energetically equivalent to leaving your refrigerator door open for seven years. A fossil-fuel apologist argues that solar energy is a pie-in-the-sky dream promulgated by na"ive environmentalists, because there's nowhere near enough solar energy to meet humankind's energy demand. A group advocating shutdown of the Vermont Yankee nuclear plant claims that 70% of its electrical energy is lost in transmission lines. Around the world, thousands agitate for climate action, under the numerical banner ``350.'' Neither the environmentalist, the fossil-fuel apologist, the antinuclear activists, nor most of those marching under the ``350'' banner can back up their assertions with quantitative arguments. Yet questions about energy and its environmental impacts almost always require quantitative answers. Physics can help! This poster gives some cogent examples, based on the newly published 2^nd edition of the author's textbook Energy, Environment, and Climate.

  13. Primary enzyme quantitation

    DOEpatents

    Saunders, G.C.

    1982-03-04

    The disclosure relates to the quantitation of a primary enzyme concentration by utilizing a substrate for the primary enzyme labeled with a second enzyme which is an indicator enzyme. Enzyme catalysis of the substrate occurs and results in release of the indicator enzyme in an amount directly proportional to the amount of primary enzyme present. By quantifying the free indicator enzyme one determines the amount of primary enzyme present.

  14. Quantitation of signal transduction.

    PubMed

    Krauss, S; Brand, M D

    2000-12-01

    Conventional qualitative approaches to signal transduction provide powerful ways to explore the architecture and function of signaling pathways. However, at the level of the complete system, they do not fully depict the interactions between signaling and metabolic pathways and fail to give a manageable overview of the complexity that is often a feature of cellular signal transduction. Here, we introduce a quantitative experimental approach to signal transduction that helps to overcome these difficulties. We present a quantitative analysis of signal transduction during early mitogen stimulation of lymphocytes, with steady-state respiration rate as a convenient marker of metabolic stimulation. First, by inhibiting various key signaling pathways, we measure their relative importance in regulating respiration. About 80% of the input signal is conveyed via identifiable routes: 50% through pathways sensitive to inhibitors of protein kinase C and MAP kinase and 30% through pathways sensitive to an inhibitor of calcineurin. Second, we quantify how each of these pathways differentially stimulates functional units of reactions that produce and consume a key intermediate in respiration: the mitochondrial membrane potential. Both the PKC and calcineurin routes stimulate consumption more strongly than production, whereas the unidentified signaling routes stimulate production more than consumption, leading to no change in membrane potential despite increased respiration rate. The approach allows a quantitative description of the relative importance of signal transduction pathways and the routes by which they activate a specific cellular process. It should be widely applicable.

  15. Enabling Technologies for Ceramic Hot Section Components

    SciTech Connect

    Venkat Vedula; Tania Bhatia

    2009-04-30

    Silicon-based ceramics are attractive materials for use in gas turbine engine hot sections due to their high temperature mechanical and physical properties as well as lower density than metals. The advantages of utilizing ceramic hot section components include weight reduction, and improved efficiency as well as enhanced power output and lower emissions as a result of reducing or eliminating cooling. Potential gas turbine ceramic components for industrial, commercial and/or military high temperature turbine applications include combustor liners, vanes, rotors, and shrouds. These components require materials that can withstand high temperatures and pressures for long duration under steam-rich environments. For Navy applications, ceramic hot section components have the potential to increase the operation range. The amount of weight reduced by utilizing a lighter gas turbine can be used to increase fuel storage capacity while a more efficient gas turbine consumes less fuel. Both improvements enable a longer operation range for Navy ships and aircraft. Ceramic hot section components will also be beneficial to the Navy's Growth Joint Strike Fighter (JSF) and VAATE (Versatile Affordable Advanced Turbine Engines) initiatives in terms of reduced weight, cooling air savings, and capability/cost index (CCI). For DOE applications, ceramic hot section components provide an avenue to achieve low emissions while improving efficiency. Combustors made of ceramic material can withstand higher wall temperatures and require less cooling air. Ability of the ceramics to withstand high temperatures enables novel combustor designs that have reduced NO{sub x}, smoke and CO levels. In the turbine section, ceramic vanes and blades do not require sophisticated cooling schemes currently used for metal components. The saved cooling air could be used to further improve efficiency and power output. The objectives of this contract were to develop technologies critical for ceramic hot section

  16. Technology-enabled Airborne Spacing and Merging

    NASA Technical Reports Server (NTRS)

    Hull, James; Barmore, Bryan; Abbott, Tetence

    2005-01-01

    Over the last several decades, advances in airborne and groundside technologies have allowed the Air Traffic Service Provider (ATSP) to give safer and more efficient service, reduce workload and frequency congestion, and help accommodate a critically escalating traffic volume. These new technologies have included advanced radar displays, and data and communication automation to name a few. In step with such advances, NASA Langley is developing a precision spacing concept designed to increase runway throughput by enabling the flight crews to manage their inter-arrival spacing from TRACON entry to the runway threshold. This concept is being developed as part of NASA s Distributed Air/Ground Traffic Management (DAG-TM) project under the Advanced Air Transportation Technologies Program. Precision spacing is enabled by Automatic Dependent Surveillance-Broadcast (ADS-B), which provides air-to-air data exchange including position and velocity reports; real-time wind information and other necessary data. On the flight deck, a research prototype system called Airborne Merging and Spacing for Terminal Arrivals (AMSTAR) processes this information and provides speed guidance to the flight crew to achieve the desired inter-arrival spacing. AMSTAR is designed to support current ATC operations, provide operationally acceptable system-wide increases in approach spacing performance and increase runway throughput through system stability, predictability and precision spacing. This paper describes problems and costs associated with an imprecise arrival flow. It also discusses methods by which Air Traffic Controllers achieve and maintain an optimum interarrival interval, and explores means by which AMSTAR can assist in this pursuit. AMSTAR is an extension of NASA s previous work on in-trail spacing that was successfully demonstrated in a flight evaluation at Chicago O Hare International Airport in September 2002. In addition to providing for precision inter-arrival spacing, AMSTAR

  17. Enabling Wireless Avionics Intra-Communications

    NASA Technical Reports Server (NTRS)

    Torres, Omar; Nguyen, Truong; Mackenzie, Anne

    2016-01-01

    The Electromagnetics and Sensors Branch of NASA Langley Research Center (LaRC) is investigating the potential of an all-wireless aircraft as part of the ECON (Efficient Reconfigurable Cockpit Design and Fleet Operations using Software Intensive, Networked and Wireless Enabled Architecture) seedling proposal, which is funded by the Convergent Aeronautics Solutions (CAS) project, Transformative Aeronautics Concepts (TAC) program, and NASA Aeronautics Research Institute (NARI). The project consists of a brief effort carried out by a small team in the Electromagnetic Environment Effects (E3) laboratory with the intention of exposing some of the challenges faced by a wireless communication system inside the reflective cavity of an aircraft and to explore potential solutions that take advantage of that environment for constructive gain. The research effort was named EWAIC for "Enabling Wireless Aircraft Intra-communications." The E3 laboratory is a research facility that includes three electromagnetic reverberation chambers and equipment that allow testing and generation of test data for the investigation of wireless systems in reflective environments. Using these chambers, the EWAIC team developed a set of tests and setups that allow the intentional variation of intensity of a multipath field to reproduce the environment of the various bays and cabins of large transport aircraft. This setup, in essence, simulates an aircraft environment that allows the investigation and testing of wireless communication protocols that can effectively be used as a tool to mitigate some of the risks inherent to an aircraft wireless system for critical functions. In addition, the EWAIC team initiated the development of a computational modeling tool to illustrate the propagation of EM waves inside the reflective cabins and bays of aircraft and to obtain quantifiable information regarding the degradation of signals in aircraft subassemblies. The nose landing gear of a UAV CAD model was used

  18. Willing and Enabled: The Academic Outcomes of a Tertiary Enabling Program in Regional Australia

    ERIC Educational Resources Information Center

    Andrewartha, Lisa; Harvey, Andrew

    2014-01-01

    This paper examines the achievement levels of students undertaking the Tertiary Enabling Program (TEP) at La Trobe University. The TEP is an alternative pathway program that traverses multiple institutions, campuses, and disciplinary areas, and is designed to prepare a diverse student cohort for tertiary study. The Program integrates several…

  19. Agile SE Enablers and Quantification Project: Identification, Characterization, and Evaluation Criteria for Systems Engineering Agile Enablers

    DTIC Science & Technology

    2015-01-16

    Development of the evaluation white paper .......................................................................................... 7 3 References...enablers as they are identified. The ultimate result of the process is an evaluation white paper supporting one of three decisions: 1. not likely to...be effective, 2. possibly suitable but more research is needed, or 3. definitely suitable and expedited transition is recommended. This paper

  20. Nanomaterials Enabled Dye-sensitized Solar Cells

    NASA Astrophysics Data System (ADS)

    Dong, Pei

    Dye sensitized solar cells (DSCs), as the third generation of solar cells, have attracted tremendous attention for their unique properties. The semi-transparent nature, low-cost, environmental friendliness, and convenient manufacturing conditions of this generation of solar cells are promising aspects of DSCs that make them competitive in their future applications. However, much improvement in many aspects of DSCs' is required for the realization of its full potential. In this thesis, various nanomaterials, such as graphene, multi wall carbon nanotubes, vertically aligned single wall carbon nanotubes, hybrid structures and etc, have been used to improve the performance of DSCs. First, the application of graphene covered metal grids as transparent conductive electrodes in DSCs is explored. It is demonstrated that the mechanical properties of these flexible hybrid transparent electrodes, in both bending and stretching tests, are better than their oxide-based counter parts. Moreover, different kinds of carbon nanotubes, for instance vertically aligned single wall carbon nanotubes, have been used as a replacement for traditional platinum counter electrodes, in both iodine electrolyte, and sulfide-electrolyte. Further, a flexible, seamlessly connected, 3-dimensional vertically-aligned few wall carbon nanotubes graphene hybrid structures on Ni foil as DSCs' counter electrodes improve their efficiency significantly. All these nanomaterials enabled DSCs architectures achieve a comparable or better performance than standard brittle platinum/fluorine doped tin oxide combination. The large surface area of such nanomaterials in addition to the high electrical conductivity and their mechanical robustness provides a platform for significant enhancements in DSCs' performance.

  1. Ion Implant Enabled 2x Lithography

    NASA Astrophysics Data System (ADS)

    Martin, Patrick M.; Godet, Ludovic; Cheung, Andrew; de Cock, Gael; Hatem, Chris

    2011-01-01

    Ion implantation has many applications in microelectronics beyond doping. The broad range of species available combined with the ability to precisely control dose, angle, and energy offers compelling advantages for use in precision material modification. The application to lithography has been reported elsewhere. Integrating ion implantation into the lithography process enables scaling the feature size requirements beyond the 15 nm node with a simplified double patterning sequence. In addition, ion implant may be used to remove line edge roughness, providing tremendous advantages to meet extreme lithography imaging requirements and provide additional device stability. We examine several species (e.g. Si, Ar, etc.) and the effect of energy and impact angle on several commercially available 193 nm immersion photoresists using a Varian VIISta® single wafer high current ion implanter. The treated photoresist will be evaluated for stability in an integrated double patterning application with ion implant used to freeze the primary image. We report on critical dimension impact, pattern integrity, optical property modification, and adhesion. We analyze the impact of line edge roughness improvement beyond the work of C. Struck including the power spectral distribution. TGA and FTIR Spectroscopy results for the implanted photoresist materials will also be included.

  2. Smart Sensors Enable Smart Air Conditioning Control

    PubMed Central

    Cheng, Chin-Chi; Lee, Dasheng

    2014-01-01

    In this study, mobile phones, wearable devices, temperature and human motion detectors are integrated as smart sensors for enabling smart air conditioning control. Smart sensors obtain feedback, especially occupants' information, from mobile phones and wearable devices placed on human body. The information can be used to adjust air conditioners in advance according to humans' intentions, in so-called intention causing control. Experimental results show that the indoor temperature can be controlled accurately with errors of less than ±0.1 °C. Rapid cool down can be achieved within 2 min to the optimized indoor capacity after occupants enter a room. It's also noted that within two-hour operation the total compressor output of the smart air conditioner is 48.4% less than that of the one using On-Off control. The smart air conditioner with wearable devices could detect the human temperature and activity during sleep to determine the sleeping state and adjusting the sleeping function flexibly. The sleeping function optimized by the smart air conditioner with wearable devices could reduce the energy consumption up to 46.9% and keep the human health. The presented smart air conditioner could provide a comfortable environment and achieve the goals of energy conservation and environmental protection. PMID:24961213

  3. Enabling electroweak baryogenesis through dark matter

    NASA Astrophysics Data System (ADS)

    Lewicki, Marek; Rindler-Daller, Tanja; Wells, James D.

    2016-06-01

    We study the impact on electroweak baryogenesis from a swifter cosmological expansion induced by dark matter. We detail the experimental bounds that one can place on models that realize it, and we investigate the modifications of these bounds that result from a non-standard cosmological history. The modifications can be sizeable if the expansion rate of the Universe increases by several orders of magnitude. We illustrate the impact through the example of scalar field dark matter, which can alter the cosmological history enough to enable a strong-enough first-order phase transition in the Standard Model when it is supplemented by a dimension six operator directly modifying the Higgs boson potential. We show that due to the modified cosmological history, electroweak baryogenesis can be realized, while keeping deviations of the triple Higgs coupling below HL-LHC sensitivies. The required scale of new physics to effectuate a strong-enough first order phase transition can change by as much as twenty percent as the expansion rate increases by six orders of magnitude.

  4. Bandwidth Enabled Flight Operations: Examining the Possibilities

    NASA Technical Reports Server (NTRS)

    Pisanich, Greg; Renema, Fritz; Clancy, Dan (Technical Monitor)

    2002-01-01

    The Bandwidth Enabled Flight Operations project is a research effort at the NASA Ames Research Center to investigate the use of satellite communications to improve aviation safety and capacity. This project is a follow on to the AeroSAPIENT Project, which demonstrated methods for transmitting high bandwidth data in various configurations. For this research, we set a goal to nominally use only 10 percent of the available bandwidth demonstrated by AeroSAPIENT or projected by near-term technology advances. This paper describes the results of our research, including available satellite bandwidth, commercial and research efforts to provide these services, and some of the limiting factors inherent with this communications medium. It also describes our investigation into the needs of the stakeholders (Airlines, Pilots, Cabin Crews, ATC, Maintenance, etc). The paper also describes our development of low-cost networked flight deck and airline operations center simulations that were used to demonstrate two application areas: Providing real time weather information to the commercial flight deck, and enhanced crew monitoring and control for airline operations centers.

  5. Smart sensors enable smart air conditioning control.

    PubMed

    Cheng, Chin-Chi; Lee, Dasheng

    2014-06-24

    In this study, mobile phones, wearable devices, temperature and human motion detectors are integrated as smart sensors for enabling smart air conditioning control. Smart sensors obtain feedback, especially occupants' information, from mobile phones and wearable devices placed on human body. The information can be used to adjust air conditioners in advance according to humans' intentions, in so-called intention causing control. Experimental results show that the indoor temperature can be controlled accurately with errors of less than ±0.1 °C. Rapid cool down can be achieved within 2 min to the optimized indoor capacity after occupants enter a room. It's also noted that within two-hour operation the total compressor output of the smart air conditioner is 48.4% less than that of the one using On-Off control. The smart air conditioner with wearable devices could detect the human temperature and activity during sleep to determine the sleeping state and adjusting the sleeping function flexibly. The sleeping function optimized by the smart air conditioner with wearable devices could reduce the energy consumption up to 46.9% and keep the human health. The presented smart air conditioner could provide a comfortable environment and achieve the goals of energy conservation and environmental protection.

  6. Nanocrystal-enabled solid state bonding.

    SciTech Connect

    San Diego State University, San Diego, CA; Puskar, Joseph David; Tikare, Veena; Garcia Cardona, Cristina; Reece, Mark; Brewer, Luke N.; Holm, Elizabeth Ann

    2010-10-01

    In this project, we performed a preliminary set of sintering experiments to examine nanocrystal-enabled diffusion bonding (NEDB) in Ag-on-Ag and Cu-on-Cu using Ag nanoparticles. The experimental test matrix included the effects of material system, temperature, pressure, and particle size. The nanoparticle compacts were bonded between plates using a customized hot press, tested in shear, and examined post mortem using microscopy techniques. NEDB was found to be a feasible mechanism for low-temperature, low-pressure, solid-state bonding of like materials, creating bonded interfaces that were able to support substantial loads. The maximum supported shear strength varied substantially within sample cohorts due to variation in bonded area; however, systematic variation with fabrication conditions was also observed. Mesoscale sintering simulations were performed in order to understand whether sintering models can aid in understanding the NEDB process. A pressure-assisted sintering model was incorporated into the SPPARKS kinetic Monte Carlo sintering code. Results reproduce most of the qualitative behavior observed in experiments, indicating that simulation can augment experiments during the development of the NEDB process. Because NEDB offers a promising route to low-temperature, low-pressure, solid-state bonding, we recommend further research and development with a goal of devising new NEDB bonding processes to support Sandia's customers.

  7. Water: A Critical Material Enabling Space Exploration

    NASA Technical Reports Server (NTRS)

    Pickering, Karen D.

    2014-01-01

    Water is one of the most critical materials in human spaceflight. The availability of water defines the duration of a space mission; the volume of water required for a long-duration space mission becomes too large, heavy, and expensive for launch vehicles to carry. Since the mission duration is limited by the amount of water a space vehicle can carry, the capability to recycle water enables space exploration. In addition, water management in microgravity impacts spaceflight in other respects, such as the recent emergency termination of a spacewalk caused by free water in an astronaut's spacesuit helmet. A variety of separation technologies are used onboard spacecraft to ensure that water is always available for use, and meets the stringent water quality required for human space exploration. These separation technologies are often adapted for use in a microgravity environment, where water behaves in unique ways. The use of distillation, membrane processes, ion exchange and granular activated carbon will be reviewed. Examples of microgravity effects on operations will also be presented. A roadmap for future technologies, needed to supply water resources for the exploration of Mars, will also be reviewed.

  8. Glass ceramic ZERODUR enabling nanometer precision

    NASA Astrophysics Data System (ADS)

    Jedamzik, Ralf; Kunisch, Clemens; Nieder, Johannes; Westerhoff, Thomas

    2014-03-01

    The IC Lithography roadmap foresees manufacturing of devices with critical dimension of < 20 nm. Overlay specification of single digit nanometer asking for nanometer positioning accuracy requiring sub nanometer position measurement accuracy. The glass ceramic ZERODUR® is a well-established material in critical components of microlithography wafer stepper and offered with an extremely low coefficient of thermal expansion (CTE), the tightest tolerance available on market. SCHOTT is continuously improving manufacturing processes and it's method to measure and characterize the CTE behavior of ZERODUR® to full fill the ever tighter CTE specification for wafer stepper components. In this paper we present the ZERODUR® Lithography Roadmap on the CTE metrology and tolerance. Additionally, simulation calculations based on a physical model are presented predicting the long term CTE behavior of ZERODUR® components to optimize dimensional stability of precision positioning devices. CTE data of several low thermal expansion materials are compared regarding their temperature dependence between - 50°C and + 100°C. ZERODUR® TAILORED 22°C is full filling the tight CTE tolerance of +/- 10 ppb / K within the broadest temperature interval compared to all other materials of this investigation. The data presented in this paper explicitly demonstrates the capability of ZERODUR® to enable the nanometer precision required for future generation of lithography equipment and processes.

  9. Enabler for the agile virtual enterprise

    NASA Astrophysics Data System (ADS)

    Fuerst, Karl; Schmidt, Thomas; Wippel, Gerald

    2001-10-01

    In this presentation, a new approach for a flexible low-cost Internet extended enterprise (project FLoCI-EE) will be presented. FLoCI-EE is a project in the fifth framework program of the European commission with 8 partners from 4 countries, which started in January 2001 and will be finished in December 2003. The main objective of FLoCI-EE is the development of a software prototype, which enables flexible enterprise cooperation with the aim to design, manufacture and sell products commonly, independent of enterprise borderlines. The needed IT-support includes functions of product data management (PDM), enterprise resource planning (ERP), supply chain management (SCM) and customer relationship management (CRM). Especially for small and medium sized enterprises, existing solutions are too expensive and inflexible to be of use under current turbulent market conditions. The second part of this paper covers the item Web Services, because in the role-specific support approach of FLoCI-EE, there are user- interface-components, which are tailored for specific roles in an enterprise. These components integrate automatically the services of the so-called basic-components, and the externally offered Web Services like UDDI.

  10. Barriers and enablers to academic health leadership.

    PubMed

    Bharwani, Aleem; Kline, Theresa; Patterson, Margaret; Craighead, Peter

    2017-02-06

    Purpose This study sought to identify the barriers and enablers to leadership enactment in academic health-care settings. Design/methodology/approach Semi-structured interviews ( n = 77) with programme stakeholders (medical school trainees, university leaders, clinical leaders, medical scientists and directors external to the medical school) were conducted, and the responses content-analysed. Findings Both contextual and individual factors were identified as playing a role in affecting academic health leadership enactment that has an impact on programme development, success and maintenance. Contextual factors included sufficient resources allocated to the programme, opportunities for learners to practise leadership skills, a competent team around the leader once that person is in place, clear expectations for the leader and a culture that fosters open communication. Contextual barriers included highly bureaucratic structures, fear-of-failure and non-trusting cultures and inappropriate performance systems. Programmes were advised to select participants based on self-awareness, strong communication skills and an innovative thinking style. Filling specific knowledge and skill gaps, particularly for those not trained in medical school, was viewed as essential. Ineffective decision-making styles and tendencies to get involved in day-to-day activities were barriers to the development of academic health leaders. Originality/value Programmes designed to develop academic health-care leaders will be most effective if they develop leadership at all levels; ensure that the organisation's culture, structure and processes reinforce positive leadership practices; and recognise the critical role of teams in supporting its leaders.

  11. Enabling scientific workflows in virtual reality

    USGS Publications Warehouse

    Kreylos, O.; Bawden, G.; Bernardin, T.; Billen, M.I.; Cowgill, E.S.; Gold, R.D.; Hamann, B.; Jadamec, M.; Kellogg, L.H.; Staadt, O.G.; Sumner, D.Y.

    2006-01-01

    To advance research and improve the scientific return on data collection and interpretation efforts in the geosciences, we have developed methods of interactive visualization, with a special focus on immersive virtual reality (VR) environments. Earth sciences employ a strongly visual approach to the measurement and analysis of geologic data due to the spatial and temporal scales over which such data ranges, As observations and simulations increase in size and complexity, the Earth sciences are challenged to manage and interpret increasing amounts of data. Reaping the full intellectual benefits of immersive VR requires us to tailor exploratory approaches to scientific problems. These applications build on the visualization method's strengths, using both 3D perception and interaction with data and models, to take advantage of the skills and training of the geological scientists exploring their data in the VR environment. This interactive approach has enabled us to develop a suite of tools that are adaptable to a range of problems in the geosciences and beyond. Copyright ?? 2008 by the Association for Computing Machinery, Inc.

  12. Survey of Enabling Technologies for CAPS

    NASA Technical Reports Server (NTRS)

    Antol, Jeffrey; Mazanek, Daniel D.; Koons, Robert H.

    2005-01-01

    The enabling technologies required for the development of a viable Comet/Asteroid Protection System (CAPS) can be divided into two principal areas: detection and deflection/orbit modification. With the proper funding levels, many of the technologies needed to support a CAPS architecture could be achievable within the next 15 to 20 years. In fact, many advanced detection technologies are currently in development for future in-space telescope systems such as the James Webb Space Telescope (JWST), formerly known as the Next Generation Space Telescope. It is anticipated that many of the JWST technologies would be available for application for CAPS detection concepts. Deflection/orbit modification technologies are also currently being studied as part of advanced power and propulsion research. However, many of these technologies, such as extremely high-output power systems, advanced propulsion, heat rejection, and directed energy systems, would likely be farther term in availability than many of the detection technologies. Discussed subsequently is a preliminary examination of the main technologies that have been identified as being essential to providing the element functionality defined during the CAPS conceptual study. The detailed requirements for many of the technology areas are still unknown, and many additional technologies will be identified as future in-depth studies are conducted in this area.

  13. 'Slings' enable neutrophil rolling at high shear.

    PubMed

    Sundd, Prithu; Gutierrez, Edgar; Koltsova, Ekaterina K; Kuwano, Yoshihiro; Fukuda, Satoru; Pospieszalska, Maria K; Groisman, Alex; Ley, Klaus

    2012-08-16

    Most leukocytes can roll along the walls of venules at low shear stress (1 dyn cm−2), but neutrophils have the ability to roll at tenfold higher shear stress in microvessels in vivo. The mechanisms involved in this shear-resistant rolling are known to involve cell flattening and pulling of long membrane tethers at the rear. Here we show that these long tethers do not retract as postulated, but instead persist and appear as 'slings' at the front of rolling cells. We demonstrate slings in a model of acute inflammation in vivo and on P-selectin in vitro, where P-selectin-glycoprotein-ligand-1 (PSGL-1) is found in discrete sticky patches whereas LFA-1 is expressed over the entire length on slings. As neutrophils roll forward, slings wrap around the rolling cells and undergo a step-wise peeling from the P-selectin substrate enabled by the failure of PSGL-1 patches under hydrodynamic forces. The 'step-wise peeling of slings' is distinct from the 'pulling of tethers' reported previously. Each sling effectively lays out a cell-autonomous adhesive substrate in front of neutrophils rolling at high shear stress during inflammation.

  14. Graphene-Enabled Electrodes for Electrocardiogram Monitoring

    PubMed Central

    Celik, Numan; Manivannan, Nadarajah; Strudwick, Andrew; Balachandran, Wamadeva

    2016-01-01

    The unique parameters of graphene (GN)—notably its considerable electron mobility, high surface area, and electrical conductivity—are bringing extensive attention into the wearable technologies. This work presents a novel graphene-based electrode for acquisition of electrocardiogram (ECG). The proposed electrode was fabricated by coating GN on top of a metallic layer of a Ag/AgCl electrode using a chemical vapour deposition (CVD) technique. To investigate the performance of the fabricated GN-based electrode, two types of electrodes were fabricated with different sizes to conduct the signal qualities and the skin-electrode contact impedance measurements. Performances of the GN-enabled electrodes were compared to the conventional Ag/AgCl electrodes in terms of ECG signal quality, skin–electrode contact impedance, signal-to-noise ratio (SNR), and response time. Experimental results showed the proposed GN-based electrodes produced better ECG signals, higher SNR (improved by 8%), and lower contact impedance (improved by 78%) values than conventional ECG electrodes. PMID:28335284

  15. Quantitative Genetics in the Genomics Era

    PubMed Central

    Hill, William G.

    2012-01-01

    The genetic analysis of quantitative or complex traits has been based mainly on statistical quantities such as genetic variances and heritability. These analyses continue to be developed, for example in studies of natural populations. Genomic methods are having an impact on progress and prospects. Actual relationships of individuals can be estimated enabling novel quantitative analyses. Increasing precision of linkage mapping is feasible with dense marker panels and designed stocks allowing multiple generations of recombination, and large SNP panels enable the use of genome wide association analysis utilising historical recombination. Whilst such analyses are identifying many loci for disease genes and traits such as height, typically each individually contributes a small amount of the variation. Only by fitting all SNPs without regard to significance can a high proportion be accounted for, so a classical polygenic model with near infinitesimally small effects remains a useful one. Theory indicates that a high proportion of variants will have low minor allele frequency, making detection difficult. Genomic selection, based on simultaneously fitting very dense markers and incorporating these with phenotypic data in breeding value prediction is revolutionising breeding programmes in agriculture and has a major potential role in human disease prediction. PMID:23115521

  16. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  17. Enabling performance skills: Assessment in engineering education

    NASA Astrophysics Data System (ADS)

    Ferrone, Jenny Kristina

    Current reform in engineering education is part of a national trend emphasizing student learning as well as accountability in instruction. Assessing student performance to demonstrate accountability has become a necessity in academia. In newly adopted criterion proposed by the Accreditation Board for Engineering and Technology (ABET), undergraduates are expected to demonstrate proficiency in outcomes considered essential for graduating engineers. The case study was designed as a formative evaluation of freshman engineering students to assess the perceived effectiveness of performance skills in a design laboratory environment. The mixed methodology used both quantitative and qualitative approaches to assess students' performance skills and congruency among the respondents, based on individual, team, and faculty perceptions of team effectiveness in three ABET areas: Communications Skills. Design Skills, and Teamwork. The findings of the research were used to address future use of the assessment tool and process. The results of the study found statistically significant differences in perceptions of Teamwork Skills (p < .05). When groups composed of students and professors were compared, professors were less likely to perceive student's teaming skills as effective. The study indicated the need to: (1) improve non-technical performance skills, such as teamwork, among freshman engineering students; (2) incorporate feedback into the learning process; (3) strengthen the assessment process with a follow-up plan that specifically targets performance skill deficiencies, and (4) integrate the assessment instrument and practice with ongoing curriculum development. The findings generated by this study provides engineering departments engaged in assessment activity, opportunity to reflect, refine, and develop their programs as it continues. It also extends research on ABET competencies of engineering students in an under-investigated topic of factors correlated with team

  18. Enabling a Scientific Cloud Marketplace: VGL (Invited)

    NASA Astrophysics Data System (ADS)

    Fraser, R.; Woodcock, R.; Wyborn, L. A.; Vote, J.; Rankine, T.; Cox, S. J.

    2013-12-01

    The Virtual Geophysics Laboratory (VGL) provides a flexible, web based environment where researchers can browse data and use a variety of scientific software packaged into tool kits that run in the Cloud. Both data and tool kits are published by multiple researchers and registered with the VGL infrastructure forming a data and application marketplace. The VGL provides the basic work flow of Discovery and Access to the disparate data sources and a Library for tool kits and scripting to drive the scientific codes. Computation is then performed on the Research or Commercial Clouds. Provenance information is collected throughout the work flow and can be published alongside the results allowing for experiment comparison and sharing with other researchers. VGL's "mix and match" approach to data, computational resources and scientific codes, enables a dynamic approach to scientific collaboration. VGL allows scientists to publish their specific contribution, be it data, code, compute or work flow, knowing the VGL framework will provide other components needed for a complete application. Other scientists can choose the pieces that suit them best to assemble an experiment. The coarse grain workflow of the VGL framework combined with the flexibility of the scripting library and computational toolkits allows for significant customisation and sharing amongst the community. The VGL utilises the cloud computational and storage resources from the Australian academic research cloud provided by the NeCTAR initiative and a large variety of data accessible from national and state agencies via the Spatial Information Services Stack (SISS - http://siss.auscope.org). VGL v1.2 screenshot - http://vgl.auscope.org

  19. New Catalog of Resources Enables Paleogeosciences Research

    NASA Astrophysics Data System (ADS)

    Lingo, R. C.; Horlick, K. A.; Anderson, D. M.

    2014-12-01

    The 21st century promises a new era for scientists of all disciplines, the age where cyber infrastructure enables research and education and fuels discovery. EarthCube is a working community of over 2,500 scientists and students of many Earth Science disciplines who are looking to build bridges between disciplines. The EarthCube initiative will create a digital infrastructure that connects databases, software, and repositories. A catalog of resources (databases, software, repositories) has been produced by the Research Coordination Network for Paleogeosciences to improve the discoverability of resources. The Catalog is currently made available within the larger-scope CINERGI geosciences portal (http://hydro10.sdsc.edu/geoportal/catalog/main/home.page). Other distribution points and web services are planned, using linked data, content services for the web, and XML descriptions that can be harvested using metadata protocols. The databases provide searchable interfaces to find data sets that would otherwise remain dark data, hidden in drawers and on personal computers. The software will be described in catalog entries so just one click will lead users to methods and analytical tools that many geoscientists were unaware of. The repositories listed in the Paleogeosciences Catalog contain physical samples found all across the globe, from natural history museums to the basements of university buildings. EarthCube has over 250 databases, 300 software systems, and 200 repositories which will grow in the coming year. When completed, geoscientists across the world will be connected into a productive workflow for managing, sharing, and exploring geoscience data and information that expedites collaboration and innovation within the paleogeosciences, potentially bringing about new interdisciplinary discoveries.

  20. Web enabled data management with DPM & LFC

    NASA Astrophysics Data System (ADS)

    Alvarez Ayllon, Alejandro; Beche, Alexandre; Furano, Fabrizio; Hellmich, Martin; Keeble and, Oliver; Brito Da Rocha, Ricardo

    2012-12-01

    The Disk Pool Manager (DPM) and LCG File Catalog (LFC) are two grid data management components currently used in production with more than 240 endpoints. Together with a set of grid client tools they give the users a unified view of their data, hiding most details concerning data location and access. Recently we've put a lot of effort in developing a reliable and high performance HTTP/WebDAV frontend to both our grid catalog and storage components, exposing the existing functionality to users accessing the services via standard clients - e.g. web browsers, curl - present in all operating systems, giving users a simple and straight-forward way of interaction. In addition, as other relevant grid storage components (like dCache) expose their data using the same protocol, for the first time we had the opportunity of attempting a unified view of all grid storage using HTTP. We describe the HTTP redirection mechanism used to integrate the grid catalog(s) with the multiple storage components, including details on some assumptions made to allow integration with other implementations. We describe the way we hide the details regarding site availability or catalog inconsistencies by switching the standard HTTP client automatically between multiple replicas. We also present measurements of access performance, and the relevant factors regarding replica selection - current throughput and load, geographic proximity, etc. Finally, we report on some additional work done to have this system as a viable alternative to GridFTP, providing multi-stream transfers and exploiting some additional features of WebDAV to enable third party copies - essential for managing data movements between storage systems - with equivalent performance.

  1. "Nanotechnology Enabled Advanced Industrial Heat Transfer Fluids"

    SciTech Connect

    Dr. Ganesh Skandan; Dr. Amit Singhal; Mr. Kenneth Eberts; Mr. Damian Sobrevilla; Prof. Jerry Shan; Stephen Tse; Toby Rossmann

    2008-06-12

    ABSTRACT Nanotechnology Enabled Advanced industrial Heat Transfer Fluids” Improving the efficiency of Industrial Heat Exchangers offers a great opportunity to improve overall process efficiencies in diverse industries such as pharmaceutical, materials manufacturing and food processing. The higher efficiencies can come in part from improved heat transfer during both cooling and heating of the material being processed. Additionally, there is great interest in enhancing the performance and reducing the weight of heat exchangers used in automotives in order to increase fuel efficiency. The goal of the Phase I program was to develop nanoparticle containing heat transfer fluids (e.g., antifreeze, water, silicone and hydrocarbon-based oils) that are used in transportation and in the chemical industry for heating, cooling and recovering waste heat. Much work has been done to date at investigating the potential use of nanoparticle-enhanced thermal fluids to improve heat transfer in heat exchangers. In most cases the effect in a commercial heat transfer fluid has been marginal at best. In the Phase I work, we demonstrated that the thermal conductivity, and hence heat transfer, of a fluid containing nanoparticles can be dramatically increased when subjected to an external influence. The increase in thermal conductivity was significantly larger than what is predicted by commonly used thermal models for two-phase materials. Additionally, the surface of the nanoparticles was engineered so as to have a minimal influence on the viscosity of the fluid. As a result, a nanoparticle-laden fluid was successfully developed that can lead to enhanced heat transfer in both industrial and automotive heat exchangers

  2. Quantitative Imaging in Cancer Evolution and Ecology

    PubMed Central

    Grove, Olya; Gillies, Robert J.

    2013-01-01

    Cancer therapy, even when highly targeted, typically fails because of the remarkable capacity of malignant cells to evolve effective adaptations. These evolutionary dynamics are both a cause and a consequence of cancer system heterogeneity at many scales, ranging from genetic properties of individual cells to large-scale imaging features. Tumors of the same organ and cell type can have remarkably diverse appearances in different patients. Furthermore, even within a single tumor, marked variations in imaging features, such as necrosis or contrast enhancement, are common. Similar spatial variations recently have been reported in genetic profiles. Radiologic heterogeneity within tumors is usually governed by variations in blood flow, whereas genetic heterogeneity is typically ascribed to random mutations. However, evolution within tumors, as in all living systems, is subject to Darwinian principles; thus, it is governed by predictable and reproducible interactions between environmental selection forces and cell phenotype (not genotype). This link between regional variations in environmental properties and cellular adaptive strategies may permit clinical imaging to be used to assess and monitor intratumoral evolution in individual patients. This approach is enabled by new methods that extract, report, and analyze quantitative, reproducible, and mineable clinical imaging data. However, most current quantitative metrics lack spatialness, expressing quantitative radiologic features as a single value for a region of interest encompassing the whole tumor. In contrast, spatially explicit image analysis recognizes that tumors are heterogeneous but not well mixed and defines regionally distinct habitats, some of which appear to harbor tumor populations that are more aggressive and less treatable than others. By identifying regional variations in key environmental selection forces and evidence of cellular adaptation, clinical imaging can enable us to define intratumoral

  3. Quantitative Ultrasound for Nondestructive Characterization of Engineered Tissues and Biomaterials.

    PubMed

    Dalecki, Diane; Mercado, Karla P; Hocking, Denise C

    2016-03-01

    Non-invasive, non-destructive technologies for imaging and quantitatively monitoring the development of artificial tissues are critical for the advancement of tissue engineering. Current standard techniques for evaluating engineered tissues, including histology, biochemical assays and mechanical testing, are destructive approaches. Ultrasound is emerging as a valuable tool for imaging and quantitatively monitoring the properties of engineered tissues and biomaterials longitudinally during fabrication and post-implantation. Ultrasound techniques are rapid, non-invasive, non-destructive and can be easily integrated into sterile environments necessary for tissue engineering. Furthermore, high-frequency quantitative ultrasound techniques can enable volumetric characterization of the structural, biological, and mechanical properties of engineered tissues during fabrication and post-implantation. This review provides an overview of ultrasound imaging, quantitative ultrasound techniques, and elastography, with representative examples of applications of these ultrasound-based techniques to the field of tissue engineering.

  4. Hydrologic Prediction Through Earthcube Enabled Hydrogeophysical Cyberinfrastructure

    NASA Astrophysics Data System (ADS)

    Versteeg, R. J.; Johnson, D.

    2012-12-01

    to "develop a framework to understand and predict responses of the Earth as a system— from the space-atmosphere boundary to the core, including the influences of humans and ecosystems." Effective development of hydrologic prediction tools will require the hydrogeophysical community to engage in and become conversant with the cyberinfrastructure community. In my presentation I will provide several examples of how such tools could look like, and what some of the opportunities are for getting this engagement going and develop cyberinfrastructure enabled hydrologic prediction tools.

  5. The OGC Sensor Web Enablement framework

    NASA Astrophysics Data System (ADS)

    Cox, S. J.; Botts, M.

    2006-12-01

    Sensor observations are at the core of natural sciences. Improvements in data-sharing technologies offer the promise of much greater utilisation of observational data. A key to this is interoperable data standards. The Open Geospatial Consortium's (OGC) Sensor Web Enablement initiative (SWE) is developing open standards for web interfaces for the discovery, exchange and processing of sensor observations, and tasking of sensor systems. The goal is to support the construction of complex sensor applications through real-time composition of service chains from standard components. The framework is based around a suite of standard interfaces, and standard encodings for the message transferred between services. The SWE interfaces include: Sensor Observation Service (SOS)-parameterized observation requests (by observation time, feature of interest, property, sensor); Sensor Planning Service (SPS)-tasking a sensor- system to undertake future observations; Sensor Alert Service (SAS)-subscription to an alert, usually triggered by a sensor result exceeding some value. The interface design generally follows the pattern established in the OGC Web Map Service (WMS) and Web Feature Service (WFS) interfaces, where the interaction between a client and service follows a standard sequence of requests and responses. The first obtains a general description of the service capabilities, followed by obtaining detail required to formulate a data request, and finally a request for a data instance or stream. These may be implemented in a stateless "REST" idiom, or using conventional "web-services" (SOAP) messaging. In a deployed system, the SWE interfaces are supplemented by Catalogue, data (WFS) and portrayal (WMS) services, as well as authentication and rights management. The standard SWE data formats are Observations and Measurements (O&M) which encodes observation metadata and results, Sensor Model Language (SensorML) which describes sensor-systems, Transducer Model Language (TML) which

  6. Bioblendstocks that Enable High Efficiency Engine Designs

    SciTech Connect

    McCormick, Robert L.; Fioroni, Gina M.; Ratcliff, Matthew A.; Zigler, Bradley T.; Farrell, John

    2016-11-03

    The past decade has seen a high level of innovation in production of biofuels from sugar, lipid, and lignocellulose feedstocks. As discussed in several talks at this workshop, ethanol blends in the E25 to E50 range could enable more highly efficient spark-ignited (SI) engines. This is because of their knock resistance properties that include not only high research octane number (RON), but also charge cooling from high heat of vaporization, and high flame speed. Emerging alcohol fuels such as isobutanol or mixed alcohols have desirable properties such as reduced gasoline blend vapor pressure, but also have lower RON than ethanol. These fuels may be able to achieve the same knock resistance benefits, but likely will require higher blend levels or higher RON hydrocarbon blendstocks. A group of very high RON (>150) oxygenates such as dimethyl furan, methyl anisole, and related compounds are also produced from biomass. While providing no increase in charge cooling, their very high octane numbers may provide adequate knock resistance for future highly efficient SI engines. Given this range of options for highly knock resistant fuels there appears to be a critical need for a fuel knock resistance metric that includes effects of octane number, heat of vaporization, and potentially flame speed. Emerging diesel fuels include highly branched long-chain alkanes from hydroprocessing of fats and oils, as well as sugar-derived terpenoids. These have relatively high cetane number (CN), which may have some benefits in designing more efficient CI engines. Fast pyrolysis of biomass can produce diesel boiling range streams that are high in aromatic, oxygen and acid contents. Hydroprocessing can be applied to remove oxygen and consequently reduce acidity, however there are strong economic incentives to leave up to 2 wt% oxygen in the product. This oxygen will primarily be present as low CN alkyl phenols and aryl ethers. While these have high heating value, their presence in diesel fuel

  7. Technical Assessment: Data-Enabled Technology Watch and Horizon Scanning

    DTIC Science & Technology

    2015-10-01

    current and can manage technology-enabled opportunities and challenges. All of these decisions can benefit substantially from a keen understanding of...1 Potential Benefits from Data-Enabled TW/HS...6 Leveraging Knowledge Management

  8. CURRENT STATUS OF THE IAEA'S NET ENABLED WASTE MANAGEMENT DATABASE

    SciTech Connect

    Csullog, G.W.; Pozdniakov, I.; Bellag, M.J.

    2003-02-27

    The International Atomic Energy Agency's Net Enabled Waste Management Database (NEWMDB) contains information on national radioactive waste management programs and organizations, plans and activities, relevant laws and regulations, policies and radioactive waste inventories. The NEWMDB, which was launched on the Internet July 6, 2001, is the successor to the Agency's Waste Management Database (WMDB), which was in use during the 1990's. The NEWMDB's first data collection cycle took place from July 2001 to March 2002. Agency Member State participation in the first data collection cycle was low--only 22 submissions were received. However, the first data collection cycle demonstrated that: the NEWMDB could be used to collect information on national radioactive waste management programs and radioactive waste inventories annually, the NEWMDB data can support the routine reporting of status and trends in radioactive waste management based on quantitative data, the NEWMDB can support the compilation of a consolidated, international radioactive waste inventory based on a unified waste classification scheme, the data needed to compute an indicator of sustainable development for radioactive waste management are available at the national level, NEWMDB data can be used to assess the development and implementation of national systems for radioactive waste management, and the NEWMDB can support the reporting requirements of the Joint Convention on the Safety of Spent Fuel Management and on the Safety of Radioactive Waste Management. Agency Member States that had not made data submissions in the first cycle were asked to submit data during an extension of the first cycle (July 2002--January 2003). When this paper was written, the Agency had conducted two of three international workshops to provide training for future NEWMDB data collection cycles and to compile lessons learned for the first data collection cycle. A third workshop was scheduled for January 2003. This paper provides

  9. An Investigation of Relations among Academic Enablers and Reading Outcomes

    ERIC Educational Resources Information Center

    Jenkins, Lyndsay N.; Demaray, Michelle Kilpatrick

    2015-01-01

    The current study examined the link between academic enablers and different types of reading achievement measures. Academic enablers are skills and behaviors that support, or enable, students to perform well academically, such as engagement, interpersonal skills, motivation, and study skills. The sample in this study consisted of 61 third-,…

  10. 78 FR 76603 - Enable Gas Transmission, LLC; Notice of Application

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-18

    ... Energy Regulatory Commission Enable Gas Transmission, LLC; Notice of Application Take notice that on November 26, 2013, Enable Gas Transmission, LLC (Enable) 1111 Louisiana Street, Houston, Texas 77002, filed... Gas Transmission, LLC, P.O. Box 21734 Shreveport, LA 71151 at (318) 429- 3708. Specifically,...

  11. Quantitative analysis in megageomorphology

    NASA Technical Reports Server (NTRS)

    Mayer, L.

    1985-01-01

    Megageomorphology is the study of regional topographic features and their relations to independent geomorphic variables that operate at the regional scale. These independent variables can be classified as either tectonic or climatic in nature. Quantitative megageomorphology stresses the causal relations between plate tectonic factors and landscape features or correlations between climatic factors and geomorphic processes. In addition, the cumulative effects of tectonics and climate on landscape evolution that simultaneously operate in a complex system of energy transfer is of interst. Regional topographic differentiation, say between continents and ocean floors, is largely the result of the different densities and density contrasts within the oceanic and continental lithosphere and their isostatic consequences. Regional tectonic processes that alter these lithospheric characteristics include rifting, collision, subduction, transpression and transtension.

  12. Complexity Science Framework for Big Data: Data-enabled Science

    NASA Astrophysics Data System (ADS)

    Surjalal Sharma, A.

    2016-07-01

    such new analytics can yield improved risk estimates. The challenges of scientific inference from complex and massive data are addressed by data-enabled science, also referred as the Fourth paradigm, after experiment, theory and simulation. An example of this approach is the modelling of dynamical and statistical features of natural systems, without assumptions of specific processes. An effective use of the techniques of complexity science to yield the inherent features of a system from extensive data from observations and large scale numerical simulations is evident in the case of Earth's magnetosphere. The multiscale nature of the magnetosphere makes the numerical simulations a challenge, requiring very large computing resources. The reconstruction of dynamics from observational data can however yield the inherent characteristics using typical desktop computers. Such studies for other systems are in progress. Data-enabled approach using the framework of complexity science provides new techniques for modelling and prediction using Big Data. The studies of Earth's magnetosphere, provide an example of the potential for a new approach to the development of quantitative analytic tools.

  13. Quantitative deconvolution microscopy.

    PubMed

    Goodwin, Paul C

    2014-01-01

    The light microscope is an essential tool for the study of cells, organelles, biomolecules, and subcellular dynamics. A paradox exists in microscopy whereby the higher the needed lateral resolution, the more the image is degraded by out-of-focus information. This creates a significant need to generate axial contrast whenever high lateral resolution is required. One strategy for generating contrast is to measure or model the optical properties of the microscope and to use that model to algorithmically reverse some of the consequences of high-resolution imaging. Deconvolution microscopy implements model-based methods to enable the full diffraction-limited resolution of the microscope to be exploited even in complex and living specimens.

  14. Quantitative Hyperspectral Reflectance Imaging

    PubMed Central

    Klein, Marvin E.; Aalderink, Bernard J.; Padoan, Roberto; de Bruin, Gerrit; Steemers, Ted A.G.

    2008-01-01

    Hyperspectral imaging is a non-destructive optical analysis technique that can for instance be used to obtain information from cultural heritage objects unavailable with conventional colour or multi-spectral photography. This technique can be used to distinguish and recognize materials, to enhance the visibility of faint or obscured features, to detect signs of degradation and study the effect of environmental conditions on the object. We describe the basic concept, working principles, construction and performance of a laboratory instrument specifically developed for the analysis of historical documents. The instrument measures calibrated spectral reflectance images at 70 wavelengths ranging from 365 to 1100 nm (near-ultraviolet, visible and near-infrared). By using a wavelength tunable narrow-bandwidth light-source, the light energy used to illuminate the measured object is minimal, so that any light-induced degradation can be excluded. Basic analysis of the hyperspectral data includes a qualitative comparison of the spectral images and the extraction of quantitative data such as mean spectral reflectance curves and statistical information from user-defined regions-of-interest. More sophisticated mathematical feature extraction and classification techniques can be used to map areas on the document, where different types of ink had been applied or where one ink shows various degrees of degradation. The developed quantitative hyperspectral imager is currently in use by the Nationaal Archief (National Archives of The Netherlands) to study degradation effects of artificial samples and original documents, exposed in their permanent exhibition area or stored in their deposit rooms. PMID:27873831

  15. Quantitative immunoglobulins in adulthood.

    PubMed

    Crisp, Howard C; Quinn, James M

    2009-01-01

    Although age-related changes in serum immunoglobulins are well described in childhood, alterations in immunoglobulins in the elderly are less well described and published. This study was designed to better define expected immunoglobulin ranges and differences in adults of differing decades of life. Sera from 404 patients, aged 20-89 years old were analyzed for quantitative immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA). The patients with diagnoses or medications known to affect immunoglobulin levels were identified while blinded to their immunoglobulin levels. A two-factor ANOVA was performed using decade of life and gender on both the entire sample population as well as the subset without any disease or medication expected to alter immunoglobulin levels. A literature review was also performed on all English language articles evaluating quantitative immunoglobulin levels in adults >60 years old. For the entire population, IgM was found to be higher in women when compared with men (p < 0.001) and lower in the oldest sample population compared with the youngest population (p < 0.001). For the population without diseases known to affect immunoglobulin levels, the differences in IgM with gender and age were maintained (p < or = 0.001) and IgA levels were generally higher in the older population when compared with the younger population (p = 0.009). Elderly patients without disease known to affect immunoglobulin levels have higher serum IgA levels and lower serum IgM levels. Women have higher IgM levels than men throughout life. IgG levels are not significantly altered in an older population.

  16. Tyrosine phosphoproteomics identified both co-drivers and co-targeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer

    PubMed Central

    Smith, Matthew A.; Lopez, Alex S.; Bai, Yun; Li, Jiannong; Fang, Bin; Koomen, John; Rawal, Bhupendra; Fisher, Kate J.; Chen, Y. Ann; Kitano, Michiko; Morita, Yume; Yamaguchi, Haruka; Shibata, Kiyoko; Okabe, Takafumi; Okamoto, Isamu; Nakagawa, Kazuhiko; Haura, Eric B.

    2014-01-01

    Purpose Irreversible EGFR-tyrosine kinase inhibitors (TKIs) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gate-keeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass-spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined. Results We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src-family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK-inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced anti-tumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions Our results identified both co-drivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in NSCLC patients with acquired T790M. PMID:24919575

  17. Quantitative Microbial Ecology through Stable Isotope Probing

    PubMed Central

    Mau, Rebecca L.; Schwartz, Egbert; Caporaso, J. Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J.; Liu, Cindy M.; McHugh, Theresa A.; Marks, Jane C.; Morrissey, Ember M.; Price, Lance B.

    2015-01-01

    Bacteria grow and transform elements at different rates, and as yet, quantifying this variation in the environment is difficult. Determining isotope enrichment with fine taxonomic resolution after exposure to isotope tracers could help, but there are few suitable techniques. We propose a modification to stable isotope probing (SIP) that enables the isotopic composition of DNA from individual bacterial taxa after exposure to isotope tracers to be determined. In our modification, after isopycnic centrifugation, DNA is collected in multiple density fractions, and each fraction is sequenced separately. Taxon-specific density curves are produced for labeled and nonlabeled treatments, from which the shift in density for each individual taxon in response to isotope labeling is calculated. Expressing each taxon's density shift relative to that taxon's density measured without isotope enrichment accounts for the influence of nucleic acid composition on density and isolates the influence of isotope tracer assimilation. The shift in density translates quantitatively to isotopic enrichment. Because this revision to SIP allows quantitative measurements of isotope enrichment, we propose to call it quantitative stable isotope probing (qSIP). We demonstrated qSIP using soil incubations, in which soil bacteria exhibited strong taxonomic variations in 18O and 13C composition after exposure to [18O]water or [13C]glucose. The addition of glucose increased the assimilation of 18O into DNA from [18O]water. However, the increase in 18O assimilation was greater than expected based on utilization of glucose-derived carbon alone, because the addition of glucose indirectly stimulated bacteria to utilize other substrates for growth. This example illustrates the benefit of a quantitative approach to stable isotope probing. PMID:26296731

  18. Quantitative analysis of culture using millions of digitized books.

    PubMed

    Michel, Jean-Baptiste; Shen, Yuan Kui; Aiden, Aviva Presser; Veres, Adrian; Gray, Matthew K; Pickett, Joseph P; Hoiberg, Dale; Clancy, Dan; Norvig, Peter; Orwant, Jon; Pinker, Steven; Nowak, Martin A; Aiden, Erez Lieberman

    2011-01-14

    We constructed a corpus of digitized texts containing about 4% of all books ever printed. Analysis of this corpus enables us to investigate cultural trends quantitatively. We survey the vast terrain of 'culturomics,' focusing on linguistic and cultural phenomena that were reflected in the English language between 1800 and 2000. We show how this approach can provide insights about fields as diverse as lexicography, the evolution of grammar, collective memory, the adoption of technology, the pursuit of fame, censorship, and historical epidemiology. Culturomics extends the boundaries of rigorous quantitative inquiry to a wide array of new phenomena spanning the social sciences and the humanities.

  19. Quantitative analysis of culture using millions of digitized books

    PubMed Central

    Michel, Jean-Baptiste; Shen, Yuan Kui; Aiden, Aviva P.; Veres, Adrian; Gray, Matthew K.; Pickett, Joseph P.; Hoiberg, Dale; Clancy, Dan; Norvig, Peter; Orwant, Jon; Pinker, Steven; Nowak, Martin A.; Aiden, Erez Lieberman

    2011-01-01

    We constructed a corpus of digitized texts containing about 4% of all books ever printed. Analysis of this corpus enables us to investigate cultural trends quantitatively. We survey the vast terrain of ‘culturomics’, focusing on linguistic and cultural phenomena that were reflected in the English language between 1800 and 2000. We show how this approach can provide insights about fields as diverse as lexicography, the evolution of grammar, collective memory, the adoption of technology, the pursuit of fame, censorship, and historical epidemiology. ‘Culturomics’ extends the boundaries of rigorous quantitative inquiry to a wide array of new phenomena spanning the social sciences and the humanities. PMID:21163965

  20. Quantitative numerical analysis of transient IR-experiments on buildings

    NASA Astrophysics Data System (ADS)

    Maierhofer, Ch.; Wiggenhauser, H.; Brink, A.; Röllig, M.

    2004-12-01

    Impulse-thermography has been established as a fast and reliable tool in many areas of non-destructive testing. In recent years several investigations have been done to apply active thermography to civil engineering. For quantitative investigations in this area of application, finite difference calculations have been performed for systematic studies on the influence of environmental conditions, heating power and time, defect depth and size and thermal properties of the bulk material (concrete). The comparison of simulated and experimental data enables the quantitative analysis of defects.

  1. Petascale Computing Enabling Technologies Project Final Report

    SciTech Connect

    de Supinski, B R

    2010-02-14

    The Petascale Computing Enabling Technologies (PCET) project addressed challenges arising from current trends in computer architecture that will lead to large-scale systems with many more nodes, each of which uses multicore chips. These factors will soon lead to systems that have over one million processors. Also, the use of multicore chips will lead to less memory and less memory bandwidth per core. We need fundamentally new algorithmic approaches to cope with these memory constraints and the huge number of processors. Further, correct, efficient code development is difficult even with the number of processors in current systems; more processors will only make it harder. The goal of PCET was to overcome these challenges by developing the computer science and mathematical underpinnings needed to realize the full potential of our future large-scale systems. Our research results will significantly increase the scientific output obtained from LLNL large-scale computing resources by improving application scientist productivity and system utilization. Our successes include scalable mathematical algorithms that adapt to these emerging architecture trends and through code correctness and performance methodologies that automate critical aspects of application development as well as the foundations for application-level fault tolerance techniques. PCET's scope encompassed several research thrusts in computer science and mathematics: code correctness and performance methodologies, scalable mathematics algorithms appropriate for multicore systems, and application-level fault tolerance techniques. Due to funding limitations, we focused primarily on the first three thrusts although our work also lays the foundation for the needed advances in fault tolerance. In the area of scalable mathematics algorithms, our preliminary work established that OpenMP performance of the AMG linear solver benchmark and important individual kernels on Atlas did not match the predictions of our

  2. Quantitative Techniques in Volumetric Analysis

    NASA Astrophysics Data System (ADS)

    Zimmerman, John; Jacobsen, Jerrold J.

    1996-12-01

    Quantitative Techniques in Volumetric Analysis is a visual library of techniques used in making volumetric measurements. This 40-minute VHS videotape is designed as a resource for introducing students to proper volumetric methods and procedures. The entire tape, or relevant segments of the tape, can also be used to review procedures used in subsequent experiments that rely on the traditional art of quantitative analysis laboratory practice. The techniques included are: Quantitative transfer of a solid with a weighing spoon Quantitative transfer of a solid with a finger held weighing bottle Quantitative transfer of a solid with a paper strap held bottle Quantitative transfer of a solid with a spatula Examples of common quantitative weighing errors Quantitative transfer of a solid from dish to beaker to volumetric flask Quantitative transfer of a solid from dish to volumetric flask Volumetric transfer pipet A complete acid-base titration Hand technique variations The conventional view of contemporary quantitative chemical measurement tends to focus on instrumental systems, computers, and robotics. In this view, the analyst is relegated to placing standards and samples on a tray. A robotic arm delivers a sample to the analysis center, while a computer controls the analysis conditions and records the results. In spite of this, it is rare to find an analysis process that does not rely on some aspect of more traditional quantitative analysis techniques, such as careful dilution to the mark of a volumetric flask. Figure 2. Transfer of a solid with a spatula. Clearly, errors in a classical step will affect the quality of the final analysis. Because of this, it is still important for students to master the key elements of the traditional art of quantitative chemical analysis laboratory practice. Some aspects of chemical analysis, like careful rinsing to insure quantitative transfer, are often an automated part of an instrumental process that must be understood by the

  3. Multi-omic data integration enables discovery of hidden biological regularities

    PubMed Central

    Ebrahim, Ali; Brunk, Elizabeth; Tan, Justin; O'Brien, Edward J.; Kim, Donghyuk; Szubin, Richard; Lerman, Joshua A.; Lechner, Anna; Sastry, Anand; Bordbar, Aarash; Feist, Adam M.; Palsson, Bernhard O.

    2016-01-01

    Rapid growth in size and complexity of biological data sets has led to the ‘Big Data to Knowledge' challenge. We develop advanced data integration methods for multi-level analysis of genomic, transcriptomic, ribosomal profiling, proteomic and fluxomic data. First, we show that pairwise integration of primary omics data reveals regularities that tie cellular processes together in Escherichia coli: the number of protein molecules made per mRNA transcript and the number of ribosomes required per translated protein molecule. Second, we show that genome-scale models, based on genomic and bibliomic data, enable quantitative synchronization of disparate data types. Integrating omics data with models enabled the discovery of two novel regularities: condition invariant in vivo turnover rates of enzymes and the correlation of protein structural motifs and translational pausing. These regularities can be formally represented in a computable format allowing for coherent interpretation and prediction of fitness and selection that underlies cellular physiology. PMID:27782110

  4. An evolving Mars telecommunications network to enable exploration and increase science data return

    NASA Technical Reports Server (NTRS)

    Edwards, Chad; Komarek, Tomas A.; Noreen, Gary K.; Wilson, Gregory R.

    2003-01-01

    The coming decade of Mars exploration involves a variety of unique telecommunications challenges. Increasing spatial and spectral resolution of in situ science instruments drive the need for increased bandwidth. At the same time, many innovative and low-cost in situ mission concepts are enabled by energy-efficient relay communications. In response to these needs, the Mars Exploration Program has established a plan for an evolving orbital infrastructure that can provide enhancing and enabling telecommunications services to future Mars missions. We will present the evolving capabilities of this network over the coming decade in terms of specific quantitative metrics such as data volume per sol and required lander energy per Gb of returned data for representative classes of Mars exploration spacecraft.

  5. Web-based interactive visualization in a Grid-enabled neuroimaging application using HTML5.

    PubMed

    Siewert, René; Specovius, Svenja; Wu, Jie; Krefting, Dagmar

    2012-01-01

    Interactive visualization and correction of intermediate results are required in many medical image analysis pipelines. To allow certain interaction in the remote execution of compute- and data-intensive applications, new features of HTML5 are used. They allow for transparent integration of user interaction into Grid- or Cloud-enabled scientific workflows. Both 2D and 3D visualization and data manipulation can be performed through a scientific gateway without the need to install specific software or web browser plugins. The possibilities of web-based visualization are presented along the FreeSurfer-pipeline, a popular compute- and data-intensive software tool for quantitative neuroimaging.

  6. Quantitative single-molecule imaging by confocal laser scanning microscopy.

    PubMed

    Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

    2008-11-25

    A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

  7. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  8. Quantitative environmental risk analysis

    SciTech Connect

    Klovning, J.; Nilsen, E.F.

    1995-12-31

    According to regulations relating to implementation and rise of risk analysis in the petroleum activities issued by the Norwegian Petroleum Directorate, it is mandatory for an operator on the Norwegian Continental Shelf to establish acceptance criteria for environmental risk in the activities and carry out environmental risk analysis. This paper presents a {open_quotes}new{close_quotes} method for environmental risk analysis developed by the company. The objective has been to assist the company to meet rules and regulations and to assess and describe the environmental risk in a systematic manner. In the environmental risk analysis the most sensitive biological resource in the affected area is used to assess the environmental damage. The analytical method is based on the methodology for quantitative risk analysis related to loss of life. In addition it incorporates the effect of seasonal fluctuations in the environmental risk evaluations. The paper is describing the function of the main analytical sequences exemplified through an analysis of environmental risk related to exploration drilling in an environmental sensitive area on the Norwegian Continental Shelf.

  9. Quantitative Electron Nanodiffraction.

    SciTech Connect

    Spence, John

    2015-01-30

    This Final report summarizes progress under this award for the final reporting period 2002 - 2013 in our development of quantitive electron nanodiffraction to materials problems, especially devoted to atomistic processes in semiconductors and electronic oxides such as the new artificial oxide multilayers, where our microdiffraction is complemented with energy-loss spectroscopy (ELNES) and aberration-corrected STEM imaging (9). The method has also been used to map out the chemical bonds in the important GaN semiconductor (1) used for solid state lighting, and to understand the effects of stacking sequence variations and interfaces in digital oxide superlattices (8). Other projects include the development of a laser-beam Zernike phase plate for cryo-electron microscopy (5) (based on the Kapitza-Dirac effect), work on reconstruction of molecular images using the scattering from many identical molecules lying in random orientations (4), a review article on space-group determination for the International Tables on Crystallography (10), the observation of energy-loss spectra with millivolt energy resolution and sub-nanometer spatial resolution from individual point defects in an alkali halide, a review article for the Centenary of X-ray Diffration (17) and the development of a new method of electron-beam lithography (12). We briefly summarize here the work on GaN, on oxide superlattice ELNES, and on lithography by STEM.

  10. QUANTITATIVE STUDIES OF PROSTATIC SECRETION

    PubMed Central

    Huggins, Charles; Masina, M. H.; Eichelberger, Lillian; Wharton, James D.

    1939-01-01

    A simple isolation of the prostate enabled quantitative collection of prostatic secretion in dogs over periods of months. The secretory stimulant was pilocarpine and 2 similar amounts injected with a 6 hour interval gave smaller amounts at the second testing, suggesting a fatigue effect. The prostate was not absolutely refractory since doubling the amount of alkaloid injected at the second test increased the volume to equal or exceed the preliminary secretion. The depression effect had disappeared at 24 hours. In normal dogs the secretory curves were essentially regular, with occasional prolonged rises or depressions. The amount of secretion did not bear a direct relationship to the weight of the gland in adult dogs. The germinal epithelium of the testis underwent atrophy during the first few weeks of cage life while the prostatic secretion was maintained, showing that the atrophy was differential and did not involve the cells producing the androgenic hormone. The atrophy was reversible and all dogs kept for more than 4 months showed restoration of the germ cells. A few dogs developed atrophy of the germinal cells with cessation of prostatic secretion for many weeks but with final recovery. Removal of the suprarenal glands with suprarenal insufficiency did not produce sterility. The distribution of electrolytes in the prostatic secretion differed from that in the serum-transudate system, although the concentration of osmotically active substances was the same, being made up almost entirely of sodium and chloride. The distribution was not affected by the different physiological procedures used in this study. Protein concentrations were less than 1 per cent. The rate of prostatic atrophy following castration was determined, and cessation of secretion occurred in 7 to 23 days. The restoration of prostatic fluid in castrate dogs following daily injections of testosterone propionate followed a smooth curve to form a plateau which was interrupted occasionally by

  11. Toward Accurate and Quantitative Comparative Metagenomics

    PubMed Central

    Nayfach, Stephen; Pollard, Katherine S.

    2016-01-01

    Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

  12. Quantitative nondestructive evaluation: Requirements for tomorrow's reliability

    NASA Technical Reports Server (NTRS)

    Heyman, Joseph S.

    1991-01-01

    Quantitative Nondestructive Evaluation (QNDE) is the technology of measurement, analysis, and prediction of the state of material/structural systems for safety, reliability, and mission assurance. QNDE has impact on everyday life from the cars we drive, the planes we fly, the buildings we work or live in, literally to the infrastructure of our world. Here, researchers highlight some of the new sciences and technologies that are part of a safer, cost effective tomorrow. Specific technologies that are discussed are thermal QNDE of aircraft structural integrity, ultrasonic QNDE for materials characterization, and technology spinoffs from aerospace to the medical sector. In each case, examples are given of how new requirements result in enabling measurement technologies, which in turn change the boundaries of design/practice.

  13. Implementing a Quantitative Analysis Design Tool for Future Generation Interfaces

    DTIC Science & Technology

    2012-03-01

    future MAC-enabled systems. A human-computer interaction ( HCI ) Index, originally applied to multi-function displays was applied to the prototype Vigilant...Spirit interface. A modified version of the HCI Index was successfully applied to perform a quantitative analysis of the baseline VSCS interface and...two modified interface designs. The modified HCI Index incorporates the Hick-Hyman decision time, Fitts’ Law time, and the physical actions

  14. Studying learning in the healthcare setting: the potential of quantitative diary methods.

    PubMed

    Ciere, Yvette; Jaarsma, Debbie; Visser, Annemieke; Sanderman, Robbert; Snippe, Evelien; Fleer, Joke

    2015-08-01

    Quantitative diary methods are longitudinal approaches that involve the repeated measurement of aspects of peoples' experience of daily life. In this article, we outline the main characteristics and applications of quantitative diary methods and discuss how their use may further research in the field of medical education. Quantitative diary methods offer several methodological advantages, such as measuring aspects of learning with great detail, accuracy and authenticity. Moreover, they enable researchers to study how and under which conditions learning in the health care setting occurs and in which way learning can be promoted. Hence, quantitative diary methods may contribute to theory development and the optimization of teaching methods in medical education.

  15. Quantitative Literacy: Geosciences and Beyond

    NASA Astrophysics Data System (ADS)

    Richardson, R. M.; McCallum, W. G.

    2002-12-01

    Quantitative literacy seems like such a natural for the geosciences, right? The field has gone from its origin as a largely descriptive discipline to one where it is hard to imagine failing to bring a full range of mathematical tools to the solution of geological problems. Although there are many definitions of quantitative literacy, we have proposed one that is analogous to the UNESCO definition of conventional literacy: "A quantitatively literate person is one who, with understanding, can both read and represent quantitative information arising in his or her everyday life." Central to this definition is the concept that a curriculum for quantitative literacy must go beyond the basic ability to "read and write" mathematics and develop conceptual understanding. It is also critical that a curriculum for quantitative literacy be engaged with a context, be it everyday life, humanities, geoscience or other sciences, business, engineering, or technology. Thus, our definition works both within and outside the sciences. What role do geoscience faculty have in helping students become quantitatively literate? Is it our role, or that of the mathematicians? How does quantitative literacy vary between different scientific and engineering fields? Or between science and nonscience fields? We will argue that successful quantitative literacy curricula must be an across-the-curriculum responsibility. We will share examples of how quantitative literacy can be developed within a geoscience curriculum, beginning with introductory classes for nonmajors (using the Mauna Loa CO2 data set) through graduate courses in inverse theory (using singular value decomposition). We will highlight six approaches to across-the curriculum efforts from national models: collaboration between mathematics and other faculty; gateway testing; intensive instructional support; workshops for nonmathematics faculty; quantitative reasoning requirement; and individual initiative by nonmathematics faculty.

  16. Quantitative Spectroscopy of Deneb

    NASA Astrophysics Data System (ADS)

    Schiller, Florian; Przybilla, N.

    We use the visually brightest A-type supergiant Deneb (A2 Ia) as benchmark for testing a spectro- scopic analysis technique developed for quantitative studies of BA-type supergiants. Our NLTE spectrum synthesis technique allows us to derive stellar parameters and elemental abundances with unprecedented accuracy. The study is based on a high-resolution and high-S/N spectrum obtained with the Echelle spectrograph FOCES on the Calar Alto 2.2 m telescope. Practically all inconsistencies reported in earlier studies are resolved. A self-consistent view of Deneb is thus obtained, allowing us to discuss its evolutionary state in detail by comparison with the most recent generation of evolution models for massive stars. The basic atmospheric parameters Teff = 8525 ± 75 K and log g = 1.10 ± 0.05 dex (cgs) and the distance imply the following fundamental parameters for Deneb: M spec = 17 ± 3 M⊙ , L = 1.77 ± 0.29 · 105 L⊙ and R = 192 ± 16 R⊙ . The derived He and CNO abundances indicate mixing with nuclear processed matter. The high N/C ratio of 4.64 ± 1.39 and a N/O ratio of 0.88 ± 0.07 (mass fractions) could in principle be explained by evolutionary models with initially very rapid rotation. A mass of ˜ 22 M⊙ is implied for the progenitor on the zero-age main se- quence, i.e. it was a late O-type star. Significant mass-loss has occurred, probably enhanced by pronounced centrifugal forces. The observational constraints favour a scenario for the evolu- tion of Deneb where the effects of rotational mixing may be amplified by an interaction with a magnetic field. Analogous analyses of such highly luminous BA-type supergiants will allow for precision studies of different galaxies in the Local Group and beyond.

  17. Quantitative Luminescence Imaging System

    SciTech Connect

    Batishko, C.R.; Stahl, K.A.; Fecht, B.A.

    1992-12-31

    The goal of the MEASUREMENT OF CHEMILUMINESCENCE project is to develop and deliver a suite of imaging radiometric instruments for measuring spatial distributions of chemiluminescence. Envisioned deliverables include instruments working at the microscopic, macroscopic, and life-sized scales. Both laboratory and field portable instruments are envisioned. The project also includes development of phantoms as enclosures for the diazoluminomelanin (DALM) chemiluminescent chemistry. A suite of either phantoms in a variety of typical poses, or phantoms that could be adjusted to a variety of poses, is envisioned. These are to include small mammals (rats), mid-sized mammals (monkeys), and human body parts. A complete human phantom that can be posed is a long-term goal of the development. Taken together, the chemistry and instrumentation provide a means for imaging rf dosimetry based on chemiluminescence induced by the heat resulting from rf energy absorption. The first delivered instrument, the Quantitative Luminescence Imaging System (QLIS), resulted in a patent, and an R&D Magazine 1991 R&D 100 award, recognizing it as one of the 100 most significant technological developments of 1991. The current status of the project is that three systems have been delivered, several related studies have been conducted, two preliminary human hand phantoms have been delivered, system upgrades have been implemented, and calibrations have been maintained. Current development includes sensitivity improvements to the microscope-based system; extension of the large-scale (potentially life-sized targets) system to field portable applications; extension of the 2-D large-scale system to 3-D measurement; imminent delivery of a more refined human hand phantom and a rat phantom; rf, thermal and imaging subsystem integration; and continued calibration and upgrade support.

  18. Quantitative luminescence imaging system

    NASA Astrophysics Data System (ADS)

    Batishko, C. R.; Stahl, K. A.; Fecht, B. A.

    The goal of the Measurement of Chemiluminescence project is to develop and deliver a suite of imaging radiometric instruments for measuring spatial distributions of chemiluminescence. Envisioned deliverables include instruments working at the microscopic, macroscopic, and life-sized scales. Both laboratory and field portable instruments are envisioned. The project also includes development of phantoms as enclosures for the diazoluminomelanin (DALM) chemiluminescent chemistry. A suite of either phantoms in a variety of typical poses, or phantoms that could be adjusted to a variety of poses, is envisioned. These are to include small mammals (rats), mid-sized mammals (monkeys), and human body parts. A complete human phantom that can be posed is a long-term goal of the development. Taken together, the chemistry and instrumentation provide a means for imaging rf dosimetry based on chemiluminescence induced by the heat resulting from rf energy absorption. The first delivered instrument, the Quantitative Luminescence Imaging System (QLIS), resulted in a patent, and an R&D Magazine 1991 R&D 100 award, recognizing it as one of the 100 most significant technological developments of 1991. The current status of the project is that three systems have been delivered, several related studies have been conducted, two preliminary human hand phantoms have been delivered, system upgrades have been implemented, and calibrations have been maintained. Current development includes sensitivity improvements to the microscope-based system; extension of the large-scale (potentially life-sized targets) system to field portable applications; extension of the 2-D large-scale system to 3-D measurement; imminent delivery of a more refined human hand phantom and a rat phantom; rf, thermal and imaging subsystem integration; and continued calibration and upgrade support.

  19. Quantitative proteomics in cardiovascular research: global and targeted strategies

    PubMed Central

    Shen, Xiaomeng; Young, Rebeccah; Canty, John M.; Qu, Jun

    2014-01-01

    Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation. PMID:24920501

  20. Quantitative proteomics in cardiovascular research: global and targeted strategies.

    PubMed

    Shen, Xiaomeng; Young, Rebeccah; Canty, John M; Qu, Jun

    2014-08-01

    Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here, we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation.

  1. Realtime Data to Enable Earth-Observing Sensor Web Capabilities

    NASA Astrophysics Data System (ADS)

    Seablom, M. S.

    2015-12-01

    Over the past decade NASA's Earth Science Technology Office (ESTO) has invested in new technologies for information systems to enhance the Earth-observing capabilities of satellites, aircraft, and ground-based in situ observations. One focus area has been to create a common infrastructure for coordinated measurements from multiple vantage points which could be commanded either manually or through autonomous means, such as from a numerical model. This paradigm became known as the sensor web, formally defined to be "a coherent set of heterogeneous, loosely-coupled, distributed observing nodes interconnected by a communications fabric that can collectively behave as a single dynamically adaptive and reconfigurable observing system". This would allow for adaptive targeting of rapidly evolving, transient, or variable meteorological features to improve our ability to monitor, understand, and predict their evolution. It would also enable measurements earmarked at critical regions of the atmosphere that are highly sensitive to data analysis errors, thus offering the potential for significant improvements in the predictive skill of numerical weather forecasts. ESTO's investment strategy was twofold. Recognizing that implementation of an operational sensor web would not only involve technical cost and risk but also would require changes to the culture of how flight missions were designed and operated, ESTO funded the development of a mission-planning simulator that would quantitatively assess the added value of coordinated observations. The simulator was designed to provide the capability to perform low-cost engineering and design trade studies using synthetic data generated by observing system simulation experiments (OSSEs). The second part of the investment strategy was to invest in prototype applications that implemented key features of a sensor web, with the dual goals of developing a sensor web reference architecture as well as supporting useful science activities that

  2. Understanding quantitative research: part 2.

    PubMed

    Hoare, Zoë; Hoe, Juanita

    This article, which is the second in a two-part series, provides an introduction to understanding quantitative research, basic statistics and terminology used in research articles. Understanding statistical analysis will ensure that nurses can assess the credibility and significance of the evidence reported. This article focuses on explaining common statistical terms and the presentation of statistical data in quantitative research.

  3. Workshop on quantitative dynamic stratigraphy

    SciTech Connect

    Cross, T.A.

    1988-04-01

    This document discusses the development of quantitative simulation models for the investigation of geologic systems. The selection of variables, model verification, evaluation, and future directions in quantitative dynamic stratigraphy (QDS) models are detailed. Interdisciplinary applications, integration, implementation, and transfer of QDS are also discussed. (FI)

  4. Prototype cantilevers for quantitative lateral force microscopy

    SciTech Connect

    Reitsma, Mark G.; Gates, Richard S.; Friedman, Lawrence H.; Cook, Robert F.

    2011-09-15

    Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The ''hammerhead'' cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever ''torque sensitivity'' to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.

  5. Quantitative aspects of inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Bulska, Ewa; Wagner, Barbara

    2016-10-01

    Accurate determination of elements in various kinds of samples is essential for many areas, including environmental science, medicine, as well as industry. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful tool enabling multi-elemental analysis of numerous matrices with high sensitivity and good precision. Various calibration approaches can be used to perform accurate quantitative measurements by ICP-MS. They include the use of pure standards, matrix-matched standards, or relevant certified reference materials, assuring traceability of the reported results. This review critically evaluates the advantages and limitations of different calibration approaches, which are used in quantitative analyses by ICP-MS. Examples of such analyses are provided. This article is part of the themed issue 'Quantitative mass spectrometry'.

  6. A multiplexed system for quantitative comparisons of chromatin landscapes

    PubMed Central

    van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

    2015-01-01

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

  7. Quantitative aspects of inductively coupled plasma mass spectrometry.

    PubMed

    Bulska, Ewa; Wagner, Barbara

    2016-10-28

    Accurate determination of elements in various kinds of samples is essential for many areas, including environmental science, medicine, as well as industry. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful tool enabling multi-elemental analysis of numerous matrices with high sensitivity and good precision. Various calibration approaches can be used to perform accurate quantitative measurements by ICP-MS. They include the use of pure standards, matrix-matched standards, or relevant certified reference materials, assuring traceability of the reported results. This review critically evaluates the advantages and limitations of different calibration approaches, which are used in quantitative analyses by ICP-MS. Examples of such analyses are provided.This article is part of the themed issue 'Quantitative mass spectrometry'.

  8. A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

    PubMed

    van Galen, Peter; Viny, Aaron D; Ram, Oren; Ryan, Russell J H; Cotton, Matthew J; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Carroll, Kaitlin M; Cross, Michael B; Levine, Ross L; Bernstein, Bradley E

    2016-01-07

    Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

  9. Microfluidics for High-Throughput Quantitative Studies of Early Development.

    PubMed

    Levario, Thomas J; Lim, Bomyi; Shvartsman, Stanislav Y; Lu, Hang

    2016-07-11

    Developmental biology has traditionally relied on qualitative analyses; recently, however, as in other fields of biology, researchers have become increasingly interested in acquiring quantitative knowledge about embryogenesis. Advances in fluorescence microscopy are enabling high-content imaging in live specimens. At the same time, microfluidics and automation technologies are increasing experimental throughput for studies of multicellular models of development. Furthermore, computer vision methods for processing and analyzing bioimage data are now leading the way toward quantitative biology. Here, we review advances in the areas of fluorescence microscopy, microfluidics, and data analysis that are instrumental to performing high-content, high-throughput studies in biology and specifically in development. We discuss a case study of how these techniques have allowed quantitative analysis and modeling of pattern formation in the Drosophila embryo.

  10. Uncertainty of quantitative microbiological methods of pharmaceutical analysis.

    PubMed

    Gunar, O V; Sakhno, N G

    2015-12-30

    The total uncertainty of quantitative microbiological methods, used in pharmaceutical analysis, consists of several components. The analysis of the most important sources of the quantitative microbiological methods variability demonstrated no effect of culture media and plate-count techniques in the estimation of microbial count while the highly significant effect of other factors (type of microorganism, pharmaceutical product and individual reading and interpreting errors) was established. The most appropriate method of statistical analysis of such data was ANOVA which enabled not only the effect of individual factors to be estimated but also their interactions. Considering all the elements of uncertainty and combining them mathematically the combined relative uncertainty of the test results was estimated both for method of quantitative examination of non-sterile pharmaceuticals and microbial count technique without any product. These data did not exceed 35%, appropriated for a traditional plate count methods.

  11. Quantitative multimodality imaging in cancer research and therapy.

    PubMed

    Yankeelov, Thomas E; Abramson, Richard G; Quarles, C Chad

    2014-11-01

    Advances in hardware and software have enabled the realization of clinically feasible, quantitative multimodality imaging of tissue pathophysiology. Earlier efforts relating to multimodality imaging of cancer have focused on the integration of anatomical and functional characteristics, such as PET-CT and single-photon emission CT (SPECT-CT), whereas more-recent advances and applications have involved the integration of multiple quantitative, functional measurements (for example, multiple PET tracers, varied MRI contrast mechanisms, and PET-MRI), thereby providing a more-comprehensive characterization of the tumour phenotype. The enormous amount of complementary quantitative data generated by such studies is beginning to offer unique insights into opportunities to optimize care for individual patients. Although important technical optimization and improved biological interpretation of multimodality imaging findings are needed, this approach can already be applied informatively in clinical trials of cancer therapeutics using existing tools. These concepts are discussed herein.

  12. Quantitative imaging with a mobile phone microscope.

    PubMed

    Skandarajah, Arunan; Reber, Clay D; Switz, Neil A; Fletcher, Daniel A

    2014-01-01

    Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone-based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications.

  13. Quantitative Imaging with a Mobile Phone Microscope

    PubMed Central

    Skandarajah, Arunan; Reber, Clay D.; Switz, Neil A.; Fletcher, Daniel A.

    2014-01-01

    Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone–based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications. PMID:24824072

  14. Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles

    PubMed Central

    Wissenbach, Dirk K.; Pfeiffer, Susanne E. M.; Baumann, Sven; Hofbauer, Lorenz C.; von Bergen, Martin; Kalkhof, Stefan; Rammelt, Stefan

    2016-01-01

    Bone healing involves a variety of different cell types and biological processes. Although certain key molecules have been identified, the molecular interactions of the healing progress are not completely understood. Moreover, a clinical routine for predicting the quality of bone healing after a fracture in an early phase is missing. This is mainly due to a lack of techniques to comprehensively screen for cytokines, growth factors and metabolites at their local site of action. Since all soluble molecules of interest are present in the fracture hematoma, its in-depth assessment could reveal potential markers for the monitoring of bone healing. Here, we describe an approach for sampling and quantification of cytokines and metabolites by using microdialysis, combined with solid phase extractions of proteins from wound fluids. By using a control group with an isolated soft tissue wound, we could reveal several bone defect-specific molecular features. In bone defect dialysates the neutrophil chemoattractants CXCL1, CXCL2 and CXCL3 were quantified with either a higher or earlier response compared to dialysate from soft tissue wound. Moreover, by analyzing downstream adaptions of the cells on protein level and focusing on early immune response, several proteins involved in the immune cell migration and activity could be identified to be specific for the bone defect group, e.g. immune modulators, proteases and their corresponding inhibitors. Additionally, the metabolite screening revealed different profiles between the bone defect group and the control group. In summary, we identified potential biomarkers to indicate imbalanced healing progress on all levels of analysis. PMID:27441377

  15. Battlefield Renewable Energy: A Key Joint Force Enabler

    DTIC Science & Technology

    2010-06-01

    Environment, Energy Security & Sustainability Symposium Jun 2010 Battlefield Renewable Energy A Key Joint Force Enabler Roy H. Adams III, LTC, USA...COVERED 00-00-2010 to 00-00-2010 4. TITLE AND SUBTITLE Battlefield Renewable Energy : A Key Joint Force Enabler 5a. CONTRACT NUMBER 5b. GRANT

  16. AFC-Enabled Vertical Tail System Integration Study

    NASA Technical Reports Server (NTRS)

    Mooney, Helen P.; Brandt, John B.; Lacy, Douglas S.; Whalen, Edward A.

    2014-01-01

    This document serves as the final report for the SMAAART AFC-Enabled Vertical Tail System Integration Study. Included are the ground rule assumptions which have gone into the study, layouts of the baseline and AFC-enabled configurations, critical sizing information, system requirements and architectures, and assumed system properties that result in an NPV assessment of the two candidate AFC technologies.

  17. Quantitative analysis of PET studies.

    PubMed

    Weber, Wolfgang A

    2010-09-01

    Quantitative analysis can be included relatively easily in clinical PET-imaging protocols, but in order to obtain meaningful quantitative results one needs to follow a standardized protocol for image acquisition and data analysis. Important factors to consider are the calibration of the PET scanner, the radiotracer uptake time and the approach for definition of regions of interests. Using such standardized acquisition protocols quantitative parameters of tumor metabolism or receptor status can be derived from tracer kinetic analysis and simplified approaches such as calculation of standardized uptake values (SUVs).

  18. A Primer on Quantitative Modeling.

    PubMed

    Neagu, Iulia; Levine, Erel

    2015-01-01

    Caenorhabditis elegans is particularly suitable for obtaining quantitative data about behavior, neuronal activity, gene expression, ecological interactions, quantitative traits, and much more. To exploit the full potential of these data one seeks to interpret them within quantitative models. Using two examples from the C. elegans literature we briefly explore several types of modeling approaches relevant to worm biology, and show how they might be used to interpret data, formulate testable hypotheses, and suggest new experiments. We emphasize that the choice of modeling approach is strongly dependent on the questions of interest and the type of available knowledge.

  19. Understanding quantitative research: part 1.

    PubMed

    Hoe, Juanita; Hoare, Zoë

    This article, which is the first in a two-part series, provides an introduction to understanding quantitative research, basic statistics and terminology used in research articles. Critical appraisal of research articles is essential to ensure that nurses remain up to date with evidence-based practice to provide consistent and high-quality nursing care. This article focuses on developing critical appraisal skills and understanding the use and implications of different quantitative approaches to research. Part two of this article will focus on explaining common statistical terms and the presentation of statistical data in quantitative research.

  20. Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

    PubMed Central

    Yeh, Erh-Chia; Fu, Chi-Cheng; Hu, Lucy; Thakur, Rohan; Feng, Jeffrey; Lee, Luke P.

    2017-01-01

    Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays. PMID:28345028