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Sample records for based phosphoproteomic analysis

  1. Phosphoproteomics-based systems analysis of signal transduction networks

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    Hiroko eKozuka-Hata

    2012-01-01

    Full Text Available Signal transduction systems coordinate complex cellular information to regulate biological events such as cell proliferation and differentiation. Although the accumulating evidence on widespread association of signaling molecules has revealed essential contribution of phosphorylation-dependent interaction networks to cellular regulation, their dynamic behavior is mostly yet to be analyzed. Recent technological advances regarding mass spectrometry-based quantitative proteomics have enabled us to describe the comprehensive status of phosphorylated molecules in a time-resolved manner. Computational analyses based on the phosphoproteome dynamics accelerate generation of novel methodologies for mathematical analysis of cellular signaling. Phosphoproteomics-based numerical modeling can be used to evaluate regulatory network elements from a statistical point of view. Integration with transcriptome dynamics also uncovers regulatory hubs at the transcriptional level. These omics-based computational methodologies, which have firstly been applied to representative signaling systems such as the epidermal growth factor receptor pathway, have now opened up a gate for systems analysis of signaling networks involved in immune response and cancer.

  2. SILAC-Based Temporal Phosphoproteomics

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Hekmat, Omid; Blagoev, Blagoy;

    2014-01-01

    signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated...

  3. Knowledge-Based Analysis for Detecting Key Signaling Events from Time-Series Phosphoproteomics Data.

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    Pengyi Yang

    2015-08-01

    Full Text Available Cell signaling underlies transcription/epigenetic control of a vast majority of cell-fate decisions. A key goal in cell signaling studies is to identify the set of kinases that underlie key signaling events. In a typical phosphoproteomics study, phosphorylation sites (substrates of active kinases are quantified proteome-wide. By analyzing the activities of phosphorylation sites over a time-course, the temporal dynamics of signaling cascades can be elucidated. Since many substrates of a given kinase have similar temporal kinetics, clustering phosphorylation sites into distinctive clusters can facilitate identification of their respective kinases. Here we present a knowledge-based CLUster Evaluation (CLUE approach for identifying the most informative partitioning of a given temporal phosphoproteomics data. Our approach utilizes prior knowledge, annotated kinase-substrate relationships mined from literature and curated databases, to first generate biologically meaningful partitioning of the phosphorylation sites and then determine key kinases associated with each cluster. We demonstrate the utility of the proposed approach on two time-series phosphoproteomics datasets and identify key kinases associated with human embryonic stem cell differentiation and insulin signaling pathway. The proposed approach will be a valuable resource in the identification and characterizing of signaling networks from phosphoproteomics data.

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

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    Maria Hernandez-Valladares

    2016-08-01

    Full Text Available 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.

  5. Phosphoproteomic analysis of cell-based resistance to BRAF inhibitor therapy in melanoma

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    Robert eParker

    2015-05-01

    Full Text Available 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.

  6. Systems Analysis for Interpretation of Phosphoproteomics Data

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V

    2016-01-01

    Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. Systems level analysis...... provides the best means for extracting functional insights from such types of datasets, and this has primed a rapid development of bioinformatics tools and resources over the last decade. Many of these tools are specialized databases that can be mined for annotation and pathway enrichment, whereas others...... provide a platform to generate functional protein networks and explore the relations between proteins of interest. The use of these tools requires careful consideration with regard to the input data, and the interpretation demands a critical approach. This chapter provides a summary of the most...

  7. Evaluating experimental bias and completeness in comparative phosphoproteomics analysis.

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    Jos Boekhorst

    Full Text Available Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically

  8. Interleukin-2 signaling pathway analysis by quantitative phosphoproteomics

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    Osinalde, Nerea; Moss, Helle; Arrizabalaga, Onetsine;

    2011-01-01

    by IL-2, we aimed to define the global tyrosine-phosphoproteome of IL-2 pathway in human T cell line Kit225 using high resolution mass spectrometry combined with phosphotyrosine immunoprecipitation and SILAC. The molecular snapshot at 5min of IL-2 stimulation resulted in identification of 172...... proteins among which 79 were found with increased abundance in the tyrosine-phosphorylated complexes, including several previously not reported IL-2 downstream effectors. Combinatorial site-specific phosphoproteomic analysis resulted in identification of 99 phosphorylated sites mapping to the identified...

  9. Phosphoproteomics Analysis of Endometrium in Women with or without Endometriosis

    Institute of Scientific and Technical Information of China (English)

    Hong-Mei Xu; Hai-Teng Deng; Chong-Dong Liu; Yu-Ling Chen; Zhen-Yu Zhang

    2015-01-01

    Background:The molecular mechanisms underlying the endometriosis are still not completely understood.In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets,we carried out a phosphoproteomics analysis of human endometrium.Methods:A large-scale differential phosphoproteome analysis,using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry,was performed in endometrium tissues from 8 women with or without endometriosis.Results:The phosphorylation profiling of endometrium from endometriosis patients had been obtained,and found that identified 516 proteins were modified at phosphorylation level during endometriosis.Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity.Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways,which might be deregulated during the development of endometriosis.Conclusions:That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

  10. Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

    DEFF Research Database (Denmark)

    Bøgebo, Rikke; Horn, Heiko; Olsen, Jesper V;

    2014-01-01

    -arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data...... developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets....

  11. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis

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    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J.; Ghazanfar, Shila; James, David E.; Jothi, Raja; Yang, Jean Yee Hwa

    2016-01-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the “directPA” R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org). PMID:27145998

  12. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis.

    Science.gov (United States)

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J; Ghazanfar, Shila; James, David E; Jothi, Raja; Yang, Jean Yee Hwa

    2016-07-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the "directPA" R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org). PMID:27145998

  13. Quantitative phosphoproteomic analysis of porcine muscle within 24 h postmortem

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Palmisano, Giuseppe;

    2014-01-01

    in meat quality development, a quantitative mass spectrometry-based phosphoproteomic study was performed to analyze the porcine muscle within 24h PM using dimethyl labeling combined with the TiSH phosphopeptide enrichment strategy. In total 305 unique proteins were identified, including 160...... phosphorylation levels in muscle within 24 h PM. The high phosphorylation level of heat shock proteins (HSPs) in early PM may be an adaptive response to slaughter stress and protect muscle cell from apoptosis, as observed in the serine 84 of HSP27. This work indicated that PM muscle proteins underwent significant...... and rigor mortis development in PM muscle. BIOLOGICAL SIGNIFICANCE: The manuscript describes the characterization of postmortem (PM) porcine muscle within 24 h postmortem from the perspective of protein phosphorylation using advanced phosphoproteomic techniques. In the study, the authors employed...

  14. Analytical strategies in mass spectrometry-based phosphoproteomics

    DEFF Research Database (Denmark)

    Rosenqvist, Heidi; Ye, Juanying; Jensen, Ole N

    2011-01-01

    Phosphoproteomics, the systematic study of protein phosphorylation events and cell signaling networks in cells and tissues, is a rapidly evolving branch of functional proteomics. Current phosphoproteomics research provides a large toolbox of strategies and protocols that may assist researchers to...

  15. Phosphoproteomic analysis of aged skeletal muscle.

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    Gannon, Joan; Staunton, Lisa; O'Connell, Kathleen; Doran, Philip; Ohlendieck, Kay

    2008-07-01

    One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phospho-specific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month- and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin alpha, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins. PMID:18575773

  16. In Vivo SILAC-Based Proteomics Reveals Phosphoproteome Changes during Mouse Skin Carcinogenesis

    DEFF Research Database (Denmark)

    Zanivan, Sara; Meves, Alexander; Behrendt, Kristina;

    2013-01-01

    SILAC technology in combination with high-resolution mass spectrometry (MS) can be successfully used to measure phosphoproteomes in vivo. Here, Zanivan, Mann, and colleagues have applied SILAC-based MS to investigate phosphoproteomic changes during skin carcinogenesis, using the DMBA/TPA two-stag...

  17. Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H;

    2011-01-01

    Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein phosphory......Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...... phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p...

  18. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

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    Sebastian K Wandinger

    Full Text Available 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.

  19. Phosphoproteomics-based modeling defines the regulatory mechanism underlying aberrant EGFR signaling.

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    Shinya Tasaki

    Full Text Available BACKGROUND: Mutation of the epidermal growth factor receptor (EGFR results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling. METHODOLOGY/PRINCIPAL FINDINGS: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992, one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics. CONCLUSIONS/SIGNIFICANCE: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling.

  20. Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin.

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    Alejandro Zimman

    Full Text Available 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 (dephosphorylation, 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.

  1. Quantitative phosphoproteomic analysis of prion-infected neuronal cells

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    Löwer Johannes

    2010-09-01

    Full Text Available Abstract Prion diseases or transmissible spongiform encephalopathies (TSEs are fatal diseases associated with the conversion of the cellular prion protein (PrPC to the abnormal prion protein (PrPSc. Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.

  2. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: Expanding the mycobacterial phosphoproteome catalogue

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    Suereta eFortuin

    2015-02-01

    Full Text Available Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX and Titanium dioxide (TiO2 chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only forty five of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

  3. In Vivo SILAC-Based Proteomics Reveals Phosphoproteome Changes during Mouse Skin Carcinogenesis

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    Sara Zanivan

    2013-02-01

    Full Text Available Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

  4. Phosphoproteome analysis of Lotus japonicus seeds.

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    Ino, Yoko; Ishikawa, Akiyo; Nomura, Ayako; Kajiwara, Hideyuki; Harada, Kyuya; Hirano, Hisashi

    2014-01-01

    In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase-specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM-kinase target motifs, X-X-pS/pT-Q-X-X, were detected in cotyledons. Moreover, by real-time RT-PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM-kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 (http://proteomecentral.proteomexchange.org/dataset/PXD000053).

  5. Assessment of phosphopeptide enrichment/precipitation method for LC-MS/MS based phosphoproteomic analysis of plant tissue

    DEFF Research Database (Denmark)

    Ye, Juanying; Rudashevskaya, Elena; Hansen, Thomas Aarup;

    (MSA) method was used for phosphopeptide fragmentation. The resulting fragment ion spectra were processed with Proteome Discoverer software (Thermo Electron, Bremen, Germany). Results We first investigated the global phosphorylation profile of plant plasma membrane proteins by enriching...... the phosphopeptides with IMAC, TiO2 enrichment methods prior to LTQ-Orbitrap MS analysis. 100 ug plant plasma membrane protein was used for each enrichment experiment. The data was searched against NCBI database on MASCOT server, and the results were validated by in home bioinformatic software using the A...

  6. Phosphoproteomics Analysis of Endometrium in Women with or without Endometriosis

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    Hong-Mei Xu

    2015-01-01

    Conclusions: That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

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

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    Xiuli eHu

    2015-05-01

    Full Text Available 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.

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

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    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

  9. Phosphoproteomics analysis of postmortem porcine muscle with pH decline rate and time difference

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin R; Karlsson, Anders H;

    2012-01-01

    . The sarcoplasmic and myofibrillar fractions were analyzed using gel electrophoresis in combination with a phosphoprotein specific staining. Globally, the group with fast pH decline rate had the highest phosphorylation level at PM 1 h, but lowest at PM 24 h, whereas the group with slow pH decline rate showed...... the reverse case. The phosphorylation level of 12 bands in sarcoplasmic fraction and 3 bands in myofibrillar fraction were significantly affected by the synergy effects of pH and time (p...The aim of this study was to characterize the protein phosphorylation in postmortem (PM) muscle and reveal the change during meat quality development. The gel-based phosphoproteomic analysis of PM porcine muscle was performed in three pig groups with different pH decline rates from PM 1h to 24 h...

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

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    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.

  11. Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Panpan Yu

    Full Text Available Chondroitin sulfate proteoglycans (CSPGs are major components of the extracellular matrix which mediate inhibition of axonal regeneration after injury to the central nervous system (CNS. Several neuronal receptors for CSPGs have recently been identified; however, the signaling pathways by which CSPGs restrict axonal growth are still largely unknown. In this study, we applied quantitative phosphoproteomics to investigate the global changes in protein phosphorylation induced by CSPGs in primary neurons. In combination with isobaric Tags for Relative and Absolute Quantitation (iTRAQ labeling, strong cation exchange chromatography (SCX fractionation, immobilized metal affinity chromatography (IMAC and LC-MS/MS, we identified and quantified 2214 unique phosphopeptides corresponding to 1118 phosphoproteins, with 118 changing significantly in abundance with CSPG treatment. The proteins that were regulated by CSPGs included key components of synaptic vesicle trafficking, axon guidance mediated by semaphorins, integrin signaling, cadherin signaling and EGF receptor signaling pathways. A significant number of the regulated proteins are cytoskeletal and related proteins that have been implicated in regulating neurite growth. Another highly represented protein category regulated by CSPGs is nucleic acid binding proteins involved in RNA post-transcriptional regulation. Together, by screening the overall phosphoproteome changes induced by CSPGs, this data expand our understanding of CSPG signaling, which provides new insights into development of strategies for overcoming CSPG inhibition and promoting axonal regeneration after CNS injury.

  12. Predicting kinase activity in angiotensin receptor phosphoproteomes based on sequence-motifs and interactions.

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    Rikke Bøgebo

    Full Text Available Recent progress in the understanding of seven-transmembrane receptor (7TMR signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.

  13. Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties.

    Science.gov (United States)

    Narushima, Yuta; Kozuka-Hata, Hiroko; Koyama-Nasu, Ryo; Tsumoto, Kouhei; Inoue, Jun-ichiro; Akiyama, Tetsu; Oyama, Masaaki

    2016-03-01

    Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-β receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-β receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-β receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation. PMID:26670566

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia

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    Roytrakul Sittiruk

    2011-06-01

    Full Text Available Abstract Background Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. Methods The phosphoproteome of bone marrow HSCs/CD34+ cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34+ cells were compared with HbE/β-thalassemia and normal HSCs. Results A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia. Conclusions Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia.

  16. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821: Phosphoproteomic Analysis

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    Barbora Šalovská

    2014-07-01

    Full Text Available DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs: Ataxia teleangiectasia mutated (ATM, DNA-dependent protein kinase (DNA-PK and ATM and Rad3-related kinase (ATR. Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonylphenyl-N-phenylpyrazine-2-carboxamide, has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative. Hydrophilic interaction liquid chromatography (HILIC-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

  17. Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics

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    He Qiu-Yan

    2011-06-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is usually overexpressed in nasopharyngeal carcinoma (NPC and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells. Results We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2 were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins. Conclusion The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

  18. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen;

    2011-01-01

    for protein kinase A, protein kinase C, casein kinase II and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria...

  19. Quantitative phosphoproteomics analysis of nitric oxide-responsive phosphoproteins in cotton leaf.

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    Shuli Fan

    Full Text Available Knowledge of phosphorylation events and their regulation is crucial to understanding the functional biology of plant proteins, but very little is currently known about nitric oxide-responsive phosphorylation in plants. Here, we report the first large-scale, quantitative phosphoproteome analysis of cotton (Gossypium hirsutum treated with sodium nitroprusside (nitric oxide donor by utilizing the isobaric tag for relative and absolute quantitation (iTRAQ method. A total of 1315 unique phosphopeptides, spanning 1528 non-redundant phosphorylation sites, were detected from 1020 cotton phosphoproteins. Among them, 183 phosphopeptides corresponding to 167 phosphoproteins were found to be differentially phosphorylated in response to sodium nitroprusside. Several of the phosphorylation sites that we identified, including RQxS, DSxE, TxxxxSP and SPxT, have not, to our knowledge, been reported to be protein kinase sites in other species. The phosphoproteins identified are involved in a wide range of cellular processes, including signal transduction, RNA metabolism, intracellular transport and so on. This study reveals unique features of the cotton phosphoproteome and provides new insight into the biochemical pathways that are regulated by nitric oxide.

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

    Science.gov (United States)

    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

  1. Comparative gel-based phosphoproteomics in response to signaling molecules

    KAUST Repository

    Marondedze, Claudius

    2013-09-03

    The gel-based proteomics approach is a valuable technique for studying the characteristics of proteins. This technique has diverse applications ranging from analysis of a single protein to the study of the total cellular proteins. Further, protein quality and to some extent distribution can be first assessed by means of one-dimensional gel electrophoresis and then more informatively, for comparative analysis, using the two-dimensional gel electrophoresis technique. Here, we describe how to take advantage of the availability of fluorescent dyes to stain for a selective class of proteins on the same gel for the detection of both phospho- and total proteomes. This enables the co-detection of phosphoproteins as well as total proteins from the same gel and is accomplished by utilizing two different fluorescent stains, the ProQ-Diamond, which stains only phosphorylated proteins, and Sypro Ruby, which stains the entire subset of proteins. This workflow can be applied to gain insights into the regulatory mechanisms induced by signaling molecules such as cyclic nucleotides through the quantification and subsequent identification of responsive phospho- and total proteins. © Springer Science+Business Media New York 2013.

  2. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

    Science.gov (United States)

    Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

    2016-09-13

    Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. PMID:27569059

  3. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (PhosphoProteomic Profiling

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    Ilyas Singec

    2016-09-01

    Full Text Available Controlled differentiation of human embryonic stem cells (hESCs can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs. This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families, phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt.

  4. Kinomic and phospho-proteomic analysis of breast cancer stem-like cells

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Christensen, Anne Geske Lindhard; Ehmsen, Sidse;

    Kinomic and phospho-proteomic analysis of breast cancer stem-like cells Rikke Leth-Larsen1, Anne G Christensen1, Sidse Ehmsen1, Mark Møller1, Giuseppe Palmisano2, Martin R Larsen2, Henrik J Ditzel1,3 1Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark 2Institute...... cell death, while the bulk of a tumor lacks these capacities. The resistance mechanisms may cause these cells to survive and become the source of later tumor recurrence, highlighting the need for therapeutic strategies that specifically target pathways central to these cancer stem cells. The CD44hi....../CD24-/low compartment of human breast cancer is enriched in tumor-initiating cells; however the functional heterogeneity within this subpopulation remains poorly defined. From a triple-negative breast cancer cell line we isolated and cloned CD44hi single-cells that exhibited functional heterogeneity...

  5. Phosphoproteomic Analysis of the Highly-Metastatic Hepatocellular Carcinoma Cell Line, MHCC97-H

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    Miaomiao Tian

    2015-02-01

    Full Text Available Invasion and metastasis of hepatocellular carcinoma (HCC is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis. In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2, Eukaryotic Initiation Factor 4A-III (EIF4A3, Cell Division Cycle 5-Like (CDC5L and Survival Motor Neuron Domain Containing 1 (SMNDC1, were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing.

  6. Quantitative phosphoproteomic analysis of the PI3K-regulated signaling network.

    Science.gov (United States)

    Gnad, Florian; Wallin, Jeffrey; Edgar, Kyle; Doll, Sophia; Arnott, David; Robillard, Liliane; Kirkpatrick, Donald S; Stokes, Matthew P; Vijapurkar, Ulka; Hatzivassiliou, Georgia; Friedman, Lori S; Belvin, Marcia

    2016-07-01

    The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899). PMID:27282143

  7. The Arabidopsis thaliana Cyclic-Nucleotide-Dependent Response – a Quantitative Proteomic and Phosphoproteomic Analysis

    KAUST Repository

    Alqurashi, May M.

    2013-11-01

    Protein phosphorylation governs many regulatory pathways and an increasing number of kinases, proteins that transfer phosphate groups, are in turn activated by cyclic nucleotides. One of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP), has been shown to be a second messenger in abiotic and biotic stress responses. However, little is known about the precise role of cAMP in plants and in the down-stream activation of kinases, and hence cAMP-dependent phosphorylation. To increase our understanding of the role of cAMP, proteomic and phosphoproteomic profiles of Arabidopsis thaliana suspension culture cells were analyzed before and after treatment of cells with two different concentrations of 8-Bromo-cAMP (1 µM and 100 nM) and over a time-course of one hour. A comparative quantitative analysis was undertaken using two- dimensional gel electrophoresis and the Delta 2D software (DECODON) followed by protein spot identification by tandem mass spectrometry combined with Mascot and Scaffold. Differentially expressed proteins and regulated phosphoproteins were categorized according to their biological function using bioinformatics tools. The results revealed that the treatment with 1 µM and 100 nM 8-Bromo-cAMP was sufficient to induce specific concentration- and time-dependent changes at the proteome and phosphoproteome levels. In particular, different phosphorylation patterns were observed overtime preferentially affecting proteins in a number of functional categories, notably phosphatases, proteins that remove phosphate groups. This suggests that cAMP both transiently activates and deactivates proteins through specific phosphorylation events and provides new insight into biological mechanisms and functions at the systems level.

  8. Progress in Quantitative Analysis Strategies of Phosphoproteomics%磷酸化蛋白质组定量研究策略

    Institute of Scientific and Technical Information of China (English)

    甄艳; 李春映; 施季森

    2014-01-01

    Protein phosphorylation is an important post-translational modification in organisms for the regulation of diverse biological processes. Quantitative analysis of the phosphorylated proteins is important for understanding the biological functions under internal and external factors. The early studies focus on the qualitative analysis, identification and location of protein phosphorylation sites. In recent years, with the rapid development of mass spectrometry, the quantitative analysis strategies on phosphoproteomics have been made great progress. Monitoring changes in phosphorylated proteins is capable of clarifying the processes of biological functions. In this paper, we mainly present an overview of several strategies of quantitative phosphoproteomics, such as strategies of quantitative phosphoproteomics based on 2-DE, quantitative phosphoproteomics based on gel-free MS/MS labelling and label free, etc. Quantitative analysis of protein phosphorylation would be help to better elucidate the signal transduction mechanisms of biological processes.%蛋白质磷酸化修饰是调控多样种生物过程的一种重要的翻译后修饰,定量分析内部和外部因子作用下磷酸化蛋白质的分子调控机制对理解生物功能非常重要。早期磷酸化蛋白质组研究的重点是定性检测、鉴定磷酸化蛋白质及磷酸化位点的定位。近年来,随着质谱技术的快速发展使得定量磷酸化蛋白质组研究有了很大的发展。利用磷酸化蛋白质组的定量分析监测蛋白质磷酸化的动态变化有助于阐明生物过程及其功能的实现机制。本文综述了磷酸化蛋白质组定量分析策略:基于2-DE的磷酸化蛋白质组定量策略,基于无胶的质谱磷酸化蛋白质组标记定量策略及无标记定量分析策略。磷酸化蛋白质组的定量分析将有助于更好的阐明生物的信号传导机制。

  9. Plug-and-play analysis of the human phosphoproteome by targeted high-resolution mass spectrometry.

    Science.gov (United States)

    Lawrence, Robert T; Searle, Brian C; Llovet, Ariadna; Villén, Judit

    2016-05-01

    Systematic approaches to studying cellular signaling require phosphoproteomic techniques that reproducibly measure the same phosphopeptides across multiple replicates, conditions, and time points. Here we present a method to mine information from large-scale, heterogeneous phosphoproteomics data sets to rapidly generate robust targeted mass spectrometry (MS) assays. We demonstrate the performance of our method by interrogating the IGF-1/AKT signaling pathway, showing that even rarely observed phosphorylation events can be consistently detected and precisely quantified. PMID:27018578

  10. Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins

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    Nespoulous Claude

    2012-10-01

    Full Text Available Abstract Background Genome-wide statistics established that long intrinsically disordered regions (over 30 residues are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. Results Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation. It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. Conclusions The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.

  11. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    Science.gov (United States)

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  12. Quantitative Phosphoproteomic Analysis of Arabidopsis in Response to Salt and Hydrogen Peroxide Stresses

    Institute of Scientific and Technical Information of China (English)

    Yanmei Chen

    2012-01-01

    Salinity and oxidative stresses are major factors in affecting and limiting the productivity of agricultural crops.The study of biochemical and molecular responses of plants in response to those stresses is important for crop genetics and breeding.Extensive evidence shows that reversible protein phosphorylation plays a central role in mediating stress-regulated physiological responses,but little is known about its extent and function.Mass spectrometry provides a powerful tool for the in-depth analysis of systems biology.In this study,we performed a global quantitative analysis of the Arabidopsis phosphoproteomics in response to a time course of stress treatments using 15N-metabolic labeling and subcellular fractionation approaches.In total,we found 176 phosphoproteins showed to be regulated under stresses.Nine SnRK2 kinases identified to be differentially phosphorylated at multiple serine/threonine residues in their kinase domains following stress treatments,demonstrating different temporal phosphorylation induction of the various isoforms.K+ and Na+ transporters showed coordinated phosphorylation regulation under salt stress.In particular,nuclear proteins and protein kinases have high phosphorylation site occupancy in response to stress treatment.This suggests that the wide range of signaling and cellular processes that are modulated in this study.

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

    Directory of Open Access Journals (Sweden)

    Ines Lassowskat

    2013-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  15. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.

    Science.gov (United States)

    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 FDF1(T), 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

  16. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    Science.gov (United States)

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

    2013-03-01

    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.

  17. Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús;

    2011-01-01

    bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation...

  18. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B;

    2013-01-01

    used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and...

  19. Label-free quantitative phosphoproteomic analysis reveals differentially regulated proteins and pathway in PRRSV-infected pulmonary alveolar macrophages.

    Science.gov (United States)

    Luo, Rui; Fang, Liurong; Jin, Hui; Wang, Dang; An, Kang; Xu, Ningzhi; Chen, Huanchun; Xiao, Shaobo

    2014-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine worldwide and causes significant economic losses. Through regulating the host proteins phosphorylation, PRRSV was found to manipulate the activities of several signaling molecules to regulate innate immune responses. However, the role of protein phosphorylation during PRRSV infection and the signal pathways responsible for it are relatively unknown. Here liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global phosphorylation events in PRRSV-infected pulmonary alveolar macrophages. In total, we identified 2125 unique phosphosites, of which the phosphorylation level of 292 phosphosites on 242 proteins and 373 phosphosites on 249 proteins was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional networks. Pathway analysis revealed that PRRSV-induced inflammatory cytokines production was probably due to the activation of mitogen-activated protein kinase and NF-κB signal pathway, which were regulated by several protein kinases during virus infection. Interacting network analysis indicated that altered phosphoproteins were involved in cellular assembly and organization, protein synthesis, molecular transport, and signal transduction in PRRSV infected cells. These pathways and functional networks analysis could provide direct insights into the biological significance of phosphorylation events modulated by PRRSV and may help us elucidate the pathogenic mechanisms of PRRSV infection. PMID:24533505

  20. Preparation of Polypropylene Spin Tips Filled with Immobilized Titanium(IV) Ion Monolithic Adsorbent for Robust Phosphoproteome Analysis.

    Science.gov (United States)

    Liu, Fangjie; Wan, Hao; Liu, Zhongshan; Wang, Hongwei; Mao, Jiawei; Ye, Mingliang; Zou, Hanfa

    2016-05-17

    In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol β-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 μg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells. PMID:27101427

  1. Time-resolved quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Verano-Braga, Thiago; Schwämmle, Veit; Sylvester, Marc;

    2012-01-01

    proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide...

  2. Phosphoproteomic Analysis of the Highly-Metastatic Hepatocellular Carcinoma Cell Line, MHCC97-H

    OpenAIRE

    Miaomiao Tian; Han Cheng; Zhiqiang Wang; Na Su; Zexian Liu; Changqing Sun; Bei Zhen; Xuechuan Hong; Yu Xue; Ping Xu

    2015-01-01

    Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC–MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). I...

  3. Phosphoproteomic analysis of induced resistance reveals activation of signal transduction processes by beneficial and pathogenic interaction in grapevine.

    Science.gov (United States)

    Perazzolli, Michele; Palmieri, Maria Cristina; Matafora, Vittoria; Bachi, Angela; Pertot, Ilaria

    2016-05-20

    Protein phosphorylation regulates several key processes of the plant immune system. Protein kinases and phosphatases are pivotal regulators of defense mechanisms elicited by resistance inducers. However, the phosphorylation cascades that trigger the induced resistance mechanisms in plants have not yet been deeply investigated. The beneficial fungus Trichoderma harzianum T39 (T39) induces resistance against grapevine downy mildew (Plasmopara viticola), but its efficacy could be further improved by a better understanding of the cellular regulations involved. We investigated quantitative changes in the grapevine phosphoproteome during T39-induced resistance to get an overview of regulatory mechanisms of downy mildew resistance. Immunodetection experiments revealed activation of the 45 and 49kDa kinases by T39 treatment both before and after pathogen inoculation, and the phosphoproteomic analysis identified 103 phosphopeptides that were significantly affected by the phosphorylation cascades during T39-induced resistance. Peptides affected by T39 treatment showed comparable phosphorylation levels after P. viticola inoculation, indicating activation of the microbial recognition machinery before pathogen infection. Phosphorylation profiles of proteins related to photosynthetic processes and protein ubiquitination indicated a partial overlap of cellular responses in T39-treated and control plants. However, phosphorylation changes of proteins involved in response to stimuli, signal transduction, hormone signaling, gene expression regulation, and RNA metabolism were exclusively elicited by P. viticola inoculation in T39-treated plants. These results highlighted the relevance of phosphorylation changes during T39-induced resistance and identified key regulator candidates of the grapevine defense against downy mildew. PMID:27010348

  4. Data set from the phosphoproteomic analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars

    Directory of Open Access Journals (Sweden)

    Yunfeng Li

    2015-06-01

    Full Text Available Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice–M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides.

  5. Comparative phosphoproteomic analysis of the mouse testis reveals changes in phosphopeptide abundance in response to Ppp1cc deletion

    Directory of Open Access Journals (Sweden)

    Graham MacLeod

    2014-03-01

    Full Text Available Male Ppp1cc knockout mice are infertile due to failure of spermatogenesis, thought to arise from a deficiency of the predominant testis isoform PPP1CC2. We hypothesize that substrates of the PPP1CC2 will be hyperphosphorylated in Ppp1cc mutant testes. Quantitative RT-PCR and histological data suggest a role for PPP1CC2 by 3 weeks of age in the testis. Comparative phosphoproteomic analysis identified 828 proteins phosphorylated in the 3 week mouse testis, and confidently assigned 1026 unique phosphorylation sites. Thirty-two peptides corresponding to 30 proteins were found to be more abundant in Ppp1cc mutant samples than in wild-type, representing candidate substrates of PPP1CC2.

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

    Science.gov (United States)

    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.

  7. Global investigation of interleukin-1β signaling in primary β-cells using quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Størling, Joachim; Pociot, Flemming;

    Novel Aspect: Global phosphoproteomic analysis of cytokine signaling in primary β-cells Introduction The insulin-producing β-cells of the pancreatic islets of Langerhans are targeted by aberrant immune system responses in diabetes mellitus involving cytokines, especially interleukin-1β (IL-1 β......), which initiate apoptosis of the β-cells. As only limited amounts of primary β-cells can be isolated from model organisms like mouse and rat, global phosphoproteomic analysis of these signaling events by mass spectrometry has generally been unfeasible. We have therefore developed a strategy...... in cell culture-based phosphoproteomic experiments to increase stimulus-induced phosphorylation changes by reducing the general cellular protein phosphorylation level. Therefore, further IL-1β stimulation experiments on serum-starved islets have been performed to validate the phosphorylation changes...

  8. Integrating phosphoproteomics in systems biology

    Directory of Open Access Journals (Sweden)

    Yu Liu

    2014-07-01

    Full Text Available Phosphorylation of serine, threonine and tyrosine plays significant roles in cellular signal transduction and in modifying multiple protein functions. Phosphoproteins are coordinated and regulated by a network of kinases, phosphatases and phospho-binding proteins, which modify the phosphorylation states, recognize unique phosphopeptides, or target proteins for degradation. Detailed and complete information on the structure and dynamics of these networks is required to better understand fundamental mechanisms of cellular processes and diseases. High-throughput technologies have been developed to investigate phosphoproteomes in model organisms and human diseases. Among them, mass spectrometry (MS-based technologies are the major platforms and have been widely applied, which has led to explosive growth of phosphoproteomic data in recent years. New bioinformatics tools are needed to analyze and make sense of these data. Moreover, most research has focused on individual phosphoproteins and kinases. To gain a more complete knowledge of cellular processes, systems biology approaches, including pathways and networks modeling, have to be applied to integrate all components of the phosphorylation machinery, including kinases, phosphatases, their substrates, and phospho-binding proteins. This review presents the latest developments of bioinformatics methods and attempts to apply systems biology to analyze phosphoproteomics data generated by MS-based technologies. Challenges and future directions in this field will be also discussed.

  9. Phosphoproteomics and Lung Cancer Research

    Directory of Open Access Journals (Sweden)

    William C. S. Cho

    2012-09-01

    Full Text Available Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed.

  10. Analytical strategies for phosphoproteomics

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Larsen, Martin R

    2009-01-01

    sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho-specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis...

  11. Phosphoproteomics in translational research: a sarcoma perspective.

    Science.gov (United States)

    Noujaim, J; Payne, L S; Judson, I; Jones, R L; Huang, P H

    2016-05-01

    Phosphoproteomics has been extensively used as a preclinical research tool to characterize the phosphorylated components of the cancer proteome. Advances in the field have yielded insights into new drug targets, mechanisms of disease progression and drug resistance, and biomarker discovery. However, application of this technology to clinical research has been challenging because of practical issues relating to specimen integrity and tumour heterogeneity. Beyond these limitations, phosphoproteomics has the potential to play a pivotal role in translational studies and contribute to advances in different tumour groups, including rare disease sites like sarcoma. In this review, we propose that deploying phosphoproteomic technologies in translational research may facilitate the identification of better defined predictive biomarkers for patient stratification, inform drug selection in umbrella trials and identify new combinations to overcome drug resistance. We provide an overview of current phosphoproteomic technologies, such as affinity-based assays and mass spectrometry-based approaches, and discuss their advantages and limitations. We use sarcoma as an example to illustrate the current challenges in evaluating targeted kinase therapies in clinical trials. We then highlight useful lessons from preclinical studies in sarcoma biology to demonstrate how phosphoproteomics may address some of these challenges. Finally, we conclude by offering a perspective and list the key measures required to translate and benchmark a largely preclinical technology into a useful tool for translational research. PMID:26802162

  12. Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis.

    Directory of Open Access Journals (Sweden)

    Morgan O'Hayre

    Full Text Available BACKGROUND: Chronic Lymphocytic Leukemia (CLL pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4, is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27, which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (approximately 25% of CLL patients cells examined. CONCLUSIONS/SIGNIFICANCE: Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a

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

    Science.gov (United States)

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

    2014-12-01

    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

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

    Science.gov (United States)

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

    2014-12-01

    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

  15. Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics

    Directory of Open Access Journals (Sweden)

    Frithjof eGlowinski

    2014-07-01

    Full Text Available Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS, which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12ΔCagA deletion mutant, and a P12ΔT4SS deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr phosphopeptides and analysed by nano-LC-Orbitrap MS. In total, 58 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK familiy. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

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

    Science.gov (United States)

    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. PMID:26846344

  17. Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

    Science.gov (United States)

    Ino, Yoko; Arakawa, Noriaki; Ishiguro, Hitoshi; Uemura, Hiroji; Kubota, Yoshinobu; Hirano, Hisashi; Toda, Tosifusa

    2016-04-01

    Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

  18. Quantitative phosphoproteome analysis unveils LAT as a modulator of CD3ζ and ZAP-70 tyrosine phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mogjiborahman Salek

    Full Text Available Signaling through the T cell receptor (TCR initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.

  19. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates

    DEFF Research Database (Denmark)

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima;

    2015-01-01

    importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed...

  20. Phosphoproteomics technologies and applications in plant biology research

    Directory of Open Access Journals (Sweden)

    Jinna eLi

    2015-06-01

    Full Text Available 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.

  1. Signaling network dynamics investigated by quantitative phosphoproteomics

    NARCIS (Netherlands)

    Giansanti, Piero

    2014-01-01

    This thesis describes the application of proteomics technologies to get insight into several aspects of phosphorylation signaling dynamics. The core tool in all performed experiments is mass spectrometry (MS)-based phosphoproteomics. In Chapter 1, a general introduction is given into proteomics and

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

    Directory of Open Access Journals (Sweden)

    Denis Avey

    2015-07-01

    Full Text Available 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

  3. Identifying differentially regulated subnetworks from phosphoproteomic data

    Directory of Open Access Journals (Sweden)

    Tebbe Andreas

    2010-06-01

    Full Text Available Abstract 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.

  4. Evaluation and Properties of the Budding Yeast Phosphoproteome

    OpenAIRE

    Amoutzias, G. D.; He, Y.; Lilley, K. S.; Van de Peer, Y.; Oliver, S G

    2012-01-01

    We have assembled a reliable phosphoproteomic data set for budding yeast Saccharomyces cerevisiae and have investigated its properties. Twelve publicly available phosphoproteome data sets were triaged to obtain a subset of high-confidence phosphorylation sites (p-sites), free of "noisy" phosphorylations. Analysis of this combined data set suggests that the inventory of phosphoproteins in yeast is close to completion, but that these proteins may have many undiscovered p-sites. Proteins involve...

  5. Feasibility of large-scale phosphoproteomics with higher energy collisional dissociation fragmentation

    DEFF Research Database (Denmark)

    Nagaraj, Nagarjuna; D'Souza, Rochelle C J; Cox, Juergen;

    2010-01-01

    Mass spectrometry (MS)-based proteomics now enables the analysis of thousands of phosphorylation sites in single projects. Among a wide range of analytical approaches, the combination of high resolution MS scans in an Orbitrap analyzer with low resolution MS/MS scans in a linear ion trap has prov...... large-scale phosphoproteome analysis alongside collisional induced dissociation, (CID) and electron capture/transfer dissociation (ECD/ETD)....

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  8. Quantitative phosphoproteomics to characterize signaling networks

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2012-01-01

    and quantify thousands of phosphorylations, thus providing extensive overviews of the cellular signaling networks. As a result of these developments quantitative phosphoproteomics have been applied to study processes as diverse as immunology, stem cell biology and DNA damage. Here we review the developments...... in phosphoproteomics technology that have facilitated the application of phosphoproteomics to signaling networks and introduce examples of recent system-wide applications of quantitative phosphoproteomics. Despite the great advances in phosphoproteomics technology there are still several outstanding issues and we...

  9. Recent findings and technological advances in phosphoproteomics for cells and tissues

    DEFF Research Database (Denmark)

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many...... technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed...

  10. Quantitative phosphoproteomics of CXCL12 (SDF-1 signaling.

    Directory of Open Access Journals (Sweden)

    Jason A Wojcechowskyj

    Full Text Available CXCL12 (SDF-1 is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1 infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473 and ERK2 (pY204 were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16 and AKT1S1 (pT246 by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation.

  11. Phosphoproteomic analysis of cells treated with longevity-related autophagy inducers

    DEFF Research Database (Denmark)

    Bennetzen, Martin V; Mariño, Guillermo; Pultz, Dennis;

    2012-01-01

    Macroautophagy is a self-cannibalistic process that enables cells to adapt to various stresses and maintain energy homeostasis. Additionally, autophagy is an important route for turnover of misfolded proteins and damaged organelles, with important implications in cancer, neurodegenerative diseases...... between distinct networks of post-translational modifications and provide a resource for future analysis of autophagy and cell death....

  12. Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity

    DEFF Research Database (Denmark)

    Jersie-Christensen, Rosa R.; Sultan, Abida; Olsen, Jesper V

    2016-01-01

    The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical......, and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike standard cell lysis procedures the cell harvesting is performed at high temperatures (99 °C) and without detergents and subsequent need for protein precipitation. Phosphopeptides are enriched...... artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction...

  13. Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

    Directory of Open Access Journals (Sweden)

    Qian Pei-Yuan

    2011-05-01

    Full Text Available Abstract Background The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles. Results We reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1 development, (2 cell differentiation and integrity, (3 transcription and translation, (4 metabolism, (5 protein-protein interaction and proteolysis, and (6 receptors and enzymes. Conclusion This is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with

  14. Phosphoproteome analysis during larval development and metamorphosis in the spionid polychaete Pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2011-05-25

    Background: The metamorphosis of the spionid polychaete Pseudopolydora vexillosa includes spontaneous settlement onto soft-bottom habitats and morphogenesis that can be completed in a very short time. A previous study on the total changes to the proteome during the various developmental stages of P. vexillosa suggested that little or no de novo protein synthesis occurs during metamorphosis. In this study, we used multicolor fluorescence detection of proteins in 2-D gels for differential analysis of proteins and phosphoproteins to reveal the dynamics of post-translational modification proteins in this species. A combination of affinity chromatography, 2D-PAGE, and mass spectrometry was used to identify the phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles. Results: We reproducibly detected 210, 492, and 172 phosphoproteins in pre-competent larvae, competent larvae, and newly metamorphosed juveniles, respectively. The highest percentage of phosphorylation was observed during the competent larval stage. About 64 stage-specific phosphoprotein spots were detected in the competent stage, and 32 phosphoproteins were found to be significantly differentially expressed in the three stages. We identified 38 phosphoproteins, 10 of which were differentially expressed during metamorphosis. These phosphoproteins belonged to six categories of biological processes: (1) development, (2) cell differentiation and integrity, (3) transcription and translation, (4) metabolism, (5) protein-protein interaction and proteolysis, and (6) receptors and enzymes. Conclusion: This is the first study to report changes in phosphoprotein expression patterns during the metamorphosis of the marine polychaete P. vexillosa. The higher degree of phosphorylation during the process of attaining competence to settle and metamorphose may be due to fast morphological transitions regulated by various mechanisms. Our data are consistent with previous studies showing a

  15. The use of elemental mass spectrometry in phosphoproteomic applications.

    Science.gov (United States)

    Maes, Evelyne; Tirez, Kristof; Baggerman, Geert; Valkenborg, Dirk; Schoofs, Liliane; Encinar, Jorge Ruiz; Mertens, Inge

    2016-01-01

    Reversible phosphorylation is one of the most important post-translational modifications in mammalian cells. Because this molecular switch is an important mechanism that diversifies and regulates proteins in cellular processes, knowledge about the extent and quantity of phosphorylation is very important to understand the complex cellular interplay. Although phosphoproteomics strategies are applied worldwide, they mainly include only molecular mass spectrometry (like MALDI or ESI)-based experiments. Although identification and relative quantification of phosphopeptides is straightforward with these techniques, absolute quantification is more complex and usually requires for specific isotopically phosphopeptide standards. However, the use of elemental mass spectrometry, and in particular inductively coupled plasma mass spectrometry (ICP-MS), in phosphoproteomics-based experiments, allow one to absolutely quantify phosphopeptides. Here, these phosphoproteomic applications with ICP-MS as elemental detector are reviewed. Pioneering work and recent developments in the field are both described. Additionally, the advantage of the parallel use of molecular and elemental mass spectrometry is stressed. PMID:25139451

  16. Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.

    Science.gov (United States)

    Gladilovich, Vladimir; Greifenhagen, Uta; Sukhodolov, Nikolai; Selyutin, Artem; Singer, David; Thieme, Domenika; Majovsky, Petra; Shirkin, Alexey; Hoehenwarter, Wolfgang; Bonitenko, Evgeny; Podolskaya, Ekaterina; Frolov, Andrej

    2016-04-22

    Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research. PMID:27016113

  17. The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants.

    Science.gov (United States)

    Wang, Xiaoqin; Qi, Meiyan; Li, Jingyun; Ji, Zhongzhong; Hu, Yong; Bao, Fang; Mahalingam, Ramamurthy; He, Yikun

    2014-05-01

    The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development.

  18. Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer.

    OpenAIRE

    Drake, JM; Paull, EO; Graham, NA; Lee, JK; Smith, BA; Titz, B; Stoyanova, T; Faltermeier, CM; Uzunangelov, V; Carlin,, R.; Fleming, DT; Wong, CK; Newton, Y; Sudha, S; Vashisht, AA

    2016-01-01

    We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of drugga...

  19. Quantitative phosphoproteomic analysis of signaling downstream of the prostaglandin e2/g-protein coupled receptor in human synovial fibroblasts: potential antifibrotic networks.

    Science.gov (United States)

    Gerarduzzi, Casimiro; He, QingWen; Antoniou, John; Di Battista, John A

    2014-11-01

    The Prostaglandin E2 (PGE2) signaling mechanism within fibroblasts is of growing interest as it has been shown to prevent numerous fibrotic features of fibroblast activation with limited evidence of downstream pathways. To understand the mechanisms of fibroblasts producing tremendous amounts of PGE2 with autocrine effects, we apply a strategy of combining a wide-screening of PGE2-induced kinases with quantitative phosphoproteomics. Our large-scale proteomic approach identified a PKA signal transmitted through phosphorylation of its substrates harboring the R(R/X)X(S*/T*) motif. We documented 115 substrates, of which 72 had 89 sites with a 2.5-fold phosphorylation difference in PGE2-treated cells than in untreated cells, where approximately half of such sites were defined as being novel. They were compiled by networking software to focus on highlighted activities and to associate them with a functional readout of fibroblasts. The substrates were associated with a variety of cellular functions including cytoskeletal structures (migration/motility), regulators of G-protein coupled receptor function, protein kinases, and transcriptional/translational regulators. For the first time, we extended the PGE2 pathway into an elaborate network of interconnecting phosphoproteins, providing vital information to a once restricted signalosome. These data provide new insights into eicosanoid-initiated cell signaling with regards to the regulation of fibroblast activation and the identification of new targets for evidenced-based pharmacotherapy against fibrosis. PMID:25223752

  20. Taking Aim at Shotgun Phosphoproteomics

    OpenAIRE

    Hoffert, Jason D.; Knepper, Mark A.

    2007-01-01

    Shotgun phosphoproteomics employs liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) to analyze phosphopeptides from complex protein mixtures, allowing detection and quantification of phosphorylation events on a global scale. Within the past few years, this powerful technique has been used to uncover novel phosphorylation sites as well as explore changes in protein phosphorylation during cellular signaling in various systems. This review presents a general overview of current p...

  1. The phosphoproteome analysis in PBMCs of SLE patients%系统性红斑狼疮外周血单个核细胞磷酸化蛋白质组学研究

    Institute of Scientific and Technical Information of China (English)

    程娟; 马华林; 戴勇

    2014-01-01

    Objective To investigate the aberrant expression of phosphoproteome analysis of peripheral blood mononuclear cells( PBMCs) in patients with systemic lupus erythematosus( SLE) . Lay the foundation for further re-search of mechanism and treatment in patients with SLE. Methods Phosphopeptides were enriched using TiO2 from PBMCs of patients and healthy subjects, then analyzed by automated LC-MS/MS analysis. Phosphorylation sites were identified and quantitated by MASCOT and MaxQuant. Differential expressed proteins and peptides were screened based on the bioinformatics analysis. Results 1 035 phosphorylation sites were identified from SLE com-prared with normal subjects, 618 corresponding genes were screened out in annotation proteins. Pathway analyses showed 12 signaling pathways were identified. There were the most difference phosphorylation sites in mitogen acti-vated protein kinases( MAPK) signaling pathway. Conclusion Differently phosphorylated proteins and peptides can be detected in patients with SLE, which can be used as a mechanism of reference and supplement combined with metabolic pathway, and might be used as a potential target for treatment and research of SLE.%目的:通过系统性红斑狼疮( SLE)患者外周血单个核细胞( PBMCs)的磷蛋白组学分析,为SLE的进一步机制研究及治疗奠定基础。方法收集15例SLE患者和15例健康受试者的外周血,使用 TiO2富集 PBMCs 的磷酸化肽段,进行质谱分析,然后进行磷酸化肽段和磷酸化位点鉴定,并进行生物信息学分析。结果 SLE患者与正常人存在有差异的1035个磷酸化位点,与标注蛋白对应的基因有618个。共筛选出12条代谢通路,其中丝裂原活化蛋白激酶( MAPKs)通路含差异的磷酸化位点最多。结论 SLE患者PBMCs具有差异的磷酸化蛋白质及肽段,与代谢通路一起可作为SLE发病机制研究参考和补充,并可作为治疗靶点研究。

  2. Quantitative phosphoproteomic analysis of early alterations in protein phosphorylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

    DEFF Research Database (Denmark)

    Schulz, Melanie; Brandner, Stefanie; Eberhagen, Carola;

    2013-01-01

    A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0...

  3. Characterization of the Phosphoproteome in Human Bronchoalveolar Lavage Fluid

    Directory of Open Access Journals (Sweden)

    Francesco Giorgianni

    2012-01-01

    Full Text Available Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC, LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.

  4. Systems-wide Analysis of K-Ras, Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics*

    OpenAIRE

    Gnad, Florian; Young, Amy; Zhou, Wei; Lyle, Karen; Ong, Christy C.; Matthew P. Stokes; Silva, Jeffrey C.; Belvin, Marcia; Friedman, Lori S.; Koeppen, Hartmut; Minden, Audrey; Hoeflich, Klaus P.

    2013-01-01

    Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphop...

  5. Strategies for quantitation of phosphoproteomic data

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Thingholm, Tine Engberg

    2010-01-01

    strategies in order to study the biological role of protein phosphorylation during disease progression, differentiation or during external stimulation of a cellular system. In this article, a brief summary of the most popular strategies for phosphoproteomic studies is given; however, the main focus...... will be on different quantitation strategies. Methods for metabolic labeling, chemical modification and label-free quantitation and their applicability or inapplicability in phosphoproteomic studies are discussed....

  6. Research Progress on Plant Phosphoproteome%植物磷酸化蛋白质组研究进展

    Institute of Scientific and Technical Information of China (English)

    安春鹏; 聂玉哲; 李玉花

    2011-01-01

    Based on introducing phosphoproteome enrichment technique and mass spectrometry, the research progress of phosphoproteome in recent years was summarized, and the prospect was forecasted.%在介绍磷酸化蛋白富集技术和质谱技术的基础上,综述了近年来磷酸化蛋白质组的研究进展,并对其研究前景进行了展望.

  7. Quantitative phosphoproteomics dissection of 7TM receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte Lund; Kelstrup, Christian; Lyngsø, Christina;

    2010-01-01

    performed a global quantitative phosphoproteomics analysis of the AT1R signaling network. We analyzed ligand-stimulated SILAC cells by high-resolution mass spectrometry (LTQ Orbitrap MS) and compared the phosphoproteomes of the AT1R agonist Angiotensin II and the biased agonist SII Angiotensin II, which...... only activates the Gaq protein-independent signaling.e quantified more than ten thousand phosphorylation sites of which 1183 were regulated by Angiotensin II or its analogue SII Angiotensin II. 36% of the AT1R regulated phosphorylations were regulated by SII Angiotensin II. Analysis of phosphorylation...

  8. Comparative Ser/Thr/Tyr phosphoproteomics between two Mycobacterial species: The fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG.

    Directory of Open Access Journals (Sweden)

    Kehilwe Confidence Nakedi

    2015-04-01

    Full Text Available Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized (localization probability (LP =0.75; PEP=0.01. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02 and 3.51 % for M. smegmatis and 35, 61.6 and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates.

  9. Recent findings and technological advances in phosphoproteomics for cells and tissues.

    Science.gov (United States)

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

  10. Investigation of a phosphoproteome qualitative and quantitative analysis method%一种磷酸化蛋白质组定性定量分析方法的考察

    Institute of Scientific and Technical Information of China (English)

    赵丽艳; 刘金凤; 蔡芸; 钱小红; 马守栋; 程艳芹; 纪松岗; 李明春

    2012-01-01

    目的 对用于磷酸化蛋白质的相对定量分析的一种基于双向差示凝胶电泳(2-D DIGE)技术和Pro-Q Diamond 荧光染色的方法进行考察.方法 采用DIGE技术,从Pro-Q Diamond染色后对Cy2、Cy5荧光强度的影响、Pro-QDiamond染色过程中采用的试剂对CyDye标记的蛋白质的影响、去离子水对DIGE扫描结果的影响、CyDye染料扫描的稳定性影响等方面,考察了该方法的有效性和特异性.结果 在Pro-Q Diamond染色前对DIGE凝胶进行处理的过程中,有多种因素影响荧光强度变化,以致在进行pro-Q Diamond染色后,凝胶点的荧光强度变化无法区分是染色造成的灰度值变化还是CyDye染料自身的变化,从而使通过荧光强度变化来定量检测磷酸化蛋白质变得困难.结论 不宜采用对同一DIGE凝胶进行Pro-Q Diamond再染色检测磷酸化蛋白质的方法进行比较蛋白质组学的定量分析,为能同时展示出胶上的磷酸化蛋白质和总蛋白质,可通过DIGE实验与Pro-Q Diamond分开运行,再通过DIGE扫描图谱与Pro-Q Diamond染色图谱进行匹配的方法进行改进.%Objective To investigate a phosphoproteome relative quantitative analysis method based on 2-D DIGE and fluorescence staining. Methods By the DIGE technique, the effect of Pro-Q Diamond on fluorescence intensity of Cy2 and Cy5, the effect of some reagents during the staining, the effect of deionized water on the scanning, and the scanning stability of CyDye staining were all investigated. Results Before the Pro-Q Diamond staining, the intensity fluorescence was changed during the procedure of DIGE could be affected by many factors. So the reason of the intensity changes could be due to the staining or the CyDye itself, therefore increased the difficulty of the subsequent quantitative analysis of phosphoproteins. Conclusion A relative quantitative analysis method based on 2-D DIGE following fluorescence staining was not suitable for phosphoprotein

  11. Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

    DEFF Research Database (Denmark)

    Batth, Tanveer S; Francavilla, Chiara; Olsen, Jesper V

    2014-01-01

    Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify...... thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (Hp...... 14 HpH fractions, we routinely identified over 30 000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Bahl, Justyna Maria Czarna; Jensen, Søren Skov; Larsen, Martin R;

    2008-01-01

    investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimer's disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture...

  14. Technical phosphoproteomic and bioinformatic tools useful in cancer research.

    Science.gov (United States)

    López, Elena; Wesselink, Jan-Jaap; López, Isabel; Mendieta, Jesús; Gómez-Puertas, Paulino; Muñoz, Sarbelio Rodríguez

    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

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

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Birck, Pernille; Størling, Joachim;

    2012-01-01

    , 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-¿ pathway, thereby supporting the utility of this global phosphoproteomics strategy......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...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    María Cristina Romero-Rodriguez

    2015-08-01

    Full Text Available 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 spp. 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, defence, protein folding and oxidation-reduction processes. With the exception of a putative cyclase, the other nineteen proteins had at least one orthologous phosphoprotein in A. thaliana, M. truncatula, N. tabacum and G. max. Out of the twenty 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.

  18. 小鼠肝脏磷酸化蛋白质组鉴定及磷酸化修饰激酶的分析%Mouse liver phosphoproteome methodology optimization and kinase analysis

    Institute of Scientific and Technical Information of China (English)

    林丛; 任亮亮; 姜颖; 贺福初

    2015-01-01

    Objective To analyze the construction of mouse liver phosphoproteome and phosphorylated kinases to provide useful information for integrating mouse kinase phosphorylation regulatory networks.Methods A new method was established to identify phosphoproteome from the mouse liver.First of all, liver protein was digested with trypsin before the resulting peptides were subjected to a two-step phosphopeptide enrichment and separation procedure consisting of TiO2 chro-maphy enrichment combined with high pHHPLC separation.Samples were injected onto aNanolC-Ultra-2Dplus system cou-pled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument.Then data analysis was performed to provide information of new identified phosphorylation sites of kinase.Results and Conclusion Using our efficient and high-throughput platform, we reported the identification of 5386 phosophorylation sites and 4553 phosphopeptides from 1533 proteins of the mouse liver.126 new phosphorylation sites were identified from 116 kinases, which provides valuable infor-mation for phosphorylation networks in the mouse liver.%目的:构建小鼠肝脏磷酸化蛋白质组并对磷酸化激酶进行分析,为完善小鼠激酶磷酸化调控网络提供有价值的信息。方法对正常小鼠肝组织总蛋白提取液进行FASP酶切,用TiO2富集磷酸化肽段,为降低样本的复杂度,对富集到的磷酸化肽段进行反相色谱分离后,质谱鉴定样本中的磷酸化蛋白质组,对鉴定到的磷酸化修饰的激酶进行分析,提供新鉴定到磷酸化修饰位点的信息。结果与结论成功构建了高效的鉴定小鼠肝磷酸化蛋白质组的方法,共鉴定到1533个磷酸化蛋白质,从中确认5386个磷酸化位点和4553个磷酸化肽段,其中包含116磷酸化修饰的激酶,并于发生磷酸化修饰的激酶中成功鉴定到126个新的磷酸化修饰位点,为完善小鼠肝磷酸化信号调控网络提供了有价值的信息。

  19. Characterization of early autophagy signaling by quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer Tg; Zarei, Mostafa; Sprenger, Adrian;

    2014-01-01

    Under conditions of nutrient shortage autophagy is the primary cellular mechanism ensuring availability of substrates for continuous biosynthesis. Subjecting cells to starvation or rapamycin efficiently induces autophagy by inhibiting the MTOR signaling pathway triggering increased autophagic flux....... To elucidate the regulation of early signaling events upon autophagy induction, we applied quantitative phosphoproteomics characterizing the temporal phosphorylation dynamics after starvation and rapamycin treatment. We obtained a comprehensive atlas of phosphorylation kinetics within the first 30 min upon...... induction of autophagy with both treatments affecting widely different cellular processes. The identification of dynamic phosphorylation already after 2 min demonstrates that the earliest events in autophagy signaling occur rapidly after induction. The data was subjected to extensive bioinformatics analysis...

  20. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway

    DEFF Research Database (Denmark)

    Gruhler, Albrecht; Olsen, Jesper Velgaard; Mohammed, Shabaz;

    2005-01-01

    . Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified....../MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [(13)C(6...... phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized...

  1. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J;

    2008-01-01

    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  2. Comparison of Phosphoproteomic Separation Strategies Based on Strong Cation Exchange Chromatography-Isoelectric Focusing Techniques%基于强阳离子交换色谱与等电聚焦的磷酸化蛋白质组学分离策略比较

    Institute of Scientific and Technical Information of China (English)

    隋少卉; 董俊军; 王京兰; 蔡耘; 钱小红

    2012-01-01

    Efficient pre-purification steps for the enrichment of phosphorylated proteins or phos-phopeptides are necessary for better detection of phosphorylation sites in phosphoproteomic analysis. Currently, the most common first-dimensional separation technique used is strong cation exchange (SCX). A potential alternative to SCX-based separation is to use isoelectric focusing (IEF) as a first-dimensional separation technique, which has been demonstrated recently. In this study, we present a direct comparison between SCX and IEF based on IPG strips (IPG-IEF) for the phosphoproteomic separation. The comparison experiments discussed in this study utilized standard phosphoproteins and a real sample of HepG2 cell. Then the comparison of 18O labeling phosphopeptides' stability under immobilized pH gradient gel (IPG)-IEF with under SCX was made. Fractions from both technique (SCX and IPG-IEF) were analyzed using the High Mass Accuracy LTQ-FTICR-MS/MS. The results demonstrate that SCX-LTQ-FT and IPG-IEF-LTQ-FT are useful in the phosphopeptides enrichment analysis on a large scale. And SCX-LTQ-FT is relative superior to IPG-IEF-LTQ-FT, whereas the 18 O labeling phosphopeptides'stability under SCX-LTQ-FT is relatively poor with that under IPG-IEF-LTQ-FT.%比较分析了强阳离子交换(SCX)与等电聚焦(IPG-IEF)技术在磷酸化蛋白质组学中的应用.采用3种标准磷酸化蛋白对SCX与IPG-IEF两种技术对磷酸化肽段富集的有效性进行考察.以HepG2细胞为复杂样本,考察SCX与IPG-IEF在实际样本中的应用情况.对SCX与IPG-IEF技术在18O标记的磷酸化蛋白质组定量研究中的应用情况进行考察.蛋白鉴定采用高准确度、高灵敏度、高分辨率的LTQ-FTICR-MS/MS质谱仪.实验表明:SCX和IPG- IEF在大规模磷酸化肽段分离过程中均有效;在复杂样本中,SCX技术的分离效果优于IPG- IEF; IPG- IEF的重复性好于SCX;在磷酸化蛋白质组定量分析结果表明,IPG-IEF技术的稳定性优

  3. Integrative Network Analysis of Signaling in Human CD34+ Hematopoietic Progenitor Cells by Global Phosphoproteomic Profiling Using TiO2 Enrichment Combined with 2D LC-MS/MS and Pathway Mapping

    OpenAIRE

    Guo, Hongbo; Isserlin, Ruth; Chen, Xiaoji; Wang, Weijia; Phanse, Sadhna; Zandstra, Peter W.; Bader, Gary; Paddison, Patrick; Emili, Andrew

    2013-01-01

    Protein kinase signaling regulates human hematopoietic stem/progenitor cell (HSPC) fate, yet little is known about critical pathway substrates. To address this, we have developed and applied a large-scale, empirically-optimized phosphopeptide affinity enrichment strategy with high-throughput 2D LC-MS/MS screening to evaluate the phosphoproteome of an isolated human CD34+ HSPC population. We first used hydrophilic interaction chromatography (HILIC) as a first dimension separation to separate a...

  4. 王浆高产蜜蜂咽下腺磷酸化蛋白质组分析%Phosphoproteome Analysis of Hypopharyngeal Glands of High Royal Jelly Producing Bee (Apis mellifera L.)

    Institute of Scientific and Technical Information of China (English)

    鲁小山; 韩宾; 张兰; 冯毛; 房宇; 李荣丽; 周天娥; 李建科

    2013-01-01

    [Objective]High royal jelly producing bee (Apis mellifera L.) is the unique honeybee resource in China. However, the mechanism of high royal jelly producing has not been clearly addressed. In order to reveal the significance of protein phosphorylation in hypopharyngeal gland for royal jelly synthesis and secretion, the phosphoproteome of hypopharyngeal gland of nurse bee (6-12 day) was analyzed.[Method]IMAC (immobilized metal-affinity chromatography) phosphoprotein enrichment, SCX (strong cation exchange) separation, LC-MS/MS (liquid chromatography-mass/mass) identification and bioinformatics analysis were applied to analyze phosphoproteome of hypopharyngeal gland of nurse bee.[Result]Of the identified 117 proteins in the hypopharyngeal gland, 6 of them were phosphorylated on 6 phosphopeptides and assigned 8 phosphorylated sites. They were related to protein translation and synthesis, such as 60S acidic ribosomal proteins P0, P1, P2 and 60S ribosomal protein L15, and major royal jelly proteins 1 and 7 precursor.[Conclusion]The phosphorylation modifications occurred on ribosome proteins of hypopharyngeal gland mainly contribute to the high efficiency of synthesizing and secreting of royal jelly protein. The phosphorylation of royal jelly proteins 1 and 7 maintain the reasonable ratio of calcium to phosphorus of royal jelly with the increasing yield, thus meeting the nutrition demand of fertile egg-laying queens and developing larvae. Hence, the data obtained in this study will provide new knowledge to deeply understand the mechanism how high royal jelly producing bees could produce higher amount of royal jelly at the level of protein phosphorylation.%[目的]通过对王浆高产蜜蜂(Apis mellifera L.)(浙江浆蜂)哺育蜂(6-12 d)咽下腺的磷酸化蛋白质组分析,以期探明蛋白质磷酸化修饰对王浆分泌的生物学意义。[方法]将哺育蜂咽下腺蛋白质液内酶切后,用固相金属离子亲和层析色谱法(IMAC

  5. Quantitative phosphoproteomics of proteasome inhibition in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Feng Ge

    Full Text Available BACKGROUND: The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM. Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib. CONCLUSIONS/SIGNIFICANCE: Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells.

  6. Quantitative phosphoproteomic analysis of postmortem muscle development

    DEFF Research Database (Denmark)

    Huang, Honggang

    Meat quality development is highly dependent on postmortem (PM) metabolism and rigor mortis development in PM muscle. PM glycometabolism and rigor mortis fundamentally determine most of the important qualities of raw meat, such as ultimate pH, tenderness, color and water-holding capacity. Protein...

  7. Phosphoproteomic and its application in cellular signaling network analysis%磷酸化蛋白质组学方法在信号网络解析中的应用

    Institute of Scientific and Technical Information of China (English)

    段朝军; 陈主初

    2008-01-01

    信号转导是细胞对各种外界刺激的应答反应,蛋白磷酸化或去磷酸化是信号从胞外流向胞内并导致细胞效应过程中的关键机制.磷酸化蛋白质组学(phosphoproteomics)是采用蛋白质组学的分析方法,研究细胞中所有磷酸化蛋白质及其修饰过程,从整体上观察细胞中被修饰的磷酸化蛋白质的状态及其变化,进而分析特定磷酸化修饰对生命过程的调控作用及其分子机制.

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

    Science.gov (United States)

    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. PMID:26969016

  9. A novel double-component MOAC honeycomb composite with pollen grains as a template for phosphoproteomics research.

    Science.gov (United States)

    Wang, Jiaxi; Li, Jie; Wang, Yanan; Gao, Mingxia; Zhang, Xiangmin; Deng, Chunhui

    2016-07-01

    The enrichment and separation of phosphopeptides from mixed biological samples is a technologically very significance, but highly challenging work. Current designed materials are mainly based on the broad and effective adsorptive character of metal oxide affinity chromatography (MOAC). Though significant progress has been made in the enrichment of phosphopeptides with MOAC material, there are chances for further development. In this study, a novel pollen-based MOAC honeycomb material was firstly explored in which the suitable hydrophilic channels preferentially enrich much more endogenous phosphopeptides than nonphosphopeptides or proteins while doping binary metal oxides at the atomic level and the ultra-high specific surface area have further allowed it to possess more effective active sites. Based on these unique features, the pollen-based material exhibited high selectivity for β-casein (mass ratio of β-casein/BSA, 1:1500), ultra-low detection limit (0.1fmol), desirable reusability. Moreover, the bionics MOAC composites were investigated in the enrichment of phosphopeptides from nonfat milk, human serum (male and female at the same age) and mice liver, results of which indicate the great potential of the composite for the phosphoproteome analysis of complex biological samples through the cheap and environmentally friendly process. PMID:27154659

  10. Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

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    Putty-Reddy Sudhir

    Full Text Available BACKGROUND: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. METHODOLOGY/PRINCIPAL FINDINGS: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77 using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60% of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. CONCLUSIONS/SIGNIFICANCE: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

  11. Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

    Science.gov (United States)

    Luís, Inês M.; Alexandre, Bruno M.; Oliveira, M. Margarida

    2016-01-01

    Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK. PMID

  12. Phosphoproteomic profiling of selenate-treated Alzheimer's disease model cells.

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    Ping Chen

    Full Text Available 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.

  13. Dissection of the insulin signaling pathway via quantitative phosphoproteomics

    DEFF Research Database (Denmark)

    Krüger, Marcus; Kratchmarova, Irina; Blagoev, Blagoy;

    2008-01-01

    The insulin signaling pathway is of pivotal importance in metabolic diseases, such as diabetes, and in cellular processes, such as aging. Insulin activates a tyrosine phosphorylation cascade that branches to create a complex network affecting multiple biological processes. To understand the full ...... the calcium transporting ATPase SERCA2, supporting a connection to calcium signaling. The combination of quantitative phosphoproteomics with cell culture models provides a powerful strategy to dissect the insulin signaling pathways in intact cells....

  14. Towards single-cell LC-MS phosphoproteomics

    OpenAIRE

    Polat, Ayşe Nur; Özlü, Nurhan

    2014-01-01

    Protein phosphorylation is a ubiquitous posttranslational modification, which is heavily involved in signal transduction. Misregulation of protein phosphorylation is often associated with a decrease in cell viability and complex diseases such as cancer. The dynamic and low abundant nature of phosphorylated proteins makes studying phosphoproteome a challenging task. In this review, we summarize state of the art proteomic techniques to study and quantify peptide phosphorylation in biological sy...

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

    Science.gov (United States)

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

    2010-02-01

    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 search engines.

  16. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Directory of Open Access Journals (Sweden)

    Westa Domanova

    Full Text Available In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds, making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same

  17. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies

    Science.gov (United States)

    Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  18. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    Science.gov (United States)

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

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

    Directory of Open Access Journals (Sweden)

    Matthew A Schechter

    Full Text Available 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.

  20. Celiac anti-type 2 transglutaminase antibodies induce phosphoproteome modification in intestinal epithelial Caco-2 cells.

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    Gaetana Paolella

    Full Text Available 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

  1. 巨噬细胞应激的铜绿假单胞菌磷酸化蛋白质组分析%Phosphoproteomic analysis of Pseudomonas aeruginosa strains in response to stress induced by macrophages

    Institute of Scientific and Technical Information of China (English)

    潘建义; 王长一; 叶智鸧; 陈冉; 赵辅昆

    2014-01-01

    Objective To investigate the role of protein phosphorylation in Pseudomonas aeruginosa (P.aeruginosa) strains in response to stress triggered by mouse macrophages.Methods The strong cation exchange-immobilized metal affinity chromatography (SCX-IMAC) was performed to enrich phosphopeptides.The nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) was carried out to identify and analyze phosphoproteome.Results Fourteen phosphopeptides from twelve proteins were identified within thirty-one phosphorylation sites on serine,threonine and tyrosine residues.Fifty percent of these phosphorylated proteins were membrane proteins,indicating that their phosphorylation modification was more critical for bacteria in response to the stress.In terms of biological process of Gene Ontology,these identified proteins were involved in stress response,iron transport,anaerobic respiration,response to hydrogen peroxide and signal transduction by phosphorylation,etc.Conclusion These phosphorylated proteins in P.aeruginosa strains are necessary for signal transduction and their response to harsh environment within the macrophages,such as iron limitation,hypoxia and oxidative stress.This study provides evidence for further investigation on virulence and pathogenesis of P.aeruginosa.%目的 鉴定巨噬细胞应激的铜绿假单胞菌(Pseudomonas aeruginosa)磷酸化蛋白质组,分析磷酸化修饰在该菌应答应激中的作用.方法 采用强阳离子交换层析和固相金属离子亲和层析(SCX-IMAC)富集应激细菌的磷酸化肽段,并利用纳升级液相色谱-质谱联用技术(nano LC-MS/MS)进行鉴定和分析其磷酸化蛋白质组.结果 在应激细菌中鉴定到14个磷酸化肽段,对应于12个磷酸化蛋白质,其中膜蛋白占50%,提示膜蛋白的磷酸化修饰在这一过程中具有关键作用.蛋白质功能分析显示这些磷酸化蛋白质主要涉及应激应答、铁摄取转运、厌氧呼吸、过氧化氢的应

  2. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-11-30

    Background: High doses of ionizing radiation result in biological damage, however the precise relationships between long term health effects, including cancer, and low dose exposures remain poorly understood and are currently extrapolated using high dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose dependent responses to radiation. Principle Findings: We have identified 6845 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts one hour post-exposure. Dual statistical analyses based on spectral counts and peak intensities identified 287 phosphopeptides (from 231 proteins) and 244 phosphopeptides (from 182 proteins) that varied significantly following exposure to 2 and 50 cGy respectively. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatics analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role of MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conlcusions: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provides a basis for the systems level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at

  3. Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

    Science.gov (United States)

    Sarhan, Adil R; Patel, Trushar R; Creese, Andrew J; Tomlinson, Michael G; Hellberg, Carina; Heath, John K; Hotchin, Neil A; Cunningham, Debbie L

    2016-06-01

    Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling. PMID:27074791

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

    Science.gov (United States)

    Fang, Yi; Zhang, Qian; Wang, Xin; Yang, Xue; Wang, Xiangyu; Huang, Zhen; Jiao, Yuchen; Wang, Jing

    2016-03-01

    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

  5. Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.

    Science.gov (United States)

    Zhang, Hao; Sun, Jun; Ye, Jing; Ashraf, Usama; Chen, Zheng; Zhu, Bibo; He, Wen; Xu, Qiuping; Wei, Yanming; Chen, Huanchun; Fu, Zhen F; Liu, Rong; Cao, Shengbo

    2015-12-01

    West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.

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

    Science.gov (United States)

    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

  7. Evidence for a Minimal Eukaryotic Phosphoproteome?

    NARCIS (Netherlands)

    Diks, Sander H.; Parikh, Kaushal; van der Sijde, Marijke; Joore, Jos; Ritsema, Tita; Peppelenbosch, Maikel P.

    2007-01-01

    Background. Reversible phosphorylation catalysed by kinases is probably the most important regulatory mechanism in eukaryotes. Methodology/Principal Findings. We studied the in vitro phosphorylation of peptide arrays exhibiting the majority of PhosphoBase-deposited protein sequences, by factors in c

  8. Optimization of titanium dioxide enrichment of phosphopeptides and application in the Thermoanaerobacter tengcongensis phosphoproteome analysis%二氧化钛富集磷酸肽方法优化及在腾冲嗜热厌氧菌磷酸化蛋白质组分析中的应用

    Institute of Scientific and Technical Information of China (English)

    林威; 王京兰; 应万涛; 钱小红

    2012-01-01

    Using titanium dioxide is a very good strategy for the phosphopeptide enrichment. There are many other factors can affect the enrichment efficiency, and the optimization of parameters was needed for better enrichment results. In this study, the peptide mixtures of six standard proteins were used as the model samples to evaluate and optimize the parameters such as the proportion of acetonitrile and trifluoroacetic acid in loading buffer and the TiO2-to-pep-tide ratio. The results showed that 80% (v/v) acetonitrile, 1% (v/v) trifluoroacetic acid and 40 :1 (m/m) TiO2-to-peptide ratio were the optimum parameters to obtain the best enrichment selectivity and maximum phosphopeptides identification. For the first time, the optimum enrichment conditions were applied for the phosphoproteome analysis of the Thermoanaerobacter tengcongensis, an anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengchong, China, and 25 phosphorylated proteins were identified in the preliminary experiment. The results provided a reference for further study on this organism survived under extreme environment.%为了提高二氧化钛富集磷酸肽法对磷酸肽的富集效率,以6种标准蛋白酶切肽段混合物为研究对象,对二氧化钛富集磷酸肽过程中的乙腈比例、三氟乙酸比例、二氧化钛用量等条件分别进行了优化.结果表明在乙腈含量为80% (v/v),三氟乙酸含量为1%(v/v),二氧化钛用量与需要富集肽段的质量比为40∶1的条件下,可以取得较好的富集效果.将优化后的富集方法应用于腾冲嗜热厌氧菌磷酸化蛋白质的分析,初步鉴定到25个磷酸化蛋白质,为进一步研究这种极端环境下生存的低等生物的生命活动提供了参考信息.

  9. Implications of tyrosine phosphoproteomics in cervical carcinogenesis

    Directory of Open Access Journals (Sweden)

    DeFord James

    2008-01-01

    Full Text Available Abstract Background Worldwide cervical cancer remains a leading cause of mortality from gynecologic malignancies. The link between cervical cancer and persistent infection with HPV has been established. At a molecular level little is known about the transition from the precancerous state to invasive cancer. To elucidate this process, cervical biopsies from human specimens were obtained from precancerous state to stage III disease. Methods Cervical biopsies were obtained from patients with a diagnosis of cervical cancer undergoing definitive surgery or staging operation. Biopsies were obtained from patients with precancerous lesions at the time of their excisional procedure. Control samples were obtained from patients undergoing hysterectomy for benign conditions such as fibroids. Samples were subjected to proteomic profiling using two dimensional gel electrophoresis with subsequent trypsin digestion followed by MALDI-TOF protein identification. Candidate proteins were then further studied using western blotting, immunoprecipitation and immunohistochemistry. Results Annexin A1 and DNA-PKcs were found to be differentially expressed. Phosphorylated annexin A1 was up regulated in diseased states in comparison to control and its level was strongly detected in the serum of cervical cancer patients compared to controls. DNA-PKcs was noted to be hyperphosphorylated and fragmented in cancer when compared to controls. By immunohistochemistry annexin A1 was noted in the vascular environment in cancer and certain precancerous samples. Conclusion This study suggests a probable role for protein tyrosine phosphorylation in cervical carcinogenesis. Annexin A1 and DNA-PK cs may have synergistic effects with HPV infection. Precancerous lesions that may progress to cervical cancer may be differentiated from lesions that will not base on similar immunohistochemical profile to invasive squamous cell carcinoma.

  10. Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient.

    Science.gov (United States)

    Dazert, Eva; Colombi, Marco; Boldanova, Tujana; Moes, Suzette; Adametz, David; Quagliata, Luca; Roth, Volker; Terracciano, Luigi; Heim, Markus H; Jenoe, Paul; Hall, Michael N

    2016-02-01

    Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine. PMID:26787912

  11. Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

    Science.gov (United States)

    Dazert, Eva; Colombi, Marco; Boldanova, Tujana; Moes, Suzette; Adametz, David; Quagliata, Luca; Roth, Volker; Terracciano, Luigi; Heim, Markus H.; Jenoe, Paul; Hall, Michael N.

    2016-01-01

    Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine. PMID:26787912

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

    Science.gov (United States)

    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

  13. Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes.

    Science.gov (United States)

    Lochmatter, Corinne; Fischer, Roman; Charles, Philip D; Yu, Zhanru; Powrie, Fiona; Kessler, Benedikt M

    2016-01-01

    Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn's disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation. PMID:27080861

  14. Phosphoproteomics and bioinformatics analyses of spinal cord proteins in rats with morphine tolerance.

    Directory of Open Access Journals (Sweden)

    Wen-Jinn Liaw

    Full Text Available INTRODUCTION: Morphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance. METHODS: To better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins. RESULTS: Our proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism. CONCLUSIONS: Repeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance.

  15. Comparative Proteome and Phosphoproteome Analyses during Cyprid Development of the Barnacle Balanus ( =Amphibalanus ) amphitrite

    KAUST Repository

    Zhang, Yu

    2010-06-04

    The barnacle Balanus amphitrite (=Amphibalanus amphitrite) is a major marine biofouling invertebrate worldwide. It has a complex life cycle during which the larva (called a nauplius) molts six times before transforming into the cyprid stage. The cyprid stage in B. amphitrite is the critical stage for the larval decision to attach and metamorphose. In this study, proteome and phosphoproteome alterations during cyprid development/aging and upon treatment with the antifouling agent butenolide were examined with a two-dimensional electrophoresis (2-DE) multiplexed fluorescent staining approach. Optimized protein separation strategies, including solution-phase isoelectric fractionation and narrow-pH-range 2-DE, were used in a proteomic analysis. Our results show that the differential regulation of the target proteins is highly dynamic on the levels of both protein expression and posttranslational modification. Two groups of proteins, stress-associated and energy metabolism-related proteins, are differentially expressed during cyprid development. Comparison of the control and treatment groups suggests that butenolide exerts its effects by sustaining the expression levels of these proteins. Altogether, our data suggest that proteins involved in stress regulation and energy metabolism play crucial roles in regulating larval attachment and metamorphosis of B. amphitrite. © 2010 American Chemical Society.

  16. Comprehensive analysis of signal transduction in three-dimensional ECM-based tumor cell cultures

    Directory of Open Access Journals (Sweden)

    Iris Eke

    2015-11-01

    Full Text Available Analysis of signal transduction and protein phosphorylation is fundamental to understand physiological and pathological cell behavior as well as identification of novel therapeutic targets. Despite the fact that more physiological three-dimensional cell culture assays are increasingly used, particularly proteomics and phosphoproteomics remain challenging due to easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates without compromising cell adhesion before cell lysis. The samples can be used for Western blotting as well as phosphoproteome array technology. This technique would be of interest for researchers working in all fields of biology and drug development.

  17. Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-β

    DEFF Research Database (Denmark)

    J D'Souza, Rochelle C; Knittle, Anna M; Nagaraj, Nagarjuna;

    2014-01-01

    Transforming growth factor-β (TGF-β) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic...... changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-β. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-β. We identified several proteins known to be involved in TGF-β-induced cellular processes......, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-β signaling. Although many TGF-β responses are mediated...

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

    Science.gov (United States)

    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. PMID:26792808

  19. Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome*

    Science.gov (United States)

    Johnson, Catherine; Tinti, Michele; Wood, Nicola T.; Campbell, David G.; Toth, Rachel; Dubois, Fanny; Geraghty, Kathryn M.; Wong, Barry H. C.; Brown, Laura J.; Tyler, Jennifer; Gernez, Aurélie; Chen, Shuai; Synowsky, Silvia; MacKintosh, Carol

    2011-01-01

    Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease. PMID:21725060

  20. The differential hippocampal phosphoproteome of Apodemus sylvaticus paralleling spatial memory retrieval in the Barnes maze.

    Science.gov (United States)

    Li, Lin; Csaszar, Edina; Szodorai, Edit; Patil, Sudarshan; Pollak, Arnold; Lubec, Gert

    2014-05-01

    Protein phosphorylation is a well-known and well-documented mechanism in memory processes. Although a large series of protein kinases involved in memory processes have been reported, information on phosphoproteins is limited. It was therefore the aim of the study to determine a partial and differential phosphoproteome along with the corresponding network in hippocampus of a wild caught mouse strain with excellent performance in several paradigms of spatial memory. Apodemus sylvaticus mice were trained in the Barnes maze, a non-invasive test system for spatial memory and untrained mice served as controls. Animals were sacrificed 6h following memory retrieval, hippocampi were taken, proteins extracted and in-solution digestion was carried out with subsequent iTRAQ double labelling. Phosphopeptides were enriched by a TiO2-based method and semi-quantified using two fragmentation principles on the LTQ-orbitrap Velos. In hippocampi of trained animals phosphopeptide levels representing signalling, neuronal, synaptosomal, cytoskeletal and metabolism proteins were at least twofold reduced or increased. Furthermore, a network revealing a link to pathways of ubiquitination, the androgen receptor, small GTPase Rab5 and MAPK signaling as well as synucleins was constructed. This work is relevant for interpretation of previous work and the design of future studies on protein phosphorylation in spatial memory.

  1. Quantitative Phosphoproteomics Reveals Extensive Cellular Reprogramming During HIV-1 Entry

    Science.gov (United States)

    Wojcechowskyj, Jason A.; Didigu, Chuka A.; Lee, Jessica Y.; Parrish, Nicholas F.; Sinha, Rohini; Hahn, Beatrice H.; Bushman, Frederic D.; Jensen, Shane T.; Seeholzer, Steven H.; Doms, Robert W.

    2014-01-01

    SUMMARY Receptor engagement by HIV-1 during host cell entry activates signaling pathways that can reprogram the cell for optimal viral replication. To obtain a global view of the signaling events induced during HIV-1 entry, we conducted a quantitative phosphoproteomics screen of primary human CD4+ T cell after infection with an HIV-1 strain that engages the receptors CD4 and CXCR4. We quantified 1,757 phosphorylation sites with high stringency. The abundance of 239 phosphorylation sites from 175 genes, including several proteins in pathways known to be impacted by HIV-receptor binding, changed significantly within a minute after HIV-1 exposure. Several previously uncharacterized HIV-1 host factors were also identified and confirmed through RNAi depletion studies. Surprisingly, 5 serine/arginine-rich (SR)-proteins involved in mRNA splicing, including the splicing factor SRm300 (SRRM2) were differentially phosophorylated. Mechanistic studies with SRRM2 suggest that HIV-1 modulates host cell alternative splicing machinery during entry in order to facilitate virus replication and release. PMID:23684312

  2. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    Science.gov (United States)

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. PMID:27134283

  3. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper;

    2016-01-01

    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 (OSR1, HDGF and ccdc82) as ATM...

  4. Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.

    Science.gov (United States)

    Grosstessner-Hain, Karin; Hegemann, Björn; Novatchkova, Maria; Rameseder, Jonathan; Joughin, Brian A; Hudecz, Otto; Roitinger, Elisabeth; Pichler, Peter; Kraut, Norbert; Yaffe, Michael B; Peters, Jan-Michael; Mechtler, Karl

    2011-11-01

    Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif. PMID:21857030

  5. Enhanced Phosphoproteomic Profiling Workflow For Growth Factor Signaling Analysis

    DEFF Research Database (Denmark)

    Sylvester, Marc; Burbridge, Mike; Leclerc, Gregory;

    2010-01-01

    immobilized metal affinity chromatography (SIMAC) with TiO2 enrichment. Phosphotyrosine containing peptides were enriched by immunoprecipitation. iTRAQ™ labeling enabled multiplexed quantification of changes during stimulation and comparison of different stimuli. Several fragmentation techniques were used...... transfer dissociation adds confidence in modification site assignment. The workflow is relatively simple but the integration of complementary techniques leads to a deeper insight into cellular signaling networks and the potential pharmacological intervention thereof....

  6. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sebastien; Mandacaru, Samuel C;

    2014-01-01

    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 use...

  7. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads;

    2002-01-01

    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

  8. Quantitative Phospho-proteomics to Investigate the Polo-like Kinase 1-Dependent Phospho-proteome*

    OpenAIRE

    Grosstessner-Hain, Karin; Hegemann, Björn; Novatchkova, Maria; Rameseder, Jonathan; Joughin, Brian A.; Hudecz, Otto; Roitinger, Elisabeth; Pichler, Peter; Kraut, Norbert; Yaffe, Michael B.; Peters, Jan-Michael; Mechtler, Karl

    2011-01-01

    Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with ...

  9. 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

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    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.

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

    Directory of Open Access Journals (Sweden)

    Zhengxiu Su

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Trajectory Based Traffic Analysis

    DEFF Research Database (Denmark)

    Krogh, Benjamin Bjerre; Andersen, Ove; Lewis-Kelham, Edwin;

    2013-01-01

    We present the INTRA system for interactive path-based traffic analysis. The analyses are developed in collaboration with traffic researchers and provide novel insights into conditions such as congestion, travel-time, choice of route, and traffic-flow. INTRA supports interactive point-and-click a......We present the INTRA system for interactive path-based traffic analysis. The analyses are developed in collaboration with traffic researchers and provide novel insights into conditions such as congestion, travel-time, choice of route, and traffic-flow. INTRA supports interactive point......-and-click analysis, due to a novel and efficient indexing structure. With the web-site daisy.aau.dk/its/spqdemo/we will demonstrate several analyses, using a very large real-world data set consisting of 1.9 billion GPS records (1.5 million trajectories) recorded from more than 13000 vehicles, and touching most...

  13. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. PMID:27017623

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

    International Nuclear Information System (INIS)

    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 (Mn2+)-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 Mn2+-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 of GRP78, 14

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

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhenlie [Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300 (China); Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ichihara, Sahoko [Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507 (Japan); Oikawa, Shinji [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Mie 514-8507 (Japan); Chang, Jie [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Graduate School of Regional Innovation Studies, Mie University, Tsu 514-8507 (Japan); Zhang, Lingyi [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510 (Japan); Hu, Shijie [Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300 (China); Huang, Hanlin, E-mail: huanghl@gdoh.org [Guangdong Provincial Key Laboratory of Occupational Disease Prevention and Treatment, Guangdong Province Hospital for Occupational Disease Prevention and Treatment, Guangzhou 510-300 (China); Ichihara, Gaku, E-mail: gak@rs.tus.ac.jp [Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Department of Occupational and Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda 278-8510 (Japan)

    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

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

    Directory of Open Access Journals (Sweden)

    Philipp eSpät

    2015-03-01

    Full Text Available 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 signalling, 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 towards the unbiased 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 labelling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phosphoproteome of Synechocystis to date, identifying 2,382 proteins and 183 phosphorylation events and quantifying 2,111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 hours. Among the proteins with increased phosphorylation, the PII signalling 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.

  17. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    Science.gov (United States)

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-01-01

    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. PMID:26830761

  20. Phosphoproteomics of the Dopamine Pathway Enables Discovery of Rap1 Activation as a Reward Signal In Vivo.

    Science.gov (United States)

    Nagai, Taku; Nakamuta, Shinichi; Kuroda, Keisuke; Nakauchi, Sakura; Nishioka, Tomoki; Takano, Tetsuya; Zhang, Xinjian; Tsuboi, Daisuke; Funahashi, Yasuhiro; Nakano, Takashi; Yoshimoto, Junichiro; Kobayashi, Kenta; Uchigashima, Motokazu; Watanabe, Masahiko; Miura, Masami; Nishi, Akinori; Kobayashi, Kazuto; Yamada, Kiyofumi; Amano, Mutsuki; Kaibuchi, Kozo

    2016-02-01

    Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors. PMID:26804993

  1. MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome.

    Science.gov (United States)

    Ruprecht, Benjamin; Roesli, Christoph; Lemeer, Simone; Kuster, Bernhard

    2016-05-01

    Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (∼20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides. PMID:26990019

  2. Development and Application of Aphosphoproteomic Strategy Based Ontitanium Dioxideaffinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    Mingquan Guo

    2012-01-01

    A sensitive and highly selective phosphoproteomic strategy,which allows simultaneous genome and transcriptome profiling from a single sample,has been developed by a combination of a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and a TRIzol-based protein extraction method.To establish more efficient phosphopeptide enrichment methods for complex cellular proteins,TiO2 method was comparedwith the most widely used IMAC method using model peptide mixturesbased on two criteria:one is the efficacy of the phosphopeptide enrichment (how many phosphopeptides each method can capture),and the other is the extent of exclusion of nonphosphopeptides from the pool of enriched phosphopeptides.The results indicate that the TiO2 enrichment method appears to be more effective than the IMAC method.This methodology was then further applied to the phosphoproteomic analysis of a small number of HeLa cells with and without calyculin A (CL-A) treatment.Significant increases in the number of identified phosphopeptides and phosphosites were observed in the CL-A-treated samples compared with nontreated samples as expected in the biological studies.Meanwhile,some novel phosphorylation sites,such as T19 and S20 of the myosin regulatory light chain and S116 and S379 in the heterogeneous nuclear ribonucleoprotein K transcript variant in the CL-A-treated samples,were revealed as well.Thus,the robustness,generality,and simplicity of this strategy should enable this methodology to find broad applications for phosphoproteomic analyses in systems biology,especially in cases of limited starting material requiring a systems-level analysis such as small clinical specimens.

  3. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

    Science.gov (United States)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Aloria, Kerman; Arizmendi, Jesus M; Zubiaga, Ana M; Blagoev, Blagoy; Kratchmarova, Irina

    2016-06-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies. PMID:27067055

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

    Science.gov (United States)

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

    2014-10-01

    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.

  5. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

    Science.gov (United States)

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Aloria, Kerman; Arizmendi, Jesus M; Zubiaga, Ana M; Blagoev, Blagoy; Kratchmarova, Irina

    2016-06-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.

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

    Science.gov (United States)

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

    2014-10-01

    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. PMID:25168779

  7. Picking the right tool for the job--Phosphoproteomics of egg activation.

    Science.gov (United States)

    Wessel, Gary M

    2015-12-01

    Eggs are the rarest cell in the human body, yet their study is essential for the fields of fertility, reproduction, and fetal health. Guo et al. (Proteomics 2015, 15, 4080-4095) use a "surrogate" animal to discover the phosphoproteomic pathways involved in egg activation. With datasets of several thousand phosphosites on 2500 different proteins, these investigators have defined new pathways, connections to pathways, and priorities in searches for how eggs are activated at fertilization. These results in a sea urchin are now transposable to mammals for testing on a per candidate strategy.

  8. Identification of novel protein functions and signaling mechanisms by genetics and quantitative phosphoproteomics in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Fredens, Julius; Engholm-Keller, Kasper; Møller-Jensen, Jakob;

    2014-01-01

    Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene...... knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment....

  9. System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Rigbolt, Kristoffer T.G.; Prokhorova, Tatyana; Akimov, Vyacheslav;

    2011-01-01

    To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned...... by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those...

  10. Kicking Genomic Profiling to the Curb: How Re-wiring the Phosphoproteome Can Explain Treatment Resistance in Glioma.

    Science.gov (United States)

    Lam, Fred C; Yaffe, Michael B

    2016-04-11

    In this issue of Cancer Cell, Wei et al. (2016) identify adaptive re-wiring of signaling nodes in glioma as major mechanisms of treatment resistance without genome-wide mutations. Targeting these nodes before treatment blocks resistance and underscores the importance of single-cell phosphoproteomics and network re-wiring in predicting cancer treatment responses. PMID:27070697

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

    DEFF Research Database (Denmark)

    Højlund, Kurt; Bowen, Benjamin P; Hwang, Hyonson;

    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 phosph...... skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo....

  12. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

  13. Cardiac Phosphoproteomics during Remote Ischemic Preconditioning: A Role for the Sarcomeric Z-Disk Proteins

    Directory of Open Access Journals (Sweden)

    Safa Abdul-Ghani

    2014-01-01

    Full Text Available Remote ischemic preconditioning (RIPC induced by brief ischemia/reperfusion cycles of remote organ (e.g., limb is cardioprotective. The myocardial cellular changes during RIPC responsible for this phenomenon are not currently known. The aim of this work was to identify the activation by phosphorylation of cardiac proteins following RIPC. To achieve our aim we used isobaric tandem mass tagging (TMT and reverse phase nanoliquid chromatography tandem spectrometry using a Linear Trap Quadropole (LTQ Orbitrap Velos mass spectrometer. Male C57/Bl6 mice were anesthetized by an intraperitoneal injection of Tribromoethanol. A cuff was placed around the hind limb and inflated at 200 mmHg to prevent blood flow as confirmed by Laser Doppler Flowmetry. RIPC was induced by 4 cycles of 5 min of limb ischemia followed by 5 min of reperfusion. Hearts were extracted for phosphoproteomics. We identified approximately 30 phosphoproteins that were differentially expressed in response to RIPC protocol. The levels of several phosphoproteins in the Z-disk of the sarcomere including phospho-myozenin-2 were significantly higher than control. This study describes and validates a novel approach to monitor the changes in the cardiac phosphoproteome following the cardioprotective intervention of RIPC and prior to index ischemia. The increased level of phosphorylated sarcomeric proteins suggests they may have a role in cardiac signaling during RIPC.

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

    Directory of Open Access Journals (Sweden)

    Raymond T Suhandynata

    Full Text Available 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.

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

    Science.gov (United States)

    Teets, Nicholas M; Denlinger, David L

    2016-08-01

    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. PMID:27362561

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  18. Quantitative phosphoproteomics unravels biased phosphorylation of serotonin 2A receptor at Ser280 by hallucinogenic versus nonhallucinogenic agonists.

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-05-01

    The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased

  19. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.

    Directory of Open Access Journals (Sweden)

    Feng Yang

    Full Text Available BACKGROUND: High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. PRINCIPAL FINDINGS: We have identified 7117 unique phosphopeptides (2566 phosphoproteins from control and irradiated (2 and 50 cGy primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. CONCLUSIONS: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.

  20. Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

    Directory of Open Access Journals (Sweden)

    Erick Contreras-Vallejos

    Full Text Available Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5 is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC, which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate and Grin1 (G protein regulated inducer of neurite outgrowth 1. MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.

  1. Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J.; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-01-01

    The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of

  2. Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics.

    Science.gov (United States)

    Golkowski, Martin; Shimizu-Albergine, Masami; Suh, Hyong Won; Beavo, Joseph A; Ong, Shao-En

    2016-07-01

    Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion

  3. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics

    Directory of Open Access Journals (Sweden)

    Peter F. Doubleday

    2014-04-01

    Full Text Available Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX chromatography, coupled to immobilized metal affinity chromatography (IMAC, we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain.

  4. Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma.

    Science.gov (United States)

    Wei, Wei; Shin, Young Shik; Xue, Min; Matsutani, Tomoo; Masui, Kenta; Yang, Huijun; Ikegami, Shiro; Gu, Yuchao; Herrmann, Ken; Johnson, Dazy; Ding, Xiangming; Hwang, Kiwook; Kim, Jungwoo; Zhou, Jian; Su, Yapeng; Li, Xinmin; Bonetti, Bruno; Chopra, Rajesh; James, C David; Cavenee, Webster K; Cloughesy, Timothy F; Mischel, Paul S; Heath, James R; Gini, Beatrice

    2016-04-11

    Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations. PMID:27070703

  5. A phosphoproteomics view at human pluripotent stem cells

    NARCIS (Netherlands)

    Zoumaro-Djayoon, A.D.

    2013-01-01

    Reversible protein phosphorylation has been one of the most investigated post-translational modifications (PTM) in the decade, made possible by advances in enrichment technologies and MS-based proteomics. PTMs are generally involved in the regulation of many biological processes, including processes

  6. Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse Hippocampus.

    Science.gov (United States)

    Kohtala, Samuel; Theilmann, Wiebke; Suomi, Tomi; Wigren, Henna-Kaisa; Porkka-Heiskanen, Tarja; Elo, Laura L; Rokka, Anne; Rantamäki, Tomi

    2016-06-15

    Anesthetics are widely used in medical practice and experimental research, yet the neurobiological basis governing their effects remains obscure. We have here used quantitative phosphoproteomics to investigate the protein phosphorylation changes produced by a 30 min isoflurane anesthesia in the adult mouse hippocampus. Altogether 318 phosphorylation alterations in total of 237 proteins between sham and isoflurane anesthesia were identified. Many of the hit proteins represent primary pharmacological targets of anesthetics. However, findings also enlighten the role of several other proteins-implicated in various biological processes including neuronal excitability, brain energy homeostasis, synaptic plasticity and transmission, and microtubule function-as putative (secondary) targets of anesthetics. In particular, isoflurane increases glycogen synthase kinase-3β (GSK3β) phosphorylation at the inhibitory Ser(9) residue and regulates the phosphorylation of multiple proteins downstream and upstream of this promiscuous kinase that regulate diverse biological functions. Along with confirmatory Western blot data for GSK3β and p44/42-MAPK (mitogen-activated protein kinase; reduced phosphorylation of the activation loop), we observed increased phosphorylation of microtubule-associated protein 2 (MAP2) on residues (Thr(1620,1623)) that have been shown to render its dissociation from microtubules and alterations in microtubule stability. We further demonstrate that diverse anesthetics (sevoflurane, urethane, ketamine) produce essentially similar phosphorylation changes on GSK3β, p44/p42-MAPK, and MAP2 as observed with isoflurane. Altogether our study demonstrates the potential of quantitative phosphoproteomics to study the mechanisms of anesthetics (and other drugs) in the mammalian brain and reveals how already a relatively brief anesthesia produces pronounced phosphorylation changes in multiple proteins in the central nervous system. PMID:27074656

  7. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-01-01

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively. PMID:25262027

  8. Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells.

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-10-24

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.

  9. Hand-Based Biometric Analysis

    Science.gov (United States)

    Bebis, George (Inventor); Amayeh, Gholamreza (Inventor)

    2015-01-01

    Hand-based biometric analysis systems and techniques are described which provide robust hand-based identification and verification. An image of a hand is obtained, which is then segmented into a palm region and separate finger regions. Acquisition of the image is performed without requiring particular orientation or placement restrictions. Segmentation is performed without the use of reference points on the images. Each segment is analyzed by calculating a set of Zernike moment descriptors for the segment. The feature parameters thus obtained are then fused and compared to stored sets of descriptors in enrollment templates to arrive at an identity decision. By using Zernike moments, and through additional manipulation, the biometric analysis is invariant to rotation, scale, or translation or an in put image. Additionally, the analysis utilizes re-use of commonly-seen terms in Zernike calculations to achieve additional efficiencies over traditional Zernike moment calculation.

  10. 哺乳动物精子磷酸化蛋白质组学研究进展%Resent Advances on Phosphoproteomics Researches of Mammalian Sperm Capacitation

    Institute of Scientific and Technical Information of China (English)

    张媛媛; 胡启蒙; 王亮亮; 李新红

    2012-01-01

    Studies on protein expression, modilicalion and interaction have become important in proteomics in the post-genomics era. Phosphorylation/dephosphorylation of proteins is the main mechanism of signal transduction and regulation of enzymes in sperm cells, what' s more, it plays a key role during the sperm-egg recognition and fertilization process. Researches about function of phosphorylated proteins contribute to the understanding of the molecular mechanism of sperm capacitation, hyperactive motility and acrosome reaction process. The progress in the researches of mammalian sperm phosphoproteomics was reviewed that include the phosphoproteomics technology, phosphorylated proteins identification and quantitation, function analysis benefits in finding new important fertilization related biological markers and revealing the sperm development, reproductive potential changes and molecular mechanism of fertilization.%蛋白质的表达、修饰及相互作用的研究已成为后基因组学时代蛋白质组学中的重要内容.蛋白质磷酸化和去磷酸化作为最普遍的翻译后修饰之一,是精子细胞信号转导和酶调控、表达的主要分子机制,亦是精子、卵细胞信号识别及完成受精作用的关键环节.对精子磷酸化蛋白功能的研究有助于深入理解精子的获能、超激活运动的维持、发生顶体反应及精卵结合等受精过程的分子调控机理.对哺乳动物精子磷酸化蛋白质组学的研究进展,包括动物精子磷酸化蛋白质组学研究的技术方法、磷酸化蛋白质种类的鉴定、定量及其功能分析进行了综述,为进一步发掘与受精相关的重要生物标志物,揭示精子发育、繁殖潜能变化及受精分子机理奠定基础.

  11. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    Science.gov (United States)

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  12. SILAC-based quantitative proteomic analysis of human lung cell response to copper oxide nanoparticles.

    Directory of Open Access Journals (Sweden)

    Mariola J Edelmann

    Full Text Available Copper (II oxide (CuO nanoparticles (NP are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level.

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

    Science.gov (United States)

    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. Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO(2) Chromatography Applied to Proximal EGF Signaling

    DEFF Research Database (Denmark)

    Engholm-Keller, Kasper; Hansen, Thomas Aarup; Palmisano, Giuseppe;

    2011-01-01

    Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss and thus phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy...... IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and non-phosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4,700 unique phosphopeptides, while 636 phosphosites were changing....... Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that despite having highly accurate precursor mass determination, peptides with low fragment ion scores should...

  15. Phosphoproteomic Analysis Based on Liquid Chromatography-Tandem Mass Spectrometry%基于液相色谱-串联质谱技术的磷酸化蛋白质组学分析

    Institute of Scientific and Technical Information of China (English)

    朱秀娟; 李好雨; 郭林; 赵晓璐

    2016-01-01

    本文运用稳定同位素双甲基化标记技术、固定相金属离子螯合层析(IMAC)技术并结合液相色谱-串联质谱法研究了仙台病毒感染引起的宿主细胞磷酸化蛋白质组变化.实验发现定量的4 289个磷酸化肽段有~20%的蛋白质磷酸化位点发生了显著变化;通路富集分析表明哺乳动物雷帕毒素靶蛋白(mTOR)通路和剪接体(Spliceosome)通路可能参与宿主细胞对病毒的应答;通过对显著变化磷酸化肽段进行基序分析,发现了9个代表性的磷酸化修饰位点基序.本研究方法对于深入解析抗病毒天然免疫信号通路提供了新线索.

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

    Science.gov (United States)

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

    2015-01-01

    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.

  17. The beginnings of crop phosphoproteomics: exploring early warning systems of stress.

    Directory of Open Access Journals (Sweden)

    Christof eRampitsch

    2012-07-01

    Full Text Available This review examines why a knowledge of plant protein phosphorylation events is important in devising strategies to protect crops from both biotic and abiotic stresses, and why proteomics should be included when studying stress pathways. Most of the achievements in elucidating phospho-signalling pathways in biotic and abiotic stress are reported from model systems: while these are discussed, this review attempts mainly to focus on work done with crops, with examples of achievements reported from rice, maize, wheat, grape, Brassica, tomato and soy bean after cold acclimation, hormonal and oxidative H2O2 treatment, salt stress, mechanical wounding or pathogen challenge. The challenges that remain to transfer this information into a format that can be used to protect crops against biotic and abiotic stresses are enormous. The tremendous increase in the speed and ease of DNA sequencing is poised to reveal the whole genomes of many crop species in the near future, which will facilitate phosphoproteomics and phosphogenomics research.

  18. Multiple imputations applied to the DREAM3 phosphoproteomics challenge: a winning strategy.

    Directory of Open Access Journals (Sweden)

    Nicolas Guex

    Full Text Available DREAM is an initiative that allows researchers to assess how well their methods or approaches can describe and predict networks of interacting molecules [1]. Each year, recently acquired datasets are released to predictors ahead of publication. Researchers typically have about three months to predict the masked data or network of interactions, using any predictive method. Predictions are assessed prior to an annual conference where the best predictions are unveiled and discussed. Here we present the strategy we used to make a winning prediction for the DREAM3 phosphoproteomics challenge. We used Amelia II, a multiple imputation software method developed by Gary King, James Honaker and Matthew Blackwell[2] in the context of social sciences to predict the 476 out of 4624 measurements that had been masked for the challenge. To chose the best possible multiple imputation parameters to apply for the challenge, we evaluated how transforming the data and varying the imputation parameters affected the ability to predict additionally masked data. We discuss the accuracy of our findings and show that multiple imputations applied to this dataset is a powerful method to accurately estimate the missing data. We postulate that multiple imputations methods might become an integral part of experimental design as a mean to achieve cost savings in experimental design or to increase the quantity of samples that could be handled for a given cost.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

    Science.gov (United States)

    Chong, Weng Man; Hsu, Shih-Chin; Kao, Wei-Ting; Lo, Chieh-Wen; Lee, Kuan-Ying; Shao, Jheng-Syuan; Chen, Yi-Hung; Chang, Justin; Chen, Steve S-L; Yu, Ming-Jiun

    2016-02-19

    The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. PMID:26702051

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

    Science.gov (United States)

    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. PMID:26824392

  2. Effects of poly-ether B on proteome and phosphoproteome expression in biofouling Balanus amphitrite cyprids

    KAUST Repository

    Dash, Swagatika

    2012-04-01

    Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds. © 2012 Copyright Taylor and Francis Group, LLC.

  3. Certification-Based Process Analysis

    Science.gov (United States)

    Knight, Russell L.

    2013-01-01

    Space mission architects are often challenged with knowing which investment in technology infusion will have the highest return. Certification-based analysis (CBA) gives architects and technologists a means to communicate the risks and advantages of infusing technologies at various points in a process. Various alternatives can be compared, and requirements based on supporting streamlining or automation can be derived and levied on candidate technologies. CBA is a technique for analyzing a process and identifying potential areas of improvement. The process and analysis products are used to communicate between technologists and architects. Process means any of the standard representations of a production flow; in this case, any individual steps leading to products, which feed into other steps, until the final product is produced at the end. This sort of process is common for space mission operations, where a set of goals is reduced eventually to a fully vetted command sequence to be sent to the spacecraft. Fully vetting a product is synonymous with certification. For some types of products, this is referred to as verification and validation, and for others it is referred to as checking. Fundamentally, certification is the step in the process where one insures that a product works as intended, and contains no flaws.

  4. ANALYSIS-BASED SPARSE RECONSTRUCTION WITH SYNTHESIS-BASED SOLVERS

    OpenAIRE

    Cleju, Nicolae; Jafari, Maria,; Plumbley, Mark D.

    2012-01-01

    Analysis based reconstruction has recently been introduced as an alternative to the well-known synthesis sparsity model used in a variety of signal processing areas. In this paper we convert the analysis exact-sparse reconstruction problem to an equivalent synthesis recovery problem with a set of additional constraints. We are therefore able to use existing synthesis-based algorithms for analysis-based exact-sparse recovery. We call this the Analysis-By-Synthesis (ABS) approach. We evaluate o...

  5. 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

    NARCIS (Netherlands)

    Resjö, S.; Ali, A.; Meijer, H.J.G.; Seidl, M.F.; Snel, B.; Sandin, M.; Levander, F.; Govers, F.; Andreasson, E.

    2014-01-01

    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

  6. 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

    NARCIS (Netherlands)

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

    2014-01-01

    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

  7. Exploring signal transduction networks using mass spectrometry-based proteomics

    NARCIS (Netherlands)

    Meijer, L.A.T.

    2012-01-01

    Mass spectrometry (MS)-based proteomics can be used to answer a diversity of biological questions. In this thesis, we describe the application of several MS-based proteomics approaches to get insight into several aspects of signal transduction. In Chapter 2, quantitative global phosphoproteomics are

  8. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence.

    Science.gov (United States)

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-04-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 (K48M) ) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5-3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 (K48M) under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 (K48M) mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 (K48M) , mpk6, and PTP1 (S7AS8A) under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  9. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    Science.gov (United States)

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. PMID:27196784

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

    DEFF Research Database (Denmark)

    Kempf, Stefan; Janik, Dirk; Barjaktarovic, Zarko;

    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 cumula......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...... 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....

  11. Feature-based sentiment analysis with ontologies

    OpenAIRE

    Taner, Berk

    2011-01-01

    Sentiment analysis is a topic that many researchers work on. In recent years, new research directions under sentiment analysis appeared. Feature-based sentiment analysis is one such topic that deals not only with finding sentiment in a sentence but providing a more detailed analysis on a given domain. In the beginning researchers focused on commercial products and manually generated list of features for a product. Then they tried to generate a feature-based approach to attach sentiments to th...

  12. Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice

    Science.gov (United States)

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A.; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C.; Kulkarni, Ashok B.; González-Billault, Christian

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5−/− embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5−/− brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  13. Phosphoproteomic analysis of differential expression of AGS cellular proteins in response to Helicobanter pyiori infection%幽门螺杆菌感染后人胃腺癌上皮细胞微量磷酸化蛋白质差异分析

    Institute of Scientific and Technical Information of China (English)

    肖迪; 宋衍燕; 赵飞; 何利华; 孟凡亮; 张慧芳; 张建中

    2009-01-01

    Objective To research the differential expression of trace phosphorylated proteins in human gastric adenocarcinoma epithelial (AGS) cells infected by Helicobacter pylori. Methods H. pylori 26695 strain infected AGS cells 4 h and AGS cells was cultivated for 4 h as a comparison. The proteins of AGS and comparison AGS cells were extracted. Their phosphorylated proteins were enriched by metal ion af-finity adsorption enrichment techniques. After desalinated and purified the phosphorylated proteins samples were separated by 2-dimensional polyacrylamide gel electrophoresis (2-DE) technique. Computer assisted image analysis was used to analyze the differential proteomic expression. The significantly differentially ex-pressed proteins were unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF). Results Fifteen kinds of proteins were down-regulated, 4 kinds of new proteins were observed, 1 kind of proteins were up-regulated, 1 kind of proteins unexpression. The 21 proteins that were significantly differentially expressed , including cellular calcium ion homeostasis, transcription, interpretation, protein folding and transport, ribosomal assembly, centrosome replication, chromosome stability, cellular structure, cellular proliferation and apoptosis. Conclusion H. priori can cause a wide range change to human gastric adenocarcinoma epithelial cell protein pheshorylation. This change character has great significance to further comprehensive understanding of the pathogenesis of H. pylori.%目的 研究幽门螺杆菌(Hdicobacterpylori)作用后人胃腺癌上皮细胞(AGS)中微量磷酸化蛋白的变化情况.方法 采用金属离子亲和吸附富集技术富集幽门螺杆菌与AGS细胞相互作用以及AGS细胞的磷酸化蛋白,除盐纯化后利用二维凝胶电泳技术分离磷酸化蛋白,MALDI-TOF/TOF质谱答定确认蛋白.结果 幽门螺杆菌作用后AGS细胞有21种

  14. Unraveling the dynamics of sphingolipid metabolism by mass spectrometry based (phospho)proteomics

    NARCIS (Netherlands)

    Lebesgue, N.F.M.

    2016-01-01

    Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules with imbalances affecting cellular function and contributing to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is m

  15. Chemical phosphoproteomics and development of bisubstrate based inhibitors of protein kinase C isozymes

    NARCIS (Netherlands)

    Poot, A.J.

    2010-01-01

    Protein phosphorylation is one of the most abundant post-translational modifications. Phosphorylation is important for the function of the protein, for example it regulates enzyme activity, signal transduction and cell division. Protein phosphorylation plays a central role in virtually all crucial c

  16. Proteomic and phosphoproteomic analysis of signalling by adhesion and growth factor receptors in mammary epithelial cells

    OpenAIRE

    Paul, Nikki

    2014-01-01

    Cell adhesion and communication are essential for tissue morphogenesis and repair in healthy multicellular organisms. However, dysregulation of these processes can drive disease progression in conditions such as cancer. Selective cell adhesion to the extracellular matrix is mediated by integrins, a family of transmembrane receptors that compartmentalise signalling and organise the cytoskeleton. Adhesion receptors provide spatial cues to cells to allow them to respond to growth factor and cyto...

  17. Online automated in vivo zebrafish phosphoproteomics: from large-scale analysis down to a single embryo.

    NARCIS (Netherlands)

    Lemeer, S.; Pinkse, M.W.H.; Mohammed, S.; van Breukelen, B.; den Hertog, J.; Slijper, M.; Heck, A.

    2008-01-01

    In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modi

  18. Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms.

    Science.gov (United States)

    Gnad, Florian; Doll, Sophia; Song, Kyung; Stokes, Matthew P; Moffat, John; Liu, Bonnie; Arnott, David; Wallin, Jeffrey; Friedman, Lori S; Hatzivassiliou, Georgia; Belvin, Marcia

    2016-07-01

    The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908). PMID:27273156

  19. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene;

    2008-01-01

    Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes...

  20. Pareto analysis based on records

    CERN Document Server

    Doostparast, M

    2012-01-01

    Estimation of the parameters of an exponential distribution based on record data has been treated by Samaniego and Whitaker (1986) and Doostparast (2009). Recently, Doostparast and Balakrishnan (2011) obtained optimal confidence intervals as well as uniformly most powerful tests for one- and two-sided hypotheses concerning location and scale parameters based on record data from a two-parameter exponential model. In this paper, we derive optimal statistical procedures including point and interval estimation as well as most powerful tests based on record data from a two-parameter Pareto model. For illustrative purpose, a data set on annual wages of a sample production-line workers in a large industrial firm is analyzed using the proposed procedures.

  1. ROAn, a ROOT based Analysis Framework

    CERN Document Server

    Lauf, Thomas

    2013-01-01

    The ROOT based Offline and Online Analysis (ROAn) framework was developed to perform data analysis on data from Depleted P-channel Field Effect Transistor (DePFET) detectors, a type of active pixel sensors developed at the MPI Halbleiterlabor (HLL). ROAn is highly flexible and extensible, thanks to ROOT's features like run-time type information and reflection. ROAn provides an analysis program which allows to perform configurable step-by-step analysis on arbitrary data, an associated suite of algorithms focused on DePFET data analysis, and a viewer program for displaying and processing online or offline detector data streams. The analysis program encapsulates the applied algorithms in objects called steps which produce analysis results. The dependency between results and thus the order of calculation is resolved automatically by the program. To optimize algorithms for studying detector effects, analysis parameters are often changed. Such changes of input parameters are detected in subsequent analysis runs and...

  2. TEXTURE ANALYSIS BASED IRIS RECOGNITION

    OpenAIRE

    GÜRKAN, Güray; AKAN, Aydın

    2012-01-01

    In this paper, we present a new method for personal identification, based on iris patterns. The method composed of iris image acquisition, image preprocessing, feature extraction and finally decision stages. Normalized iris images are vertically log-sampled and filtered by circular symmetric Gabor filters. The output of filters are windowed and mean absolute deviation of pixels in the window are calculated as the feature vectors. The proposed  method has the desired properties of an iris reco...

  3. Excel-Based Business Analysis

    CERN Document Server

    Anari, Ali

    2012-01-01

    ai"The trend is your friend"is a practical principle often used by business managers, who seek to forecast future sales, expenditures, and profitability in order to make production and other operational decisions. The problem is how best to identify and discover business trends and utilize trend information for attaining objectives of firms.This book contains an Excel-based solution to this problem, applying principles of the authors' "profit system model" of the firm that enables forecasts of trends in sales, expenditures, profits and other business variables. The program,

  4. Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage

    DEFF Research Database (Denmark)

    Batth, Tanveer S; Olsen, Jesper V

    2016-01-01

    peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions...

  5. JAVA based LCD Reconstruction and Analysis Tools

    International Nuclear Information System (INIS)

    We summarize the current status and future developments of the North American Group's Java-based system for studying physics and detector design issues at a linear collider. The system is built around Java Analysis Studio (JAS) an experiment-independent Java-based utility for data analysis. Although the system is an integrated package running in JAS, many parts of it are also standalone Java utilities

  6. Java based LCD reconstruction and analysis tools

    International Nuclear Information System (INIS)

    We summarize the current status and future developments of the North American Group's Java-based system for studying physics and detector design issues at a linear collider. The system is built around Java Analysis Studio (JAS) an experiment-independent Java-based utility for data analysis. Although the system is an integrated package running in JAS, many parts of it are also standalone Java utilities

  7. Reliability analysis of software based safety functions

    International Nuclear Information System (INIS)

    The methods applicable in the reliability analysis of software based safety functions are described in the report. Although the safety functions also include other components, the main emphasis in the report is on the reliability analysis of software. The check list type qualitative reliability analysis methods, such as failure mode and effects analysis (FMEA), are described, as well as the software fault tree analysis. The safety analysis based on the Petri nets is discussed. The most essential concepts and models of quantitative software reliability analysis are described. The most common software metrics and their combined use with software reliability models are discussed. The application of software reliability models in PSA is evaluated; it is observed that the recent software reliability models do not produce the estimates needed in PSA directly. As a result from the study some recommendations and conclusions are drawn. The need of formal methods in the analysis and development of software based systems, the applicability of qualitative reliability engineering methods in connection to PSA and the need to make more precise the requirements for software based systems and their analyses in the regulatory guides should be mentioned. (orig.). (46 refs., 13 figs., 1 tab.)

  8. 小鼠肝脏磷酸化蛋白质组的二维液相色谱分离%Two dimensional liquid phase chromatographic fractionation of phosphoproteome of mouse liver

    Institute of Scientific and Technical Information of China (English)

    黎永明; 陈腾祥; 杨丽萍; 刘亚伟; 姜勇

    2005-01-01

    Objective To fractionate phosphoproteome of mouse liver by two-dimensional (2D) liquid phase chromatography fractionation. Methods Phosphoproteins were extracted from lysates of normal mice livers by phosphate metal affinity chromatography (PMAC) resin. The phosphoproteins were exchanged by start buffer and separated by chromatofocusing in the first dimension. Then the fractions between pH 8.5 and pH 4.0 were separated by non-porous silica (NPS) reverse-phase high performance liquid chromatography (RP-HPLC). Finally, the UV maps were converted into gel-like maps by ProteoVue software. Results Phosphoproteins of mouse liver were successfully extracted and fractionated by two dimensional liquid phase chromatographic fractionation after concentration and desalt. Then pI/UV map of mouse liver phosphoproteome was successfully set-up. There are 16 fractions between pH 8.5 and pH 4.0 after chromatofocusing in the first dimension and the UV maps of each fraction were converted into pI/UV gel-like maps. Conclusions Combination of technique of phosphoproteins enrichment and 2-D liquid phase chromatographic fractionation is an effective approach to research phosphoproteome and the key base for further identification and investigation of phosphoproteins.%目的利用二维液相色谱法分离小鼠肝脏磷酸化蛋白质组.方法取正常小鼠肝脏,裂解肝脏后利用磷酸盐金属亲和层析(PMAC)树脂提取磷酸化蛋白.将磷酸化蛋白用初始缓冲液置换后,进行一维色谱聚焦分离,再将一维收集的pH值在8.5至4.0之间的组分分别进行二维无孔硅胶反相高效液相色谱(RP-HPLC)分离.最后利用ProteoVue软件将二维UV图转换成胶图进行分析.结果成功提取了小鼠肝脏磷酸化蛋白,并在浓缩除盐后通过二维液相色谱分离成功建立小鼠肝脏磷酸化蛋白质组pI/UV图谱.其中,一维色谱聚焦分离pH值在8.5至4.0之间共收集16个组分,每个组分的二维UV图转换成p

  9. Desalting of phosphopeptides by tandem polypyrrole-c18 reverse phase micropipette tip (TMTip{sub PPY-C18}) based on hybrid electrostatic, {Pi}-{Pi} stacking and hydrophobic interactions for mass spectrometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Shi; Wang Xiaoli; Fu Jieying; Hu Xuejiao; Xiao Xiao; Huang Lulu; Zhou Youe [Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079 (China); Zhong Hongying, E-mail: hyzhong@mail.ccnu.edu.cn [Key Laboratory of Pesticides and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, Hubei 430079 (China)

    2012-04-29

    Highlights: Black-Right-Pointing-Pointer A new micropipette tip TMTip{sub PPY-C18} was developed for desalting of phosphopeptides. Black-Right-Pointing-Pointer TMTip{sub PPY-C18} is based on polypyrrole in tandem with C18 chromatographic material. Black-Right-Pointing-Pointer TMTip{sub PPY-C18} combines electrostatic, {Pi}-{Pi} stacking and hydrophobic interactions. Black-Right-Pointing-Pointer TMTip{sub PPY-C18} can be used in both acidic and basic experimental conditions. - Abstract: Desalting and concentration of peptides using reverse phase (RP) C18 chromatographic material based on hydrophobic interaction is a routine approach used in mass spectrometry (MS)-based proteomics. However, MS detection of small hydrophilic peptides, in particular, phosphopeptides that bear multiple negative charges, is challenging due to the insufficient binding to C18 stationary phase. We described here the development of a new desalting method that takes the unique properties of polypyrrole (PPY). The presence of positively charged nitrogen atoms under acidic conditions and polyunsaturated bonds in polypyrrole provide a prospect for enhanced adsorption of phosphopeptides or hydrophilic peptides through extra electrostatic and {Pi}-{Pi} stacking interactions in addition to hydrophobic interactions. In tandem with reversed phase C18 chromatographic material, the new type of desalting method termed as TMTip{sub PPY-C18} can significantly improve the MS detection of phosphopeptides with multiple phosphate groups and other small hydrophilic peptides. It has been applied to not only tryptic digest of model proteins but also the analysis of complex lysates of zebrafish eggs. The number of detected phosphate groups on a peptide ranged from 1 to 6. Particularly, polypyrrole based method can also be used in basic condition. Thus it provides a useful means to handle peptides that may not be detectable in acidic condition. It can be envisioned that the TMTip{sub PPY-C18} should be able to

  10. Analysis of Enhanced Associativity Based Routing Protocol

    Directory of Open Access Journals (Sweden)

    Said A. Shaar

    2006-01-01

    Full Text Available This study introduces an analysis to the performance of the Enhanced Associativity Based Routing protocol (EABR based on two factors; Operation complexity (OC and Communication Complexity (CC. OC can be defined as the number of steps required in performing a protocol operation, while CC can be defined as the number of messages exchanged in performing a protocol operation[1]. The values represent the worst-case analysis. The EABR has been analyzed based on CC and OC and the results have been compared with another routing technique called ABR. The results have shown that EABR can perform better than ABR in many circumstances during the route reconstruction.

  11. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2 and IL-15 in T-lymphocytes

    Directory of Open Access Journals (Sweden)

    Nerea Osinalde

    2015-12-01

    Full Text Available This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled “Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics” [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129.

  12. Analysis of a Chaotic Memristor Based Oscillator

    Directory of Open Access Journals (Sweden)

    F. Setoudeh

    2014-01-01

    Full Text Available A chaotic oscillator based on the memristor is analyzed from a chaos theory viewpoint. Sensitivity to initial conditions is studied by considering a nonlinear model of the system, and also a new chaos analysis methodology based on the energy distribution is presented using the Discrete Wavelet Transform (DWT. Then, using Advance Design System (ADS software, implementation of chaotic oscillator based on the memristor is considered. Simulation results are provided to show the main points of the paper.

  13. Analysis of a Chaotic Memristor Based Oscillator

    OpenAIRE

    F. Setoudeh; Khaki Sedigh, A.; Dousti, M

    2014-01-01

    A chaotic oscillator based on the memristor is analyzed from a chaos theory viewpoint. Sensitivity to initial conditions is studied by considering a nonlinear model of the system, and also a new chaos analysis methodology based on the energy distribution is presented using the Discrete Wavelet Transform (DWT). Then, using Advance Design System (ADS) software, implementation of chaotic oscillator based on the memristor is considered. Simulation results are provided to show the main points of t...

  14. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Sánchez-Quiles, Virginia; Akimov, Vyacheslav;

    2015-01-01

    This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide....... The data supplied in this article is related to the research work entitled "Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics" [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129....

  15. A Comprehensive Analysis of the Dynamic Response to Aphidicolin-Mediated Replication Stress Uncovers Targets for ATM and ATMIN

    Directory of Open Access Journals (Sweden)

    Abdelghani Mazouzi

    2016-04-01

    Full Text Available The cellular response to replication stress requires the DNA-damage-responsive kinase ATM and its cofactor ATMIN; however, the roles of this signaling pathway following replication stress are unclear. To identify the functions of ATM and ATMIN in response to replication stress, we utilized both transcriptomics and quantitative mass-spectrometry-based phosphoproteomics. We found that replication stress induced by aphidicolin triggered widespread changes in both gene expression and protein phosphorylation patterns. These changes gave rise to distinct early and late replication stress responses. Furthermore, our analysis revealed previously unknown targets of ATM and ATMIN downstream of replication stress. We demonstrate ATMIN-dependent phosphorylation of H2AX and of CRMP2, a protein previously implicated in Alzheimer’s disease but not in the DNA damage response. Overall, our dataset provides a comprehensive resource for discovering the cellular responses to replication stress and, potentially, associated pathologies.

  16. Epoch-based analysis of speech signals

    Indian Academy of Sciences (India)

    B Yegnanarayana; Suryakanth V Gangashetty

    2011-10-01

    Speech analysis is traditionally performed using short-time analysis to extract features in time and frequency domains. The window size for the analysis is fixed somewhat arbitrarily, mainly to account for the time varying vocal tract system during production. However, speech in its primary mode of excitation is produced due to impulse-like excitation in each glottal cycle. Anchoring the speech analysis around the glottal closure instants (epochs) yields significant benefits for speech analysis. Epoch-based analysis of speech helps not only to segment the speech signals based on speech production characteristics, but also helps in accurate analysis of speech. It enables extraction of important acoustic-phonetic features such as glottal vibrations, formants, instantaneous fundamental frequency, etc. Epoch sequence is useful to manipulate prosody in speech synthesis applications. Accurate estimation of epochs helps in characterizing voice quality features. Epoch extraction also helps in speech enhancement and multispeaker separation. In this tutorial article, the importance of epochs for speech analysis is discussed, and methods to extract the epoch information are reviewed. Applications of epoch extraction for some speech applications are demonstrated.

  17. Texture-based analysis of COPD

    DEFF Research Database (Denmark)

    Sørensen, Lauge Emil Borch Laurs; Nielsen, Mads; Lo, Pechin Chien Pau;

    2012-01-01

    This study presents a fully automatic, data-driven approach for texture-based quantitative analysis of chronic obstructive pulmonary disease (COPD) in pulmonary computed tomography (CT) images. The approach uses supervised learning where the class labels are, in contrast to previous work, based on...... subsequently applied to classify 200 independent images from the same screening trial. The texture-based measure was significantly better at discriminating between subjects with and without COPD than were the two most common quantitative measures of COPD in the literature, which are based on density. The...

  18. Cloud Based Development Issues: A Methodical Analysis

    Directory of Open Access Journals (Sweden)

    Sukhpal Singh

    2012-11-01

    Full Text Available Cloud based development is a challenging task for various software engineering projects, especifically for those which demand extraordinary quality, reusability and security along with general architecture. In this paper we present a report on a methodical analysis of cloud based development problems published in major computer science and software engineering journals and conferences organized by various researchers. Research papers were collected from different scholarly databases using search engines within a particular period of time. A total of 89 research papers were analyzed in this methodical study and we categorized into four classes according to the problems addressed by them. The majority of the research papers focused on quality (24 papers associated with cloud based development and 16 papers focused on analysis and design. By considering the areas focused by existing authors and their gaps, untouched areas of cloud based development can be discovered for future research works.

  19. Polyphase Order Analysis Based on Convolutional Approach

    Directory of Open Access Journals (Sweden)

    M. Drutarovsky

    1999-06-01

    Full Text Available The condition of rotating machines can be determined by measuring of periodic frequency components in the vibration signal which are directly related to the (typically changing rotational speed. Classical spectrum analysis with a constant sampling frequency is not an appropriate analysis method because of spectral smearing. Spectral analysis of vibration signal sampled synchronously with the angle of rotation, known as order analysis, suppress spectral smearing even with variable rotational speed. The paper presents optimised algorithm for polyphase order analysis based on non power of two DFT algorithm efficiently implemented by chirp FFT algorithm. Proposed algorithm decreases complexity of digital resampling algorithm, which is the most complex part of complete spectral order algorithm.

  20. Security Analysis of Discrete Logarithm Based Cryptosystems

    Institute of Scientific and Technical Information of China (English)

    WANG Yuzhu; LIAO Xiaofeng

    2006-01-01

    Discrete logarithm based cryptosystems have subtle problems that make the schemes vulnerable. This paper gives a comprehensive listing of security issues in the systems and analyzes three classes of attacks which are based on mathematical structure of the group which is used in the schemes, the disclosed information of the subgroup and implementation details respectively. The analysis will, in turn, allow us to motivate protocol design and implementation decisions.

  1. Abstraction based Analysis and Arbiter Synthesis

    DEFF Research Database (Denmark)

    Ernits, Juhan-Peep; Yi, Wang

    2004-01-01

    The work focuses on the analysis of an example of synchronous systems containing FIFO buffers, registers and memory interconnected by several private and shared busses. The example used in this work is based on a Terma radar system memory interface case study from the IST AMETIST project....

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

    Science.gov (United States)

    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. PMID:21730355

  3. Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

    KAUST Repository

    Qian, Pei Yuan

    2010-09-08

    Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid-phase IEF sample prefractionation combined with 2-DE and MALDI-TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium-binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

  4. Ontology-Based Analysis of Microarray Data.

    Science.gov (United States)

    Giuseppe, Agapito; Milano, Marianna

    2016-01-01

    The importance of semantic-based methods and algorithms for the analysis and management of biological data is growing for two main reasons. From a biological side, knowledge contained in ontologies is more and more accurate and complete, from a computational side, recent algorithms are using in a valuable way such knowledge. Here we focus on semantic-based management and analysis of protein interaction networks referring to all the approaches of analysis of protein-protein interaction data that uses knowledge encoded into biological ontologies. Semantic approaches for studying high-throughput data have been largely used in the past to mine genomic and expression data. Recently, the emergence of network approaches for investigating molecular machineries has stimulated in a parallel way the introduction of semantic-based techniques for analysis and management of network data. The application of these computational approaches to the study of microarray data can broad the application scenario of them and simultaneously can help the understanding of disease development and progress.

  5. Network-based analysis of proteomic profiles

    KAUST Repository

    Wong, Limsoon

    2016-01-26

    Mass spectrometry (MS)-based proteomics is a widely used and powerful tool for profiling systems-wide protein expression changes. It can be applied for various purposes, e.g. biomarker discovery in diseases and study of drug responses. Although RNA-based high-throughput methods have been useful in providing glimpses into the underlying molecular processes, the evidences they provide are indirect. Furthermore, RNA and corresponding protein levels have been known to have poor correlation. On the other hand, MS-based proteomics tend to have consistency issues (poor reproducibility and inter-sample agreement) and coverage issues (inability to detect the entire proteome) that need to be urgently addressed. In this talk, I will discuss how these issues can be addressed by proteomic profile analysis techniques that use biological networks (especially protein complexes) as the biological context. In particular, I will describe several techniques that we have been developing for network-based analysis of proteomics profile. And I will present evidence that these techniques are useful in identifying proteomics-profile analysis results that are more consistent, more reproducible, and more biologically coherent, and that these techniques allow expansion of the detected proteome to uncover and/or discover novel proteins.

  6. Web-based pre-Analysis Tools

    CERN Document Server

    Moskalets, Tetiana

    2014-01-01

    The project consists in the initial development of a web based and cloud computing services to allow students and researches to perform fast and very useful cut-based pre-analysis on a browser, using real data and official Monte-Carlo simulations (MC). Several tools are considered: ROOT files filter, JavaScript Multivariable Cross-Filter, JavaScript ROOT browser and JavaScript Scatter-Matrix Libraries. Preliminary but satisfactory results have been deployed online for test and future upgrades.

  7. TEST COVERAGE ANALYSIS BASED ON PROGRAM SLICING

    Institute of Scientific and Technical Information of China (English)

    Chen Zhenqiang; Xu Baowen; Guanjie

    2003-01-01

    Coverage analysis is a structural testing technique that helps to eliminate gaps in atest suite and determines when to stop testing. To compute test coverage, this letter proposes anew concept coverage about variables, based on program slicing. By adding powers accordingto their importance, the users can focus on the important variables to obtain higher test coverage.The letter presents methods to compute basic coverage based on program structure graphs. Inmost cases, the coverage obtained in the letter is bigger than that obtained by a traditionalmeasure, because the coverage about a variable takes only the related codes into account.

  8. Quantum entanglement analysis based on abstract interpretation

    OpenAIRE

    Perdrix, Simon

    2008-01-01

    Entanglement is a non local property of quantum states which has no classical counterpart and plays a decisive role in quantum information theory. Several protocols, like the teleportation, are based on quantum entangled states. Moreover, any quantum algorithm which does not create entanglement can be efficiently simulated on a classical computer. The exact role of the entanglement is nevertheless not well understood. Since an exact analysis of entanglement evolution induces an exponential sl...

  9. XML-based analysis interface for particle physics data analysis

    International Nuclear Information System (INIS)

    The letter emphasizes on an XML-based interface and its framework for particle physics data analysis. The interface uses a concise XML syntax to describe, in data analysis, the basic tasks: event-selection, kinematic fitting, particle identification, etc. and a basic processing logic: the next step goes on if and only if this step succeeds. The framework can perform an analysis without compiling by loading the XML-interface file, setting p in run-time and running dynamically. An analysis coding in XML instead of C++, easy-to-understood arid use, effectively reduces the work load, and enables users to carry out their analyses quickly. The framework has been developed on the BESⅢ offline software system (BOSS) with the object-oriented C++ programming. These functions, required by the regular tasks and the basic processing logic, are implemented with both standard modules or inherited from the modules in BOSS. The interface and its framework have been tested to perform physics analysis. (authors)

  10. Chapter 11. Community analysis-based methods

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Y.; Wu, C.H.; Andersen, G.L.; Holden, P.A.

    2010-05-01

    Microbial communities are each a composite of populations whose presence and relative abundance in water or other environmental samples are a direct manifestation of environmental conditions, including the introduction of microbe-rich fecal material and factors promoting persistence of the microbes therein. As shown by culture-independent methods, different animal-host fecal microbial communities appear distinctive, suggesting that their community profiles can be used to differentiate fecal samples and to potentially reveal the presence of host fecal material in environmental waters. Cross-comparisons of microbial communities from different hosts also reveal relative abundances of genetic groups that can be used to distinguish sources. In increasing order of their information richness, several community analysis methods hold promise for MST applications: phospholipid fatty acid (PLFA) analysis, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP), cloning/sequencing, and PhyloChip. Specific case studies involving TRFLP and PhyloChip approaches demonstrate the ability of community-based analyses of contaminated waters to confirm a diagnosis of water quality based on host-specific marker(s). The success of community-based MST for comprehensively confirming fecal sources relies extensively upon using appropriate multivariate statistical approaches. While community-based MST is still under evaluation and development as a primary diagnostic tool, results presented herein demonstrate its promise. Coupled with its inherently comprehensive ability to capture an unprecedented amount of microbiological data that is relevant to water quality, the tools for microbial community analysis are increasingly accessible, and community-based approaches have unparalleled potential for translation into rapid, perhaps real-time, monitoring platforms.

  11. Ovarian Cancer Proteomic, Phosphoproteomic, and Glycoproteomic Data Released - Office of Cancer Clinical Proteomics Research

    Science.gov (United States)

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have just released a comprehensive dataset of the proteomic analysis of high grade serous ovarian tumor samples,

  12. Optimal depth-based regional frequency analysis

    Directory of Open Access Journals (Sweden)

    H. Wazneh

    2013-06-01

    Full Text Available Classical methods of regional frequency analysis (RFA of hydrological variables face two drawbacks: (1 the restriction to a particular region which can lead to a loss of some information and (2 the definition of a region that generates a border effect. To reduce the impact of these drawbacks on regional modeling performance, an iterative method was proposed recently, based on the statistical notion of the depth function and a weight function φ. This depth-based RFA (DBRFA approach was shown to be superior to traditional approaches in terms of flexibility, generality and performance. The main difficulty of the DBRFA approach is the optimal choice of the weight function ϕ (e.g., φ minimizing estimation errors. In order to avoid a subjective choice and naïve selection procedures of φ, the aim of the present paper is to propose an algorithm-based procedure to optimize the DBRFA and automate the choice of ϕ according to objective performance criteria. This procedure is applied to estimate flood quantiles in three different regions in North America. One of the findings from the application is that the optimal weight function depends on the considered region and can also quantify the region's homogeneity. By comparing the DBRFA to the canonical correlation analysis (CCA method, results show that the DBRFA approach leads to better performances both in terms of relative bias and mean square error.

  13. Optimal depth-based regional frequency analysis

    Directory of Open Access Journals (Sweden)

    H. Wazneh

    2013-01-01

    Full Text Available Classical methods of regional frequency analysis (RFA of hydrological variables face two drawbacks: (1 the restriction to a particular region which can correspond to a loss of some information and (2 the definition of a region that generates a border effect. To reduce the impact of these drawbacks on regional modeling performance, an iterative method was proposed recently. The proposed method is based on the statistical notion of the depth function and a weight function φ. This depth-based RFA (DBRFA approach was shown to be superior to traditional approaches in terms of flexibility, generality and performance. The main difficulty of the DBRFA approach is the optimal choice of the weight function φ (e.g. φ minimizing estimation errors. In order to avoid subjective choice and naïve selection procedures of φ, the aim of the present paper is to propose an algorithm-based procedure to optimize the DBRFA and automate the choice of φ according to objective performance criteria. This procedure is applied to estimate flood quantiles in three different regions in North America. One of the findings from the application is that the optimal weight function depends on the considered region and can also quantify the region homogeneity. By comparing the DBRFA to the canonical correlation analysis (CCA method, results show that the DBRFA approach leads to better performances both in terms of relative bias and mean square error.

  14. Gait correlation analysis based human identification.

    Science.gov (United States)

    Chen, Jinyan

    2014-01-01

    Human gait identification aims to identify people by a sequence of walking images. Comparing with fingerprint or iris based identification, the most important advantage of gait identification is that it can be done at a distance. In this paper, silhouette correlation analysis based human identification approach is proposed. By background subtracting algorithm, the moving silhouette figure can be extracted from the walking images sequence. Every pixel in the silhouette has three dimensions: horizontal axis (x), vertical axis (y), and temporal axis (t). By moving every pixel in the silhouette image along these three dimensions, we can get a new silhouette. The correlation result between the original silhouette and the new one can be used as the raw feature of human gait. Discrete Fourier transform is used to extract features from this correlation result. Then, these features are normalized to minimize the affection of noise. Primary component analysis method is used to reduce the features' dimensions. Experiment based on CASIA database shows that this method has an encouraging recognition performance. PMID:24592144

  15. Gait Correlation Analysis Based Human Identification

    Directory of Open Access Journals (Sweden)

    Jinyan Chen

    2014-01-01

    Full Text Available Human gait identification aims to identify people by a sequence of walking images. Comparing with fingerprint or iris based identification, the most important advantage of gait identification is that it can be done at a distance. In this paper, silhouette correlation analysis based human identification approach is proposed. By background subtracting algorithm, the moving silhouette figure can be extracted from the walking images sequence. Every pixel in the silhouette has three dimensions: horizontal axis (x, vertical axis (y, and temporal axis (t. By moving every pixel in the silhouette image along these three dimensions, we can get a new silhouette. The correlation result between the original silhouette and the new one can be used as the raw feature of human gait. Discrete Fourier transform is used to extract features from this correlation result. Then, these features are normalized to minimize the affection of noise. Primary component analysis method is used to reduce the features’ dimensions. Experiment based on CASIA database shows that this method has an encouraging recognition performance.

  16. Multifractal Time Series Analysis Based on Detrended Fluctuation Analysis

    Science.gov (United States)

    Kantelhardt, Jan; Stanley, H. Eugene; Zschiegner, Stephan; Bunde, Armin; Koscielny-Bunde, Eva; Havlin, Shlomo

    2002-03-01

    In order to develop an easily applicable method for the multifractal characterization of non-stationary time series, we generalize the detrended fluctuation analysis (DFA), which is a well-established method for the determination of the monofractal scaling properties and the detection of long-range correlations. We relate the new multifractal DFA method to the standard partition function-based multifractal formalism, and compare it to the wavelet transform modulus maxima (WTMM) method which is a well-established, but more difficult procedure for this purpose. We employ the multifractal DFA method to determine if the heartrhythm during different sleep stages is characterized by different multifractal properties.

  17. Rweb:Web-based Statistical Analysis

    Directory of Open Access Journals (Sweden)

    Jeff Banfield

    1999-03-01

    Full Text Available Rweb is a freely accessible statistical analysis environment that is delivered through the World Wide Web (WWW. It is based on R, a well known statistical analysis package. The only requirement to run the basic Rweb interface is a WWW browser that supports forms. If you want graphical output you must, of course, have a browser that supports graphics. The interface provides access to WWW accessible data sets, so you may run Rweb on your own data. Rweb can provide a four window statistical computing environment (code input, text output, graphical output, and error information through browsers that support Javascript. There is also a set of point and click modules under development for use in introductory statistics courses.

  18. Electric Equipment Diagnosis based on Wavelet Analysis

    Directory of Open Access Journals (Sweden)

    Stavitsky Sergey A.

    2016-01-01

    Full Text Available Due to electric equipment development and complication it is necessary to have a precise and intense diagnosis. Nowadays there are two basic ways of diagnosis: analog signal processing and digital signal processing. The latter is more preferable. The basic ways of digital signal processing (Fourier transform and Fast Fourier transform include one of the modern methods based on wavelet transform. This research is dedicated to analyzing characteristic features and advantages of wavelet transform. This article shows the ways of using wavelet analysis and the process of test signal converting. In order to carry out this analysis, computer software Mathcad was used and 2D wavelet spectrum for a complex function was created.

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

    Science.gov (United States)

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

    2016-02-01

    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. PMID:26689635

  20. Combined Phosphoproteomics and Bioinformatics Strategy in Deciphering Drug Resistant Related Pathways in Triple Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    Xinyu Deng

    2014-01-01

    Full Text Available Because of the absence of a clear therapeutic target for triple negative breast cancer (TNBC, conventional chemotherapy is the only available systemic treatment option for these patients. Despite chemotherapy treatment, TNBC patients still have worse prognosis when compared with other breast cancer patients. The study is to investigate unique phosphorylated proteins expressed in chemoresistant TNBC cell lines. In the current study, twelve TNBC cell lines were subjected to drug sensitivity assays against chemotherapy drugs docetaxel, doxorubicin, gemcitabine, and cisplatin. Based on their half maximal inhibitory concentrations, four resistant and two sensitive cell lines were selected for further analysis. The phosphopeptides from these cells were enriched with TiO2 beads and fractionated using strong cation exchange. 1,645 phosphoprotein groups and 9,585 unique phosphopeptides were identified by a high throughput LC-MS/MS system LTQ-Orbitrap. The phosphopeptides were further filtered with Ascore system and 1,340 phosphoprotein groups, 2,760 unique phosphopeptides, and 4,549 unique phosphosites were identified. Our study suggested that differentially phosphorylated Cdk5, PML, AP-1, and HSF-1 might work together to promote vimentin induced epithelial to mesenchymal transition (EMT in the drug resistant cells. EGFR and HGF were also shown to be involved in this process.

  1. Combined phosphoproteomics and bioinformatics strategy in deciphering drug resistant related pathways in triple negative breast cancer.

    Science.gov (United States)

    Deng, Xinyu; Kohanfars, Morris; Hsu, Huan Ming; Souda, Puneet; Capri, Joe; Whitelegge, Julian P; Chang, Helena R

    2014-01-01

    Because of the absence of a clear therapeutic target for triple negative breast cancer (TNBC), conventional chemotherapy is the only available systemic treatment option for these patients. Despite chemotherapy treatment, TNBC patients still have worse prognosis when compared with other breast cancer patients. The study is to investigate unique phosphorylated proteins expressed in chemoresistant TNBC cell lines. In the current study, twelve TNBC cell lines were subjected to drug sensitivity assays against chemotherapy drugs docetaxel, doxorubicin, gemcitabine, and cisplatin. Based on their half maximal inhibitory concentrations, four resistant and two sensitive cell lines were selected for further analysis. The phosphopeptides from these cells were enriched with TiO2 beads and fractionated using strong cation exchange. 1,645 phosphoprotein groups and 9,585 unique phosphopeptides were identified by a high throughput LC-MS/MS system LTQ-Orbitrap. The phosphopeptides were further filtered with Ascore system and 1,340 phosphoprotein groups, 2,760 unique phosphopeptides, and 4,549 unique phosphosites were identified. Our study suggested that differentially phosphorylated Cdk5, PML, AP-1, and HSF-1 might work together to promote vimentin induced epithelial to mesenchymal transition (EMT) in the drug resistant cells. EGFR and HGF were also shown to be involved in this process. PMID:25478227

  2. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells.

    Science.gov (United States)

    van den Biggelaar, Maartje; Hernández-Fernaud, Juan Ramon; van den Eshof, Bart L; Neilson, Lisa J; Meijer, Alexander B; Mertens, Koen; Zanivan, Sara

    2014-03-20

    Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function. PMID:24501219

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

    Science.gov (United States)

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

    2016-02-01

    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.

  4. Arabic Interface Analysis Based on Cultural Markers

    Directory of Open Access Journals (Sweden)

    Mohammadi Akheela Khanum

    2012-01-01

    Full Text Available This study examines the Arabic interface design elements that are largely influenced by the cultural values. Cultural markers are examined in websites from educational, business, and media. Cultural values analysis is based on Geert Hofstedes cultural dimensions. The findings show that there are cultural markers which are largely influenced by the culture and that the Hofstedes score for Arab countries is partially supported by the website design components examined in this study. Moderate support was also found for the long term orientation, for which Hoftsede has no score.

  5. Similarity-based pattern analysis and recognition

    CERN Document Server

    Pelillo, Marcello

    2013-01-01

    This accessible text/reference presents a coherent overview of the emerging field of non-Euclidean similarity learning. The book presents a broad range of perspectives on similarity-based pattern analysis and recognition methods, from purely theoretical challenges to practical, real-world applications. The coverage includes both supervised and unsupervised learning paradigms, as well as generative and discriminative models. Topics and features: explores the origination and causes of non-Euclidean (dis)similarity measures, and how they influence the performance of traditional classification alg

  6. Arabic Interface Analysis Based on Cultural Markers

    CERN Document Server

    Khanum, Mohammadi Akheela; Chaurasia, Mousmi A

    2012-01-01

    This study examines the Arabic interface design elements that are largely influenced by the cultural values. Cultural markers are examined in websites from educational, business, and media. Cultural values analysis is based on Geert Hofstede's cultural dimensions. The findings show that there are cultural markers which are largely influenced by the culture and that the Hofstede's score for Arab countries is partially supported by the website design components examined in this study. Moderate support was also found for the long term orientation, for which Hoftsede has no score.

  7. Constructing storyboards based on hierarchical clustering analysis

    Science.gov (United States)

    Hasebe, Satoshi; Sami, Mustafa M.; Muramatsu, Shogo; Kikuchi, Hisakazu

    2005-07-01

    There are growing needs for quick preview of video contents for the purpose of improving accessibility of video archives as well as reducing network traffics. In this paper, a storyboard that contains a user-specified number of keyframes is produced from a given video sequence. It is based on hierarchical cluster analysis of feature vectors that are derived from wavelet coefficients of video frames. Consistent use of extracted feature vectors is the key to avoid a repetition of computationally-intensive parsing of the same video sequence. Experimental results suggest that a significant reduction in computational time is gained by this strategy.

  8. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    Science.gov (United States)

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  9. Motion Analysis Based on Invertible Rapid Transform

    Directory of Open Access Journals (Sweden)

    J. Turan

    1999-06-01

    Full Text Available This paper presents the results of a study on the use of invertible rapid transform (IRT for the motion estimation in a sequence of images. Motion estimation algorithms based on the analysis of the matrix of states (produced in the IRT calculation are described. The new method was used experimentally to estimate crowd and traffic motion from the image data sequences captured at railway stations and at high ways in large cities. The motion vectors may be used to devise a polar plot (showing velocity magnitude and direction for moving objects where the dominant motion tendency can be seen. The experimental results of comparison of the new motion estimation methods with other well known block matching methods (full search, 2D-log, method based on conventional (cross correlation (CC function or phase correlation (PC function for application of crowd motion estimation are also presented.

  10. Visibility Graph Based Time Series Analysis.

    Directory of Open Access Journals (Sweden)

    Mutua Stephen

    Full Text Available Network based time series analysis has made considerable achievements in the recent years. By mapping mono/multivariate time series into networks, one can investigate both it's microscopic and macroscopic behaviors. However, most proposed approaches lead to the construction of static networks consequently providing limited information on evolutionary behaviors. In the present paper we propose a method called visibility graph based time series analysis, in which series segments are mapped to visibility graphs as being descriptions of the corresponding states and the successively occurring states are linked. This procedure converts a time series to a temporal network and at the same time a network of networks. Findings from empirical records for stock markets in USA (S&P500 and Nasdaq and artificial series generated by means of fractional Gaussian motions show that the method can provide us rich information benefiting short-term and long-term predictions. Theoretically, we propose a method to investigate time series from the viewpoint of network of networks.

  11. Curvelet based offline analysis of SEM images.

    Directory of Open Access Journals (Sweden)

    Syed Hamad Shirazi

    Full Text Available Manual offline analysis, of a scanning electron microscopy (SEM image, is a time consuming process and requires continuous human intervention and efforts. This paper presents an image processing based method for automated offline analyses of SEM images. To this end, our strategy relies on a two-stage process, viz. texture analysis and quantification. The method involves a preprocessing step, aimed at the noise removal, in order to avoid false edges. For texture analysis, the proposed method employs a state of the art Curvelet transform followed by segmentation through a combination of entropy filtering, thresholding and mathematical morphology (MM. The quantification is carried out by the application of a box-counting algorithm, for fractal dimension (FD calculations, with the ultimate goal of measuring the parameters, like surface area and perimeter. The perimeter is estimated indirectly by counting the boundary boxes of the filled shapes. The proposed method, when applied to a representative set of SEM images, not only showed better results in image segmentation but also exhibited a good accuracy in the calculation of surface area and perimeter. The proposed method outperforms the well-known Watershed segmentation algorithm.

  12. Glycocapture-based proteomics for secretome analysis.

    Science.gov (United States)

    Lai, Zon W; Nice, Edouard C; Schilling, Oliver

    2013-02-01

    Protein glycosylation represents the most abundant extracellular posttranslational modification in multicellular organisms. These glycoproteins unequivocally comprise the major biomolecules involved in extracellular processes, such as growth factors, signaling proteins for cellular communication, enzymes, and proteases for on- and off-site processing. It is now known that altered protein glycosylation is a hallmark event in many different pathologies. Glycoproteins are found mostly in the so-called secretome, which comprises classically and nonclassically secreted proteins and protein fragments that are released from the cell surface through ectodomain shedding. Due to biological complexity and technical difficulty, comparably few studies have taken an in-depth investigation of cellular secretomes using system-wide approaches. The cellular secretomes are considered to be a valuable source of therapeutic targets and novel biomarkers. It is not surprising that many existing biomarkers, including biomarkers for breast, ovarian, prostate, and colorectal cancers are glycoproteins. Focused analysis of secreted glycoproteins could thus provide valuable information for early disease diagnosis, and surveillance. Furthermore, since most secreted proteins are glycosylated and glycosylation predominantly targets secreted proteins, the glycan/sugar moiety itself can be used as a chemical "handle" for the targeted analysis of cellular secretomes, thereby reducing sample complexity and allowing detection of low abundance proteins in proteomic workflows. This review will focus on various glycoprotein enrichment strategies that facilitate proteomics-based technologies for the quantitative analysis of cell secretomes and cell surface proteomes.

  13. Watermark Resistance Analysis Based On Linear Transformation

    Directory of Open Access Journals (Sweden)

    N.Karthika Devi

    2012-06-01

    Full Text Available Generally, digital watermark can be embedded in any copyright image whose size is not larger than it. The watermarking schemes can be classified into two categories: spatial domain approach or transform domain approach. Previous works have shown that the transform domain scheme is typically more robust to noise, common image processing, and compression when compared with the spatial transform scheme. Improvements in performance of watermarking schemes can be obtained by exploiting the characteristics of the human visual system (HVS in the watermarking process. We propose a linear transformation based watermarking algorithm. The watermarking bits are embedded into cover image to produce watermarked image. The efficiency of watermark is checked using pre-defined attacks. Attack resistance analysis is done using BER (Bit Error Rate calculation. Finally, the Quality of the watermarked image can be obtained.

  14. Visual Similarity Based Document Layout Analysis

    Institute of Scientific and Technical Information of China (English)

    Di Wen; Xiao-Qing Ding

    2006-01-01

    In this paper, a visual similarity based document layout analysis (DLA) scheme is proposed, which by using clustering strategy can adaptively deal with documents in different languages, with different layout structures and skew angles. Aiming at a robust and adaptive DLA approach, the authors first manage to find a set of representative filters and statistics to characterize typical texture patterns in document images, which is through a visual similarity testing process.Texture features are then extracted from these filters and passed into a dynamic clustering procedure, which is called visual similarity clustering. Finally, text contents are located from the clustered results. Benefit from this scheme, the algorithm demonstrates strong robustness and adaptability in a wide variety of documents, which previous traditional DLA approaches do not possess.

  15. Voxel-Based LIDAR Analysis and Applications

    Science.gov (United States)

    Hagstrom, Shea T.

    One of the greatest recent changes in the field of remote sensing is the addition of high-quality Light Detection and Ranging (LIDAR) instruments. In particular, the past few decades have been greatly beneficial to these systems because of increases in data collection speed and accuracy, as well as a reduction in the costs of components. These improvements allow modern airborne instruments to resolve sub-meter details, making them ideal for a wide variety of applications. Because LIDAR uses active illumination to capture 3D information, its output is fundamentally different from other modalities. Despite this difference, LIDAR datasets are often processed using methods appropriate for 2D images and that do not take advantage of its primary virtue of 3-dimensional data. It is this problem we explore by using volumetric voxel modeling. Voxel-based analysis has been used in many applications, especially medical imaging, but rarely in traditional remote sensing. In part this is because the memory requirements are substantial when handling large areas, but with modern computing and storage this is no longer a significant impediment. Our reason for using voxels to model scenes from LIDAR data is that there are several advantages over standard triangle-based models, including better handling of overlapping surfaces and complex shapes. We show how incorporating system position information from early in the LIDAR point cloud generation process allows radiometrically-correct transmission and other novel voxel properties to be recovered. This voxelization technique is validated on simulated data using the Digital Imaging and Remote Sensing Image Generation (DIRSIG) software, a first-principles based ray-tracer developed at the Rochester Institute of Technology. Voxel-based modeling of LIDAR can be useful on its own, but we believe its primary advantage is when applied to problems where simpler surface-based 3D models conflict with the requirement of realistic geometry. To

  16. Interactive analysis of geodata based intelligence

    Science.gov (United States)

    Wagner, Boris; Eck, Ralf; Unmüessig, Gabriel; Peinsipp-Byma, Elisabeth

    2016-05-01

    When a spatiotemporal events happens, multi-source intelligence data is gathered to understand the problem, and strategies for solving the problem are investigated. The difficulties arising from handling spatial and temporal intelligence data represent the main problem. The map might be the bridge to visualize the data and to get the most understand model for all stakeholders. For the analysis of geodata based intelligence data, a software was developed as a working environment that combines geodata with optimized ergonomics. The interaction with the common operational picture (COP) is so essentially facilitated. The composition of the COP is based on geodata services, which are normalized by international standards of the Open Geospatial Consortium (OGC). The basic geodata are combined with intelligence data from images (IMINT) and humans (HUMINT), stored in a NATO Coalition Shared Data Server (CSD). These intelligence data can be combined with further information sources, i.e., live sensors. As a result a COP is generated and an interaction suitable for the specific workspace is added. This allows the users to work interactively with the COP, i.e., searching with an on board CSD client for suitable intelligence data and integrate them into the COP. Furthermore, users can enrich the scenario with findings out of the data of interactive live sensors and add data from other sources. This allows intelligence services to contribute effectively to the process by what military and disaster management are organized.

  17. Operating cost analysis of anaesthesia: Activity based costing (ABC analysis

    Directory of Open Access Journals (Sweden)

    Majstorović Branislava M.

    2011-01-01

    Full Text Available Introduction. Cost of anaesthesiology represent defined measures to determine a precise profile of expenditure estimation of surgical treatment, which is important regarding planning of healthcare activities, prices and budget. Objective. In order to determine the actual value of anaestesiological services, we started with the analysis of activity based costing (ABC analysis. Methods. Retrospectively, in 2005 and 2006, we estimated the direct costs of anestesiological services (salaries, drugs, supplying materials and other: analyses and equipment. of the Institute of Anaesthesia and Resuscitation of the Clinical Centre of Serbia. The group included all anesthetized patients of both sexes and all ages. We compared direct costs with direct expenditure, “each cost object (service or unit” of the Republican Health-care Insurance. The Summary data of the Departments of Anaesthesia documented in the database of the Clinical Centre of Serbia. Numerical data were utilized and the numerical data were estimated and analyzed by computer programs Microsoft Office Excel 2003 and SPSS for Windows. We compared using the linear model of direct costs and unit costs of anaesthesiological services from the Costs List of the Republican Health-care Insurance. Results. Direct costs showed 40% of costs were spent on salaries, (32% on drugs and supplies, and 28% on other costs, such as analyses and equipment. The correlation of the direct costs of anaestesiological services showed a linear correlation with the unit costs of the Republican Healthcare Insurance. Conclusion. During surgery, costs of anaesthesia would increase by 10% the surgical treatment cost of patients. Regarding the actual costs of drugs and supplies, we do not see any possibility of costs reduction. Fixed elements of direct costs provide the possibility of rationalization of resources in anaesthesia.

  18. Temporal expression-based analysis of metabolism.

    Directory of Open Access Journals (Sweden)

    Sara B Collins

    Full Text Available Metabolic flux is frequently rerouted through cellular metabolism in response to dynamic changes in the intra- and extra-cellular environment. Capturing the mechanisms underlying these metabolic transitions in quantitative and predictive models is a prominent challenge in systems biology. Progress in this regard has been made by integrating high-throughput gene expression data into genome-scale stoichiometric models of metabolism. Here, we extend previous approaches to perform a Temporal Expression-based Analysis of Metabolism (TEAM. We apply TEAM to understanding the complex metabolic dynamics of the respiratorily versatile bacterium Shewanella oneidensis grown under aerobic, lactate-limited conditions. TEAM predicts temporal metabolic flux distributions using time-series gene expression data. Increased predictive power is achieved by supplementing these data with a large reference compendium of gene expression, which allows us to take into account the unique character of the distribution of expression of each individual gene. We further propose a straightforward method for studying the sensitivity of TEAM to changes in its fundamental free threshold parameter θ, and reveal that discrete zones of distinct metabolic behavior arise as this parameter is changed. By comparing the qualitative characteristics of these zones to additional experimental data, we are able to constrain the range of θ to a small, well-defined interval. In parallel, the sensitivity analysis reveals the inherently difficult nature of dynamic metabolic flux modeling: small errors early in the simulation propagate to relatively large changes later in the simulation. We expect that handling such "history-dependent" sensitivities will be a major challenge in the future development of dynamic metabolic-modeling techniques.

  19. Temporal expression-based analysis of metabolism.

    Science.gov (United States)

    Collins, Sara B; Reznik, Ed; Segrè, Daniel

    2012-01-01

    Metabolic flux is frequently rerouted through cellular metabolism in response to dynamic changes in the intra- and extra-cellular environment. Capturing the mechanisms underlying these metabolic transitions in quantitative and predictive models is a prominent challenge in systems biology. Progress in this regard has been made by integrating high-throughput gene expression data into genome-scale stoichiometric models of metabolism. Here, we extend previous approaches to perform a Temporal Expression-based Analysis of Metabolism (TEAM). We apply TEAM to understanding the complex metabolic dynamics of the respiratorily versatile bacterium Shewanella oneidensis grown under aerobic, lactate-limited conditions. TEAM predicts temporal metabolic flux distributions using time-series gene expression data. Increased predictive power is achieved by supplementing these data with a large reference compendium of gene expression, which allows us to take into account the unique character of the distribution of expression of each individual gene. We further propose a straightforward method for studying the sensitivity of TEAM to changes in its fundamental free threshold parameter θ, and reveal that discrete zones of distinct metabolic behavior arise as this parameter is changed. By comparing the qualitative characteristics of these zones to additional experimental data, we are able to constrain the range of θ to a small, well-defined interval. In parallel, the sensitivity analysis reveals the inherently difficult nature of dynamic metabolic flux modeling: small errors early in the simulation propagate to relatively large changes later in the simulation. We expect that handling such "history-dependent" sensitivities will be a major challenge in the future development of dynamic metabolic-modeling techniques. PMID:23209390

  20. Automatic malware analysis an emulator based approach

    CERN Document Server

    Yin, Heng

    2012-01-01

    Malicious software (i.e., malware) has become a severe threat to interconnected computer systems for decades and has caused billions of dollars damages each year. A large volume of new malware samples are discovered daily. Even worse, malware is rapidly evolving becoming more sophisticated and evasive to strike against current malware analysis and defense systems. Automatic Malware Analysis presents a virtualized malware analysis framework that addresses common challenges in malware analysis. In regards to this new analysis framework, a series of analysis techniques for automatic malware analy

  1. Proteomic and phosphoproteomic comparison of human ES and iPS cells.

    Science.gov (United States)

    Phanstiel, Douglas H; Brumbaugh, Justin; Wenger, Craig D; Tian, Shulan; Probasco, Mitchell D; Bailey, Derek J; Swaney, Danielle L; Tervo, Mark A; Bolin, Jennifer M; Ruotti, Victor; Stewart, Ron; Thomson, James A; Coon, Joshua J

    2011-01-01

    Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

  2. Node-based analysis of species distributions

    DEFF Research Database (Denmark)

    Borregaard, Michael Krabbe; Rahbek, Carsten; Fjeldså, Jon;

    2014-01-01

    The integration of species distributions and evolutionary relationships is one of the most rapidly moving research fields today and has led to considerable advances in our understanding of the processes underlying biogeographical patterns. Here, we develop a set of metrics, the specific overrepre......The integration of species distributions and evolutionary relationships is one of the most rapidly moving research fields today and has led to considerable advances in our understanding of the processes underlying biogeographical patterns. Here, we develop a set of metrics, the specific...... with case studies on two groups with well-described biogeographical histories: a local-scale community data set of hummingbirds in the North Andes, and a large-scale data set of the distribution of all species of New World flycatchers. The node-based analysis of these two groups generates a set...... of intuitively interpretable patterns that are consistent with current biogeographical knowledge.Importantly, the results are statistically tractable, opening many possibilities for their use in analyses of evolutionary, historical and spatial patterns of species diversity. The method is implemented...

  3. Fast and Efficient IMAC Protocol for Phosphopeptide enrichment for phosphoproteomic Studies via LC-MS/MS

    OpenAIRE

    McKennan, C.; Spruce, L.; Seeholzer, S; Ding, H.

    2014-01-01

    Recent developments in first dimension High-Performance Liquid Chromatography (HPLC) separation of complex peptide mixtures, followed by a subsequent immobilized metal ion affinity chromatography (IMAC) for phosphopeptide enrichment have shown great promise in both selectivity and quantification of phosphopeptides via LC-MS/MS analysis. The first dimension HPLC, such as hydrophilic interaction chromatography (HILIC) or high pH Reverse Phase chromatography, was employed for its being orthogona...

  4. A Translation Case Analysis Based on Skopos Theory

    Institute of Scientific and Technical Information of China (English)

    盖孟姣

    2015-01-01

    This paper is a translation case analysis based on Skopos Theory.This paper choose President Xi’s New Year congratulations of 2015 as analysis text and gives the case analysis.This paper focuses on translating the text based on Skopos Theory.

  5. Polyphase Order Analysis Based on Convolutional Approach

    OpenAIRE

    M. Drutarovsky

    1999-01-01

    The condition of rotating machines can be determined by measuring of periodic frequency components in the vibration signal which are directly related to the (typically changing) rotational speed. Classical spectrum analysis with a constant sampling frequency is not an appropriate analysis method because of spectral smearing. Spectral analysis of vibration signal sampled synchronously with the angle of rotation, known as order analysis, suppress spectral smearing even with variable rotational ...

  6. Battle through signaling between wheat and the fungal pathogen Septoria tritici revealed by proteomics and phosphoproteomics

    DEFF Research Database (Denmark)

    Yang, Fen; Braga, Marcella Nunes de Melo; Larsen, Martin Røssel;

    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......-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...

  7. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina;

    2010-01-01

    and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R......-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha...

  8. ANALYSIS OF CIRCUIT TOLERANCE BASED ON RANDOM SET THEORY

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Monte Carlo Analysis has been an accepted method for circuit tolerance analysis,but the heavy computational complexity has always prevented its applications.Based on random set theory,this paper presents a simple and flexible tolerance analysis method to estimate circuit yield.It is the alternative to Monte Carlo analysis,but reduces the number of calculations dramatically.

  9. A Requirements Analysis Model Based on QFD

    Institute of Scientific and Technical Information of China (English)

    TANG Zhi-wei; Nelson K.H.Tang

    2004-01-01

    The enterprise resource planning (ERP) system has emerged to offer an integrated IT solution and more and more enterprises are increasing by adopting this system and regarding it as an important innovation. However, there is already evidence of high failure risks in ERP project implementation, one major reason is poor analysis of the requirements for system implementation. In this paper, the importance of requirements analysis for ERP project implementation is highlighted, and a requirements analysis model by applying quality function deployment (QFD) is presented, which will support to conduct requirements analysis for ERP project.

  10. Scope-Based Method Cache Analysis

    DEFF Research Database (Denmark)

    Huber, Benedikt; Hepp, Stefan; Schoeberl, Martin

    2014-01-01

    , as it requests memory transfers at well-defined instructions only. In this article, we present a new cache analysis framework that generalizes and improves work on cache persistence analysis. The analysis demonstrates that a global view on the cache behavior permits the precise analyses of caches which are hard......The quest for time-predictable systems has led to the exploration of new hardware architectures that simplify analysis and reasoning in the temporal domain, while still providing competitive performance. For the instruction memory, the method cache is a conceptually attractive solution...

  11. Informatics tools for the analysis and assignment of phosphorylation status in proteomics

    OpenAIRE

    Lee, Dave

    2015-01-01

    Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subseq...

  12. Implementation of Ultraviolet Photodissociation on a Benchtop Q Exactive Mass Spectrometer and Its Application to Phosphoproteomics.

    Science.gov (United States)

    Fort, Kyle L; Dyachenko, Andrey; Potel, Clement M; Corradini, Eleonora; Marino, Fabio; Barendregt, Arjan; Makarov, Alexander A; Scheltema, Richard A; Heck, Albert J R

    2016-02-16

    Proteomics applications performed on the popular benchtop Q Exactive Orbitrap mass spectrometer have so far relied exclusively on higher collision-energy dissociation (HCD) fragmentation for peptide sequencing. While this fragmentation technique is applicable to a wide range of biological questions, it also has limitations, and all questions cannot be addressed equally well. Here, we demonstrate that the fragmentation capabilities of the Q Exactive mass spectrometer can be extended with ultraviolet photodissociation (UVPD) fragmentation, complete with synchronization triggering to make it compatible with liquid chromatography (LC)/tandem mass spectrometry (MS/MS) workflows. We show that UVPD not only is directly compatible with LC/MS workflows but also, when combined with these workflows, can result in higher database scores and increased identification rates for complex samples as compared to HCD methods. UVPD as a fragmentation technique offers prompt, high-energy fragmentation, which can potentially lead to improved analyses of labile post-translational modifications. Techniques like HCD result in substantial amounts of modification losses, competing with fragmentation pathways that provide information-rich ion fragments. We investigate here the utility of UVPD for identification of phosphorylated peptides and find that UVPD fragmentation reduces the extent of labile modification loss by up to ∼60%. Collectively, when integrated into a complete workflow on the Q Exactive Orbitrap, UVPD provides distinct advantages to the analysis of post-translational modifications and is a powerful and complementary addition to the proteomic toolbox. PMID:26760441

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

    Science.gov (United States)

    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

  14. Web Based Distributed Coastal Image Analysis System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This project develops Web based distributed image analysis system processing the Moderate Resolution Imaging Spectroradiometer (MODIS) data to provide decision...

  15. Phosphoproteomics study based on in vivo inhibition reveals sites of calmodulin-dependent protein kinase II regulation in the heart

    NARCIS (Netherlands)

    Scholten, A.; Preisinger, C.; Corradini, E.; Bourgonje, V.J.A.; Hennrich, M.L.; van Veen, T.A.B.; Swaminathan, P.D.; Joiner, M.L.; Vos, M.A.; Anderson, M.E.; Heck, A.J.R.

    2013-01-01

    Background The multifunctional Ca2+‐ and calmodulin‐dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful

  16. Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

    KAUST Repository

    Rayapuram, Naganand

    2014-04-04

    Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

  17. 磷蛋白组的研究技术及其进展%Advances in Analysis Techniques of Phosphoproteome

    Institute of Scientific and Technical Information of China (English)

    杨珺; 邹全明; 蔡绍皙; 郭刚; 朱永红

    2003-01-01

    真核细胞中蛋白质磷酸化是一个重要事件.真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等.磷蛋白组的研究涉及磷蛋白的分离和鉴定,磷酸化残基定位和定量分析.由于蛋白质磷酸化是一个动态过程,在细胞中磷蛋白含量低,磷酸化位点可变,且磷酸肽的质谱信号常常会受到抑制,所以磷蛋白的分析存在更多的困难.本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况.目前,质谱仍然是核心的鉴定技术,寻找更好富集方法是最大的挑战.定量蛋白组学是对蛋白质的差异表达进行精确的定量分析.目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定,以及磷酸位点的定位和定量分析.随着样品分离技术和相关仪器的发展,磷酸蛋白快速、准确、全面分析鉴定将能够实现.

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

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone;

    2016-01-01

    . New insights into phosphorylation-dependent signaling networks that impact on nuclear proteins and controls adipocyte differentiation and cell fate. Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency...

  19. Canonical analysis based on mutual information

    DEFF Research Database (Denmark)

    Nielsen, Allan Aasbjerg; Vestergaard, Jacob Schack

    2015-01-01

    combinations with the information theoretical measure mutual information (MI). We term this type of analysis canonical information analysis (CIA). MI allows for the actual joint distribution of the variables involved and not just second order statistics. While CCA is ideal for Gaussian data, CIA facilitates......Canonical correlation analysis (CCA) is an established multi-variate statistical method for finding similarities between linear combinations of (normally two) sets of multivariate observations. In this contribution we replace (linear) correlation as the measure of association between the linear...

  20. Pathway-Based Functional Analysis of Metagenomes

    Science.gov (United States)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  1. Analysis of Task-based Syllabus

    Institute of Scientific and Technical Information of China (English)

    马进胜

    2011-01-01

    Task-based language teaching is very popular in the modem English teaching.It is based on the Task-based Syllabus.Taskbased Syllabus focuses on the learners' communicative competence,which stresses learning by doing.From the theoretical assumption and definitions of the task,the paper analysizes the components of the task,then points out the merits and demerits of the syllabus.By this means the paper may give some tips to teachers and students when they use the tsk-based language teaching.

  2. Market Based Analysis of Power System Interconnections

    OpenAIRE

    Obuševs, A; Turcik, M; Oļeiņikova, I; Junghāns, G

    2011-01-01

    Analysis in this Article is focused on usage of transmission grid under liberalized market with implicit transmission capacity allocation method, e.g. Nordic market. Attention is paid on fundamental changes in transmission utilization and its economical effective operation. For interconnection and power flow analysis and losses calculation model of Nordic grid was developed and transmission losses calculation method was created. Given approach will improve economical efficiency of system oper...

  3. Gender-Based Analysis On-Line Dialogue. Final Report.

    Science.gov (United States)

    2001

    An online dialogue on gender-based analysis (GBA) was held from February 15 to March 7, 2001. Invitations and a background paper titled "Why Gender-Based Analysis?" were sent to 350 women's organizations and individuals throughout Canada. Efforts were made to ensure that aboriginal and Metis women, visible minority women, and women with special…

  4. Science Based Governance? EU Food Regulation Submitted to Risk Analysis

    NARCIS (Netherlands)

    Szajkowska, A.; Meulen, van der B.M.J.

    2014-01-01

    Anna Szajkowska and Bernd van der Meulen analyse in their contribution, Science Based Governance? EU Food Regulation Submitted to Risk Analysis, the scope of application of risk analysis and the precautionary principle in EU food safety regulation. To what extent does this technocratic, science-base

  5. Transect based analysis versus area based analysis to quantify shoreline displacement: spatial resolution issues.

    Science.gov (United States)

    Anfuso, Giorgio; Bowman, Dan; Danese, Chiara; Pranzini, Enzo

    2016-10-01

    Field surveys, aerial photographs, and satellite images are the most commonly employed sources of data to analyze shoreline position, which are further compared by area based analysis (ABA) or transect based analysis (TBA) methods. The former is performed by computing the mean shoreline displacement for the identified coastal segments, i.e., dividing the beach area variation by the segment length; the latter is based on the measurement of the distance between each shoreline at set points along transects. The present study compares, by means of GIS tools, the ABA and TBA methods by computing shoreline displacements recorded on two stretches of the Tuscany coast (Italy): the beaches of Punta Ala, a linear coast without shore protection structures, and the one at Follonica, which is irregular due to the presence of groins and detached breakwaters. Surveys were carried out using a differential global positioning system (DGPS) in RTK mode. For each site, a 4800-m-long coastal segment was analyzed and divided into ninety-six 50-m-long sectors for which changes were computed using both the ABA and TBA methods. Sectors were progressively joined to have a length of 100, 200, 400, and 800 m to examine how this influenced results. ABA and TBA results are highly correlated for transect distance and sector length up to 100 m at both investigated locations. If longer transects are considered, the two methods still produce good correlated data on the smooth shoreline (i.e. at Punta Ala), but correlation became significantly lower on the irregular shoreline (i.e., at Follonica). PMID:27640163

  6. SSI Analysis for Base-Isolated Nuclear Power Plants

    International Nuclear Information System (INIS)

    Safety of NPPs much higher than other structures is required. An earthquake is one of the most important parameters which govern safety of NPPs among external events. Application of base isolation system for NPPs can reduce the risk for earthquakes. At present, a soil structure interaction(SSI) analysis is essential in seismic design of NPPs in consideration of ground structure interaction. In the seismic analysis of the base-isolated NPP, it is restrictive to consider nonlinear properties of seismic isolation bearings due to linear analysis of SSI analysis programs such as SASSI. Thus, in this study, SSI analyses are performed using an iterative approach considering material nonlinearity of isolators. By performing the SSI analysis using an iterative approach, nonlinear properties of isolators can be considered. The results of the SSI analysis show that the response of the base-isolated NPP with base isolation systems is significantly reduced horizontally

  7. Performance Analysis Based on Timing Simulation

    DEFF Research Database (Denmark)

    Nielsen, Christian Dalsgaard; Kishinevsky, Michael

    1994-01-01

    Determining the cycle time and a critical cycle is a fundamental problem in the analysis of concurrent systems. We solve this problemusing timing simulation of an underlying Signal Graph (an extension of Marked Graphs). For a Signal Graph with n vertices and m arcs our algorithm has the polynomia...... time complexity O(b2m), where b is the number of vertices with initially marked in-arcs (typically b≪n). The algorithm has a clear semantic and a low descriptive complexity. We illustrate the use of the algorithm by applying it to performance analysis of asynchronous circuits.......Determining the cycle time and a critical cycle is a fundamental problem in the analysis of concurrent systems. We solve this problemusing timing simulation of an underlying Signal Graph (an extension of Marked Graphs). For a Signal Graph with n vertices and m arcs our algorithm has the polynomial...

  8. Antenna trajectory error analysis in backprojection-based SAR images

    Science.gov (United States)

    Wang, Ling; Yazıcı, Birsen; Yanik, H. Cagri

    2014-06-01

    We present an analysis of the positioning errors in Backprojection (BP)-based Synthetic Aperture Radar (SAR) images due to antenna trajectory errors for a monostatic SAR traversing a straight linear trajectory. Our analysis is developed using microlocal analysis, which can provide an explicit quantitative relationship between the trajectory error and the positioning error in BP-based SAR images. The analysis is applicable to arbitrary trajectory errors in the antenna and can be extended to arbitrary imaging geometries. We present numerical simulations to demonstrate our analysis.

  9. Environmentally based Cost-Benefit Analysis

    International Nuclear Information System (INIS)

    The fundamentals of the basic elements of a new comprehensive economic assessment, MILA, developed in Sweden with inspiration from the Total Cost Assessment-model are presented. The core of the MILA approach is an expanded cost and benefit inventory. But MILA also includes a complementary addition of an internal waste stream analysis, a tool for evaluation of environmental conflicts in monetary terms, an extended time horizon and direct allocation of costs and revenues to products and processes. However, MILA does not ensure profitability for environmentally sound projects. Essentially, MILA is an approach of refining investment and profitability analysis of a project, investment or product. 109 refs., 38 figs

  10. A Goal based methodology for HAZOP analysis

    DEFF Research Database (Denmark)

    Rossing, Netta Liin; Lind, Morten; Jensen, Niels;

    2010-01-01

    for implementation into a computer aided reasoning tool for HAZOP studies to perform root cause and consequence analysis. Such a tool will facilitate finding causes far away from the site of the deviation. A Functional HAZOP Assistant is proposed and investigated in a HAZOP study of an industrial scale Indirect...

  11. 磷酸化蛋白质组学富集策略进展及其在疾病研究中的应用%Advances in enrichment strategies for phosphoproteomics and appIication of phosphoproteomics in disease research

    Institute of Scientific and Technical Information of China (English)

    武维新; 严佳; 谭喜莹; 李博; 苏梦翔; 严方; 狄斌

    2016-01-01

    Protein phosphorylation is one of the most common post-translational modifications (PTMs)in various organisms,which plays critical roles in the regulation of intracellular biological processes,such as cell prolifera-tion,signal transduction,metabolismis and tumorigenesis.However,the low abundance of phosphoprotein in the biological systems poses significant challenges of current analytical techniques.In order to further understand the phosphoproteomics,the roles of phosphorylated proteins in life process,discovery of biomarkers,diagnosis and treatment of disease,enrichment strategies of high efficiency have been developed,including the design of new nanomaterials and combination of a variety of analytical methods,et al.In this paper,we reviewed the develop-ment of enrichment strategies for phosphoproteomics and application of phosphoproteomics in disease.%蛋白质磷酸化是生物体内重要的翻译后修饰,几乎参与调节着细胞增殖、信号转导、新陈代谢、肿瘤发生等过程在内的所有生命活动。然而,磷酸化蛋白丰度低等问题限制了磷酸化蛋白质组学的发展。为了全面分析磷酸化蛋白质组,深入了解磷酸化蛋白在生命过程中的作用,发现生物标志物,帮助疾病的诊疗,高效的富集策略不断被开发出来,包括设计新型纳米材料提高检测灵敏度和特异性,合并多种分析方法增大富集容量等。本文回顾了近年来磷酸化蛋白质组学研究策略中的富集策略进展及其在疾病研究中的应用。

  12. Pathway-based Analysis Tools for Complex Diseases: A Review

    Directory of Open Access Journals (Sweden)

    Lv Jin

    2014-10-01

    Full Text Available Genetic studies are traditionally based on single-gene analysis. The use of these analyses can pose tremendous challenges for elucidating complicated genetic interplays involved in complex human diseases. Modern pathway-based analysis provides a technique, which allows a comprehensive understanding of the molecular mechanisms underlying complex diseases. Extensive studies utilizing the methods and applications for pathway-based analysis have significantly advanced our capacity to explore large-scale omics data, which has rapidly accumulated in biomedical fields. This article is a comprehensive review of the pathway-based analysis methods—the powerful methods with the potential to uncover the biological depths of the complex diseases. The general concepts and procedures for the pathway-based analysis methods are introduced and then, a comprehensive review of the major approaches for this analysis is presented. In addition, a list of available pathway-based analysis software and databases is provided. Finally, future directions and challenges for the methodological development and applications of pathway-based analysis techniques are discussed. This review will provide a useful guide to dissect complex diseases.

  13. Encounter-based worms: Analysis and Defense

    CERN Document Server

    Tanachaiwiwat, Sapon

    2007-01-01

    Encounter-based network is a frequently-disconnected wireless ad-hoc network requiring immediate neighbors to store and forward aggregated data for information disseminations. Using traditional approaches such as gateways or firewalls for deterring worm propagation in encounter-based networks is inappropriate. We propose the worm interaction approach that relies upon automated beneficial worm generation aiming to alleviate problems of worm propagations in such networks. To understand the dynamic of worm interactions and its performance, we mathematically model worm interactions based on major worm interaction factors including worm interaction types, network characteristics, and node characteristics using ordinary differential equations and analyze their effects on our proposed metrics. We validate our proposed model using extensive synthetic and trace-driven simulations. We find that, all worm interaction factors significantly affect the pattern of worm propagations. For example, immunization linearly decrea...

  14. Accelerator based techniques for aerosol analysis

    International Nuclear Information System (INIS)

    At the 3 MV Tandetron accelerator of the LABEC laboratory of INFN (Florence, Italy) an external beam facility is fully dedicated to PIXE-PIGE measurements of elemental composition of atmospheric aerosols. Examples regarding recent monitoring campaigns, performed in urban and remote areas, both on a daily basis and with high time resolution, as well as with size selection, will be presented. It will be evidenced how PIXE can provide unique information in aerosol studies or can play a complementary role to traditional chemical analysis. Finally a short presentation of 14C analysis of the atmospheric aerosol by Accelerator Mass Spectrometry (AMS) for the evaluation of the contributions from either fossil fuel combustion or modern sources (wood burning, biogenic activity) will be given. (author)

  15. Symbolic Analysis of OTRAs-Based Circuits

    Directory of Open Access Journals (Sweden)

    C. Sánchez-López

    2011-04-01

    Full Text Available A new nullor-based model to describe the behavior of Operational Transresistance Amplifiers (OTRAs is introduced.The new model is composed of four nullors and three grounded resistors. As a consequence, standard nodal analysiscan be applied to compute fully-symbolic small-signal characteristics of OTRA-based analog circuits, and the nullorbasedOTRAs model can be used in CAD tools. In this manner, the fully-symbolic transfer functions of severalapplication circuits, such as filters and oscillators can easily be approximated.

  16. Structural Analysis of Plate Based Tensegrity Structures

    DEFF Research Database (Denmark)

    Hald, Frederik; Kirkegaard, Poul Henning; Damkilde, Lars

    2013-01-01

    Plate tensegrity structures combine tension cables with a cross laminated timber plate and can then form e.g. a roof structure. The topology of plate tensegrity structures is investigated through a parametric investigation. Plate tensegrity structures are investigated, and a method for...... determination of the structures pre-stresses is used. A parametric investigation is performed to determine a more optimized form of the plate based tensegrity structure. Conclusions of the use of plate based tensegrity in civil engineering and further research areas are discussed....

  17. Crime prevention: more evidence-based analysis.

    Science.gov (United States)

    Garrido Genovés, Vicente; Farrington, David P; Welsh, Brandon C

    2008-02-01

    This paper introduces a new section of Psicothema dedicated to the evidence-based approach to crime prevention. Along with an original sexual-offender-treatment programme implemented in Spain, this section presents four systematic reviews of important subjects in the criminological arena, such as sexual offender treatment, the well-known programme, the effectiveness of custodial versus non-custodial sanctions in reoffending and the fight against terrorism. We also highlight some of the focal points that scientists, practitioners and governments should take into account in order to support this evidence-based viewpoint of crime prevention.

  18. Thanatophoric dysplasia: case-based bioethical analysis

    OpenAIRE

    Edgar Abarca López; Alejandra Rodríguez Torres; Donovan Casas Patiño; Esteban Espíndola Benítez

    2014-01-01

    This paper presents a case report of thanatophoric displasia diagnosed in the prenatal period using ultrasound standards. The course of the case pregnancy, birth process, and postnatal period is described. This report invites bioethical analysis using its principles, appealing to human dignity, diversity and otherness, particularly in the mother-child dyad and their family. An early diagnosis allows parental support as they face the course of this condition and its potentially fatal outcome.

  19. Thanatophoric dysplasia: case-based bioethical analysis

    Directory of Open Access Journals (Sweden)

    Edgar Abarca López

    2014-04-01

    Full Text Available This paper presents a case report of thanatophoric displasia diagnosed in the prenatal period using ultrasound standards. The course of the case pregnancy, birth process, and postnatal period is described. This report invites bioethical analysis using its principles, appealing to human dignity, diversity and otherness, particularly in the mother-child dyad and their family. An early diagnosis allows parental support as they face the course of this condition and its potentially fatal outcome.

  20. Movement Pattern Analysis Based on Sequence Signatures

    Directory of Open Access Journals (Sweden)

    Seyed Hossein Chavoshi

    2015-09-01

    Full Text Available Increased affordability and deployment of advanced tracking technologies have led researchers from various domains to analyze the resulting spatio-temporal movement data sets for the purpose of knowledge discovery. Two different approaches can be considered in the analysis of moving objects: quantitative analysis and qualitative analysis. This research focuses on the latter and uses the qualitative trajectory calculus (QTC, a type of calculus that represents qualitative data on moving point objects (MPOs, and establishes a framework to analyze the relative movement of multiple MPOs. A visualization technique called sequence signature (SESI is used, which enables to map QTC patterns in a 2D indexed rasterized space in order to evaluate the similarity of relative movement patterns of multiple MPOs. The applicability of the proposed methodology is illustrated by means of two practical examples of interacting MPOs: cars on a highway and body parts of a samba dancer. The results show that the proposed method can be effectively used to analyze interactions of multiple MPOs in different domains.

  1. Chip based electroanalytical systems for cell analysis

    DEFF Research Database (Denmark)

    Spegel, C.; Heiskanen, A.; Skjolding, L.H.D.;

    2008-01-01

    This review with 239 references has as its aim to give the reader an introduction to the kinds of methods used for developing microchip based electrode systems as well as to cover the existing literature on electroanalytical systems where microchips play a crucial role for 'nondestructive...

  2. Dynamic Chest Image Analysis: Model-Based Perfusion Analysis in Dynamic Pulmonary Imaging

    OpenAIRE

    Kiuru Aaro; Kormano Martti; Svedström Erkki; Liang Jianming; Järvi Timo

    2003-01-01

    The "Dynamic Chest Image Analysis" project aims to develop model-based computer analysis and visualization methods for showing focal and general abnormalities of lung ventilation and perfusion based on a sequence of digital chest fluoroscopy frames collected with the dynamic pulmonary imaging technique. We have proposed and evaluated a multiresolutional method with an explicit ventilation model for ventilation analysis. This paper presents a new model-based method for pulmonary perfusion ana...

  3. Desiccant-Based Preconditioning Market Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, J.

    2001-01-11

    A number of important conclusions can be drawn as a result of this broad, first-phase market evaluation. The more important conclusions include the following: (1) A very significant market opportunity will exist for specialized outdoor air-handling units (SOAHUs) as more construction and renovation projects are designed to incorporate the recommendations made by the ASHRAE 62-1989 standard. Based on this investigation, the total potential market is currently $725,000,000 annually (see Table 6, Sect. 3). Based on the market evaluations completed, it is estimated that approximately $398,000,000 (55%) of this total market could be served by DBC systems if they were made cost-effective through mass production. Approximately $306,000,000 (42%) of the total can be served by a non-regenerated, desiccant-based total recovery approach, based on the information provided by this investigation. Approximately $92,000,000 (13%) can be served by a regenerated desiccant-based cooling approach (see Table 7, Sect. 3). (2) A projection of the market selling price of various desiccant-based SOAHU systems was prepared using prices provided by Trane for central-station, air-handling modules currently manufactured. The wheel-component pricing was added to these components by SEMCO. This resulted in projected pricing for these systems that is significantly less than that currently offered by custom suppliers (see Table 4, Sect. 2). Estimated payback periods for all SOAHU approaches were quite short when compared with conventional over-cooling and reheat systems. Actual paybacks may vary significantly depending on site-specific considerations. (3) In comparing cost vs benefit of each SOAHU approach, it is critical that the total system design be evaluated. For example, the cost premium of a DBC system is very significant when compared to a conventional air handling system, yet the reduced chiller, boiler, cooling tower, and other expense often equals or exceeds this premium, resulting in a

  4. Google glass based immunochromatographic diagnostic test analysis

    Science.gov (United States)

    Feng, Steve; Caire, Romain; Cortazar, Bingen; Turan, Mehmet; Wong, Andrew; Ozcan, Aydogan

    2015-03-01

    Integration of optical imagers and sensors into recently emerging wearable computational devices allows for simpler and more intuitive methods of integrating biomedical imaging and medical diagnostics tasks into existing infrastructures. Here we demonstrate the ability of one such device, the Google Glass, to perform qualitative and quantitative analysis of immunochromatographic rapid diagnostic tests (RDTs) using a voice-commandable hands-free software-only interface, as an alternative to larger and more bulky desktop or handheld units. Using the built-in camera of Glass to image one or more RDTs (labeled with Quick Response (QR) codes), our Glass software application uploads the captured image and related information (e.g., user name, GPS, etc.) to our servers for remote analysis and storage. After digital analysis of the RDT images, the results are transmitted back to the originating Glass device, and made available through a website in geospatial and tabular representations. We tested this system on qualitative human immunodeficiency virus (HIV) and quantitative prostate-specific antigen (PSA) RDTs. For qualitative HIV tests, we demonstrate successful detection and labeling (i.e., yes/no decisions) for up to 6-fold dilution of HIV samples. For quantitative measurements, we activated and imaged PSA concentrations ranging from 0 to 200 ng/mL and generated calibration curves relating the RDT line intensity values to PSA concentration. By providing automated digitization of both qualitative and quantitative test results, this wearable colorimetric diagnostic test reader platform on Google Glass can reduce operator errors caused by poor training, provide real-time spatiotemporal mapping of test results, and assist with remote monitoring of various biomedical conditions.

  5. Web Application Comprehension Based on Dependence Analysis

    Institute of Scientific and Technical Information of China (English)

    WU Jun-hua; XU Bao-wen; JIANG Ji-xiang

    2004-01-01

    Many research indicate a lot of money and time are spent on maintaining and modifying program delivered.So the policies to support program comprehension are very important.Program comprehension is a crucial and difficult task.Insufficient design, illogical code structure, short documents will enhance the comprehensive difficulty.Developing Web application is usually a process with quick implementation and delivery.In addition, generally a Web application is coded by combining mark language statements with some embedded applets.Such programming mode affects comprehension of Web applications disadvantageously.This paper proposes a method to improving understanding Web by dependence analysis and slice technology.

  6. Knowledge-based analysis of phenotypes

    KAUST Repository

    Hoendorf, Robert

    2016-01-27

    Phenotypes are the observable characteristics of an organism, and they are widely recorded in biology and medicine. To facilitate data integration, ontologies that formally describe phenotypes are being developed in several domains. I will describe a formal framework to describe phenotypes. A formalized theory of phenotypes is not only useful for domain analysis, but can also be applied to assist in the diagnosis of rare genetic diseases, and I will show how our results on the ontology of phenotypes is now applied in biomedical research.

  7. An SQL-based approach to physics analysis

    Science.gov (United States)

    Limper, Maaike, Dr

    2014-06-01

    As part of the CERN openlab collaboration a study was made into the possibility of performing analysis of the data collected by the experiments at the Large Hadron Collider (LHC) through SQL-queries on data stored in a relational database. Currently LHC physics analysis is done using data stored in centrally produced "ROOT-ntuple" files that are distributed through the LHC computing grid. The SQL-based approach to LHC physics analysis presented in this paper allows calculations in the analysis to be done at the database and can make use of the database's in-built parallelism features. Using this approach it was possible to reproduce results for several physics analysis benchmarks. The study shows the capability of the database to handle complex analysis tasks but also illustrates the limits of using row-based storage for storing physics analysis data, as performance was limited by the I/O read speed of the system.

  8. Analysis and Protection of SIP based Services

    OpenAIRE

    Ferdous, Raihana

    2014-01-01

    Multimedia communications over IP are booming as they offer higher flexibility and more features than traditional voice and video services. IP telephony known as Voice over IP (VoIP) is one of the commercially most important emerging trends in multimedia communications over IP. Due to the flexibility and descriptive power, the Session Initiation Protocol (SIP) is becoming the root of many sessions-based applications such as VoIP and media streaming that are used by a growing number of use...

  9. Value-Based Analysis of Mobile Tagging

    OpenAIRE

    Oguzhan Aygoren; Kaan Varnali

    2011-01-01

    Innovative use of the mobile medium in delivering customer value presents unprecedented opportunities for marketers. Various types of mobile applications have evolved to provide ubiquitous and instant customer service to capitalize on this opportunity. One application is mobile tagging, a mobile-based innovative tool for convergence marketing. The accumulated academic knowledge on mobile marketing lacks consumer-centric information about this phenomenon. This paper addresses this issue and co...

  10. Analysis of Hashrate-Based Double Spending

    OpenAIRE

    Rosenfeld, Meni

    2014-01-01

    Bitcoin is the world's first decentralized digital currency. Its main technical innovation is the use of a blockchain and hash-based proof of work to synchronize transactions and prevent double-spending the currency. While the qualitative nature of this system is well understood, there is widespread confusion about its quantitative aspects and how they relate to attack vectors and their countermeasures. In this paper we take a look at the stochastic processes underlying typical attacks and th...

  11. Interest Based Financial Intermediation: Analysis and Solutions

    OpenAIRE

    Shaikh, Salman

    2012-01-01

    Interest is prohibited in all monotheist religions. Apart from religion, interest is also regarded as unjust price of money capital by pioneer secular philosophers as well as some renowned economists. However, it is argued by some economists that modern day, market driven interest rate in a competitive financial market is different from usury and that the interest based financial intermediation has served a useful purpose in allocation of resources as well as in allocation of risk, given the ...

  12. Model-based methods for linkage analysis.

    Science.gov (United States)

    Rice, John P; Saccone, Nancy L; Corbett, Jonathan

    2008-01-01

    The logarithm of an odds ratio (LOD) score method originated in a seminal article by Newton Morton in 1955. The method is broadly concerned with issues of power and the posterior probability of linkage, ensuring that a reported linkage has a high probability of being a true linkage. In addition, the method is sequential so that pedigrees or LOD curves may be combined from published reports to pool data for analysis. This approach has been remarkably successful for 50 years in identifying disease genes for Mendelian disorders. After discussing these issues, we consider the situation for complex disorders where the maximum LOD score statistic shares some of the advantages of the traditional LOD score approach, but is limited by unknown power and the lack of sharing of the primary data needed to optimally combine analytic results. We may still learn from the LOD score method as we explore new methods in molecular biology and genetic analysis to utilize the complete human DNA sequence and the cataloging of all human genes.

  13. Model Based Analysis and Test Generation for Flight Software

    Science.gov (United States)

    Pasareanu, Corina S.; Schumann, Johann M.; Mehlitz, Peter C.; Lowry, Mike R.; Karsai, Gabor; Nine, Harmon; Neema, Sandeep

    2009-01-01

    We describe a framework for model-based analysis and test case generation in the context of a heterogeneous model-based development paradigm that uses and combines Math- Works and UML 2.0 models and the associated code generation tools. This paradigm poses novel challenges to analysis and test case generation that, to the best of our knowledge, have not been addressed before. The framework is based on a common intermediate representation for different modeling formalisms and leverages and extends model checking and symbolic execution tools for model analysis and test case generation, respectively. We discuss the application of our framework to software models for a NASA flight mission.

  14. Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti(4+)-IMAC.

    Science.gov (United States)

    Piovesana, Susy; Capriotti, Anna Laura; Cavaliere, Chiara; Ferraris, Francesca; Samperi, Roberto; Ventura, Salvatore; Laganà, Aldo

    2016-02-25

    Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides. PMID:26851086

  15. Sandia National Laboratories analysis code data base

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, C.W.

    1994-11-01

    Sandia National Laboratories, mission is to solve important problems in the areas of national defense, energy security, environmental integrity, and industrial technology. The Laboratories` strategy for accomplishing this mission is to conduct research to provide an understanding of the important physical phenomena underlying any problem, and then to construct validated computational models of the phenomena which can be used as tools to solve the problem. In the course of implementing this strategy, Sandia`s technical staff has produced a wide variety of numerical problem-solving tools which they use regularly in the design, analysis, performance prediction, and optimization of Sandia components, systems and manufacturing processes. This report provides the relevant technical and accessibility data on the numerical codes used at Sandia, including information on the technical competency or capability area that each code addresses, code ``ownership`` and release status, and references describing the physical models and numerical implementation.

  16. Network Anomaly Detection Based on Wavelet Analysis

    Science.gov (United States)

    Lu, Wei; Ghorbani, Ali A.

    2008-12-01

    Signal processing techniques have been applied recently for analyzing and detecting network anomalies due to their potential to find novel or unknown intrusions. In this paper, we propose a new network signal modelling technique for detecting network anomalies, combining the wavelet approximation and system identification theory. In order to characterize network traffic behaviors, we present fifteen features and use them as the input signals in our system. We then evaluate our approach with the 1999 DARPA intrusion detection dataset and conduct a comprehensive analysis of the intrusions in the dataset. Evaluation results show that the approach achieves high-detection rates in terms of both attack instances and attack types. Furthermore, we conduct a full day's evaluation in a real large-scale WiFi ISP network where five attack types are successfully detected from over 30 millions flows.

  17. Network Anomaly Detection Based on Wavelet Analysis

    Directory of Open Access Journals (Sweden)

    Ali A. Ghorbani

    2008-11-01

    Full Text Available Signal processing techniques have been applied recently for analyzing and detecting network anomalies due to their potential to find novel or unknown intrusions. In this paper, we propose a new network signal modelling technique for detecting network anomalies, combining the wavelet approximation and system identification theory. In order to characterize network traffic behaviors, we present fifteen features and use them as the input signals in our system. We then evaluate our approach with the 1999 DARPA intrusion detection dataset and conduct a comprehensive analysis of the intrusions in the dataset. Evaluation results show that the approach achieves high-detection rates in terms of both attack instances and attack types. Furthermore, we conduct a full day's evaluation in a real large-scale WiFi ISP network where five attack types are successfully detected from over 30 millions flows.

  18. Mental EEG Analysis Based on Infomax Algorithm

    Institute of Scientific and Technical Information of China (English)

    WUXiao-pei; GuoXiao-jing; ZANGDao-xin; SHENQian

    2004-01-01

    The patterns of EEG will change with mental tasks performed by the subject. In the field of EEG signal analysis and application, the study to get the patterns of mental EEG and then to use them to classify mental tasks has the significant scientific meaning and great application value. But for the reasons of different artifacts existing in EEG, the pattern detection of EEG under normal mental states is a very difficult problem. In this paper, Independent Component Analysisis applied to EEG signals collected from performing different mental tasks. The experiment results show that when one subject performs a single mental task in different trials, the independent components of EEG are very similar. It means that the independent components can be used as the mental EEG patterns to classify the different mental tasks.

  19. Wavelet Based Fractal Analysis of Airborne Pollen

    CERN Document Server

    Degaudenzi, M E

    1999-01-01

    The most abundant biological particles in the atmosphere are pollen grains and spores. Self protection of pollen allergy is possible through the information of future pollen contents in the air. In spite of the importance of airborne pol len concentration forecasting, it has not been possible to predict the pollen concentrations with great accuracy, and about 25% of the daily pollen forecasts have resulted in failures. Previous analysis of the dynamic characteristics of atmospheric pollen time series indicate that the system can be described by a low dimensional chaotic map. We apply the wavelet transform to study the multifractal characteristics of an a irborne pollen time series. We find the persistence behaviour associated to low pollen concentration values and to the most rare events of highest pollen co ncentration values. The information and the correlation dimensions correspond to a chaotic system showing loss of information with time evolution.

  20. Computational based functional analysis of Bacillus phytases.

    Science.gov (United States)

    Verma, Anukriti; Singh, Vinay Kumar; Gaur, Smriti

    2016-02-01

    Phytase is an enzyme which catalyzes the total hydrolysis of phytate to less phosphorylated myo-inositol derivatives and inorganic phosphate and digests the undigestable phytate part present in seeds and grains and therefore provides digestible phosphorus, calcium and other mineral nutrients. Phytases are frequently added to the feed of monogastric animals so that bioavailability of phytic acid-bound phosphate increases, ultimately enhancing the nutritional value of diets. The Bacillus phytase is very suitable to be used in animal feed because of its optimum pH with excellent thermal stability. Present study is aimed to perform an in silico comparative characterization and functional analysis of phytases from Bacillus amyloliquefaciens to explore physico-chemical properties using various bio-computational tools. All proteins are acidic and thermostable and can be used as suitable candidates in the feed industry. PMID:26672917

  1. Face Recognition Based on Principal Component Analysis

    Directory of Open Access Journals (Sweden)

    Ali Javed

    2013-02-01

    Full Text Available The purpose of the proposed research work is to develop a computer system that can recognize a person by comparing the characteristics of face to those of known individuals. The main focus is on frontal two dimensional images that are taken in a controlled environment i.e. the illumination and the background will be constant. All the other methods of person’s identification and verification like iris scan or finger print scan require high quality and costly equipment’s but in face recognition we only require a normal camera giving us a 2-D frontal image of the person that will be used for the process of the person’s recognition. Principal Component Analysis technique has been used in the proposed system of face recognition. The purpose is to compare the results of the technique under the different conditions and to find the most efficient approach for developing a facial recognition system

  2. LEARNING DIFFICULTIES: AN ANALYSIS BASED ON VIGOTSKY

    Directory of Open Access Journals (Sweden)

    Adriane Cenci

    2010-06-01

    Full Text Available We aimed, along the text, to bring a reflection upon learning difficulties based on Socio-Historical Theory, relating what is observed in schools to what has been discussed about learning difficulties and the theory proposed by Vygotsky in the early XX century. We understand that children enter school carrying experiences and knowledge from their cultural group and that school ignores such knowledge very often. Then, it is in such disengagement that emerges what we started to call learning difficulties. One cannot forget to see a child as a whole – a student is a social being constituted by culture, language and specific values to which one must be attentive.

  3. Building Extraction from LIDAR Based Semantic Analysis

    Institute of Scientific and Technical Information of China (English)

    YU Jie; YANG Haiquan; TAN Ming; ZHANG Guoning

    2006-01-01

    Extraction of buildings from LIDAR data has been an active research field in recent years. A scheme for building detection and reconstruction from LIDAR data is presented with an object-oriented method which is based on the buildings' semantic rules. Two key steps are discussed: how to group the discrete LIDAR points into single objects and how to establish the buildings' semantic rules. In the end, the buildings are reconstructed in 3D form and three common parametric building models (flat, gabled, hipped) are implemented.

  4. Digital Terrain Analysis Based on DEM

    Institute of Scientific and Technical Information of China (English)

    Bi Huaxing; Li Xiaoyin; Guo Mengxia; Liu Xin; Li Jun

    2006-01-01

    The digital elevation model (DEM),an important source of information,is usually used to express a topographic surface in three dimensions and to imitate essential natural geography.DEM has been applied to physical geography,hydrology,ecology,and biology.This study analyzed digital elevation data sources and their structure,the arithmetic of terrain attribute extraction from DEM and its applications,and DEM's error and uncertainty algorithm.The Hayachinesan mountain area (in northeastern Japan) was chosen as research site,and the focus was on terrain analysis and the impacts of DEM resolution on topographic attributes,analyzed using TNTmips GIS software (MicroImage,Inc.,USA) and"Digital Map 25,000"(published by the Geographical Survey Institute of Japan in 1998).The results show that:(1) DEM is a very effective tool for terrain analysis:many terrain attributes (such as slope,aspect,slope type,watershed,and standard flow path) can be derived,and these attributes can be displayed with both image and attribute databases,with the help of GIS;(2) DEM resolution has a great influence on terrain attributes.The following details are shown:(a) DEM resolution has a significant effect on slope estimation:the average slope becomes smaller and the standard deviation becomes larger when DEM resolution changes from fine to coarse,and the different impacts of DEM resolution on different slope ranges can be classified into three gradient classes:0-10°(underestimated slope),10-35°(overestimated slope),and>35°(little impact on slope estimation);(b) DEM resolution has little effect on aspect estimation,but fiat areas become larger when DEM resolution changes from Fine to coarse;and (c) the quantity of hydrologic topography information declines as DEM resolution decreases.

  5. Trajectory Based Behavior Analysis for User Verification

    Science.gov (United States)

    Pao, Hsing-Kuo; Lin, Hong-Yi; Chen, Kuan-Ta; Fadlil, Junaidillah

    Many of our activities on computer need a verification step for authorized access. The goal of verification is to tell apart the true account owner from intruders. We propose a general approach for user verification based on user trajectory inputs. The approach is labor-free for users and is likely to avoid the possible copy or simulation from other non-authorized users or even automatic programs like bots. Our study focuses on finding the hidden patterns embedded in the trajectories produced by account users. We employ a Markov chain model with Gaussian distribution in its transitions to describe the behavior in the trajectory. To distinguish between two trajectories, we propose a novel dissimilarity measure combined with a manifold learnt tuning for catching the pairwise relationship. Based on the pairwise relationship, we plug-in any effective classification or clustering methods for the detection of unauthorized access. The method can also be applied for the task of recognition, predicting the trajectory type without pre-defined identity. Given a trajectory input, the results show that the proposed method can accurately verify the user identity, or suggest whom owns the trajectory if the input identity is not provided.

  6. IMAGE ANALYSIS BASED ON EDGE DETECTION TECHNIQUES

    Institute of Scientific and Technical Information of China (English)

    纳瑟; 刘重庆

    2002-01-01

    A method that incorporates edge detection technique, Markov Random field (MRF), watershed segmentation and merging techniques was presented for performing image segmentation and edge detection tasks. It first applies edge detection technique to obtain a Difference In Strength (DIS) map. An initial segmented result is obtained based on K-means clustering technique and the minimum distance. Then the region process is modeled by MRF to obtain an image that contains different intensity regions. The gradient values are calculated and then the watershed technique is used. DIS calculation is used for each pixel to define all the edges (weak or strong) in the image. The DIS map is obtained. This help as priority knowledge to know the possibility of the region segmentation by the next step (MRF), which gives an image that has all the edges and regions information. In MRF model,gray level l, at pixel location i, in an image X, depends on the gray levels of neighboring pixels. The segmentation results are improved by using watershed algorithm. After all pixels of the segmented regions are processed, a map of primitive region with edges is generated. The edge map is obtained using a merge process based on averaged intensity mean values. A common edge detectors that work on (MRF) segmented image are used and the results are compared. The segmentation and edge detection result is one closed boundary per actual region in the image.

  7. The Route Analysis Based On Flight Plan

    Science.gov (United States)

    Feriyanto, Nur; Saleh, Chairul; Fauzi, Achmad; Rachman Dzakiyullah, Nur; Riza Iwaputra, Kahfi

    2016-02-01

    Economic development effects use of air transportation since the business process in every aspect was increased. Many people these days was prefer using airplane because it can save time and money. This situation also effects flight routes, many airlines offer new routes to deal with competition. Managing flight routes is one of the problems that must be faced in order to find the efficient and effective routes. This paper investigates the best routes based on flight performance by determining the amount of block fuel for the Jakarta-Denpasar flight route. Moreover, in this work compares a two kinds of aircraft and tracks by calculating flight distance, flight time and block fuel. The result shows Jakarta-Denpasar in the Track II has effective and efficient block fuel that can be performed by Airbus 320-200 aircraft. This study can contribute to practice in making an effective decision, especially helping executive management of company due to selecting appropriate aircraft and the track in the flight plan based on the block fuel consumption for business operation.

  8. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj

    2009-01-01

    As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational analysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase...

  9. Choice-Based Conjoint Analysis: Classification vs. Discrete Choice Models

    Science.gov (United States)

    Giesen, Joachim; Mueller, Klaus; Taneva, Bilyana; Zolliker, Peter

    Conjoint analysis is a family of techniques that originated in psychology and later became popular in market research. The main objective of conjoint analysis is to measure an individual's or a population's preferences on a class of options that can be described by parameters and their levels. We consider preference data obtained in choice-based conjoint analysis studies, where one observes test persons' choices on small subsets of the options. There are many ways to analyze choice-based conjoint analysis data. Here we discuss the intuition behind a classification based approach, and compare this approach to one based on statistical assumptions (discrete choice models) and to a regression approach. Our comparison on real and synthetic data indicates that the classification approach outperforms the discrete choice models.

  10. Bayesian analysis for EMP damaged function based on Weibull distribution

    International Nuclear Information System (INIS)

    Weibull distribution is one of the most commonly used statistical distribution in EMP vulnerability analysis. In the paper, the EMP damage function based on Weibull distribution of solid state relays was solved by bayesian computation using gibbs sampling algorithm. (authors)

  11. Sentiment Analysis of Document Based on Annotation

    CERN Document Server

    Shukla, Archana

    2011-01-01

    I present a tool which tells the quality of document or its usefulness based on annotations. Annotation may include comments, notes, observation, highlights, underline, explanation, question or help etc. comments are used for evaluative purpose while others are used for summarization or for expansion also. Further these comments may be on another annotation. Such annotations are referred as meta-annotation. All annotation may not get equal weightage. My tool considered highlights, underline as well as comments to infer the collective sentiment of annotators. Collective sentiments of annotators are classified as positive, negative, objectivity. My tool computes collective sentiment of annotations in two manners. It counts all the annotation present on the documents as well as it also computes sentiment scores of all annotation which includes comments to obtain the collective sentiments about the document or to judge the quality of document. I demonstrate the use of tool on research paper.

  12. Analysis of Vehicle-Based Security Operations

    Energy Technology Data Exchange (ETDEWEB)

    Carter, Jason M [ORNL; Paul, Nate R [ORNL

    2015-01-01

    Vehicle-to-vehicle (V2V) communications promises to increase roadway safety by providing each vehicle with 360 degree situational awareness of other vehicles in proximity, and by complementing onboard sensors such as radar or camera in detecting imminent crash scenarios. In the United States, approximately three hundred million automobiles could participate in a fully deployed V2V system if Dedicated Short-Range Communication (DSRC) device use becomes mandatory. The system s reliance on continuous communication, however, provides a potential means for unscrupulous persons to transmit false data in an attempt to cause crashes, create traffic congestion, or simply render the system useless. V2V communications must be highly scalable while retaining robust security and privacy preserving features to meet the intra-vehicle and vehicle-to-infrastructure communication requirements for a growing vehicle population. Oakridge National Research Laboratory is investigating a Vehicle-Based Security System (VBSS) to provide security and privacy for a fully deployed V2V and V2I system. In the VBSS an On-board Unit (OBU) generates short-term certificates and signs Basic Safety Messages (BSM) to preserve privacy and enhance security. This work outlines a potential VBSS structure and its operational concepts; it examines how a vehicle-based system might feasibly provide security and privacy, highlights remaining challenges, and explores potential mitigations to address those challenges. Certificate management alternatives that attempt to meet V2V security and privacy requirements have been examined previously by the research community including privacy-preserving group certificates, shared certificates, and functional encryption. Due to real-world operational constraints, adopting one of these approaches for VBSS V2V communication is difficult. Timely misbehavior detection and revocation are still open problems for any V2V system. We explore the alternative approaches that may be

  13. Simulation based analysis of laser beam brazing

    Science.gov (United States)

    Dobler, Michael; Wiethop, Philipp; Schmid, Daniel; Schmidt, Michael

    2016-03-01

    Laser beam brazing is a well-established joining technology in car body manufacturing with main applications in the joining of divided tailgates and the joining of roof and side panels. A key advantage of laser brazed joints is the seam's visual quality which satisfies highest requirements. However, the laser beam brazing process is very complex and process dynamics are only partially understood. In order to gain deeper knowledge of the laser beam brazing process, to determine optimal process parameters and to test process variants, a transient three-dimensional simulation model of laser beam brazing is developed. This model takes into account energy input, heat transfer as well as fluid and wetting dynamics that lead to the formation of the brazing seam. A validation of the simulation model is performed by metallographic analysis and thermocouple measurements for different parameter sets of the brazing process. These results show that the multi-physical simulation model not only can be used to gain insight into the laser brazing process but also offers the possibility of process optimization in industrial applications. The model's capabilities in determining optimal process parameters are exemplarily shown for the laser power. Small deviations in the energy input can affect the brazing results significantly. Therefore, the simulation model is used to analyze the effect of the lateral laser beam position on the energy input and the resulting brazing seam.

  14. Structural Analysis Using Computer Based Methods

    Science.gov (United States)

    Dietz, Matthew R.

    2013-01-01

    The stiffness of a flex hose that will be used in the umbilical arms of the Space Launch Systems mobile launcher needed to be determined in order to properly qualify ground umbilical plate behavior during vehicle separation post T-0. This data is also necessary to properly size and design the motors used to retract the umbilical arms. Therefore an experiment was created to determine the stiffness of the hose. Before the test apparatus for the experiment could be built, the structure had to be analyzed to ensure it would not fail under given loading conditions. The design model was imported into the analysis software and optimized to decrease runtime while still providing accurate restlts and allow for seamless meshing. Areas exceeding the allowable stresses in the structure were located and modified before submitting the design for fabrication. In addition, a mock up of a deep space habitat and the support frame was designed and needed to be analyzed for structural integrity under different loading conditions. The load cases were provided by the customer and were applied to the structure after optimizing the geometry. Once again, weak points in the structure were located and recommended design changes were made to the customer and the process was repeated until the load conditions were met without exceeding the allowable stresses. After the stresses met the required factors of safety the designs were released for fabrication.

  15. Multilevel Solvers with Aggregations for Voxel Based Analysis of Geomaterials

    OpenAIRE

    Blaheta, R. (Radim); V. Sokol

    2012-01-01

    Our motivation for voxel based analysis comes from the investigation of geomaterials (geocomposites) arising from rock grouting or sealing. We use finite element analysis based on voxel data from tomography. The arising finite element systems are large scale, which motivates the use of multilevel iterative solvers or preconditioners. Among others we concentrate on multilevel Schwarz preconditioners with aggregations. The aggregations are efficient even in the case of problems with hete...

  16. Experimental Bifurcation Analysis Using Control-Based Continuation

    DEFF Research Database (Denmark)

    Bureau, Emil; Starke, Jens

    The focus of this thesis is developing and implementing techniques for performing experimental bifurcation analysis on nonlinear mechanical systems. The research centers around the newly developed control-based continuation method, which allows to systematically track branches of stable and unsta......The focus of this thesis is developing and implementing techniques for performing experimental bifurcation analysis on nonlinear mechanical systems. The research centers around the newly developed control-based continuation method, which allows to systematically track branches of stable...

  17. Architecture Level Dependency Analysis of SOA Based System through ?-Adl

    OpenAIRE

    Pawan Kumar; Ratneshwer

    2016-01-01

    A formal Architecture Description Language (ADL) provides an effective way to dependency analysis at early stage of development. ?-ADL is an ADL that represents the static and dynamic features of software services. In this paper, we describe an approach of dependency analysis of SOA (Service Oriented Architecture) based system, at architecture level, through ?-ADL. A set of algorithms are also proposed for identification of dependency relationships from a SOA based system. The proposed algori...

  18. Product Profitability Analysis Based on EVA and ABC

    OpenAIRE

    Chen Lin; Shuangyuan Wang; Zhilin Qiao

    2013-01-01

    On the purpose of maximizing shareholders’ value, the profitability analysis established on the basis oftraditional accounting earnings cannot meet the demands of providing accurate decision-making information forenterprises. Therefore, this paper implements the Activity Based Costing (ABC) and the Economic Value Added(EVA) into the traditional profitability analysis system, sets up an improved EVA-ABC based profitabilityanalysis system as well as its relative indexes, and applies it to the s...

  19. Empirical validation and comparison of models for customer base analysis

    OpenAIRE

    Persentili Batislam, Emine; Denizel, Meltem; Filiztekin, Alpay

    2007-01-01

    The benefits of retaining customers lead companies to search for means to profile their customers individually and track their retention and defection behaviors. To this end, the main issues addressed in customer base analysis are identification of customer active/inactive status and prediction of future purchase levels. We compare the predictive performance of Pareto/NBD and BG/NBD models from the customer base analysis literature — in terms of repeat purchase levels and active status — usi...

  20. Discrete Discriminant analysis based on tree-structured graphical models

    DEFF Research Database (Denmark)

    Perez de la Cruz, Gonzalo; Eslava, Guillermina

    The purpose of this paper is to illustrate the potential use of discriminant analysis based on tree{structured graphical models for discrete variables. This is done by comparing its empirical performance using estimated error rates for real and simulated data. The results show that discriminant...... analysis based on tree{structured graphical models is a simple nonlinear method competitive with, and sometimes superior to, other well{known linear methods like those assuming mutual independence between variables and linear logistic regression....

  1. Method for detecting software anomalies based on recurrence plot analysis

    Directory of Open Access Journals (Sweden)

    Michał Mosdorf

    2012-03-01

    Full Text Available Presented paper evaluates method for detecting software anomalies based on recurrence plot analysis of trace log generated by software execution. Described method for detecting software anomalies is based on windowed recurrence quantification analysis for selected measures (e.g. Recurrence rate - RR or Determinism - DET. Initial results show that proposed method is useful in detecting silent software anomalies that do not result in typical crashes (e.g. exceptions.

  2. Web-Based Trainer for Electrical Circuit Analysis

    Science.gov (United States)

    Weyten, L.; Rombouts, P.; De Maeyer, J.

    2009-01-01

    A Web-based system for training electric circuit analysis is presented in this paper. It is centered on symbolic analysis techniques and it not only verifies the student's final answer, but it also tracks and coaches him/her through all steps of his/her reasoning path. The system mimics homework assignments, enhanced by immediate personalized…

  3. PYTHON-based Physics Analysis Environment for LHCb

    CERN Document Server

    Belyaev, I; Mato, P; Barrand, G; Tsaregorodtsev, A; de Oliveira, E

    2004-01-01

    BENDER is the PYTHON based physics analysis application for LHCb. It combines the best features of the underlying GAUDI software architecture with the flexibility of the PYTHON scripting language and provides end-users with a friendly physics analysis oriented environment.

  4. Product Lifecycle Management Architecture: A Model Based Systems Engineering Analysis.

    Energy Technology Data Exchange (ETDEWEB)

    Noonan, Nicholas James [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-07-01

    This report is an analysis of the Product Lifecycle Management (PLM) program. The analysis is centered on a need statement generated by a Nuclear Weapons (NW) customer. The need statement captured in this report creates an opportunity for the PLM to provide a robust service as a solution. Lifecycles for both the NW and PLM are analyzed using Model Based System Engineering (MBSE).

  5. Benefits of Computer Based Content Analysis to Foresight

    OpenAIRE

    Kováříková, Ludmila; Grosová, Stanislava

    2014-01-01

    Purpose of the article: The present manuscript summarizes benefits of the use of computer-based content analysis in a generation phase of foresight initiatives. Possible advantages, disadvantages and limitations of the content analysis for the foresight projects are discussed as well. Methodology/methods: In order to specify the benefits and identify the limitations of the content analysis within the foresight, results of the generation phase of a particular foresight project perf...

  6. Surveillance data bases, analysis, and standardization program

    Energy Technology Data Exchange (ETDEWEB)

    Kam, F.B.K.

    1990-09-26

    The traveler presented a paper at the Seventh ASTM-EURATOM Symposium on Reactor Dosimetry and co-chaired an oral session on Computer Codes and Methods. Papers of considerable interest to the NRC Surveillance Dosimetry Program involved statistically based adjustment procedures and uncertainties. The information exchange meetings with Czechoslovakia and Hungary were very enlightening. Lack of large computers have hindered their surveillance program. They depended very highly on information from their measurement programs which were somewhat limited because of the lack of sophisticated electronics. The Nuclear Research Institute at Rez had to rely on expensive mockups of power reactor configurations to test their fluence exposures. Computers, computer codes, and updated nuclear data would advance their technology rapidly, and they were not hesitant to admit this fact. Both eastern-bloc countries said that IBM is providing an IBM 3090 for educational purposes but research and development studies would have very limited access. They were very apologetic that their currencies were not convertible, and any exchange means that they could provide services or pay for US scientists in their respective countries, but funding for their scientists in the United States, or expenses that involved payment in dollars, must come from us.

  7. Indoor air quality analysis based on Hadoop

    International Nuclear Information System (INIS)

    The air of the office environment is our research object. The data of temperature, humidity, concentrations of carbon dioxide, carbon monoxide and ammonia are collected peer one to eight seconds by the sensor monitoring system. And all the data are stored in the Hbase database of Hadoop platform. With the help of HBase feature of column-oriented store and versioned (automatically add the time column), the time-series data sets are bulit based on the primary key Row-key and timestamp. The parallel computing programming model MapReduce is used to process millions of data collected by sensors. By analysing the changing trend of parameters' value at different time of the same day and at the same time of various dates, the impact of human factor and other factors on the room microenvironment is achieved according to the liquidity of the office staff. Moreover, the effective way to improve indoor air quality is proposed in the end of this paper

  8. Indoor air quality analysis based on Hadoop

    Science.gov (United States)

    Tuo, Wang; Yunhua, Sun; Song, Tian; Liang, Yu; Weihong, Cui

    2014-03-01

    The air of the office environment is our research object. The data of temperature, humidity, concentrations of carbon dioxide, carbon monoxide and ammonia are collected peer one to eight seconds by the sensor monitoring system. And all the data are stored in the Hbase database of Hadoop platform. With the help of HBase feature of column-oriented store and versioned (automatically add the time column), the time-series data sets are bulit based on the primary key Row-key and timestamp. The parallel computing programming model MapReduce is used to process millions of data collected by sensors. By analysing the changing trend of parameters' value at different time of the same day and at the same time of various dates, the impact of human factor and other factors on the room microenvironment is achieved according to the liquidity of the office staff. Moreover, the effective way to improve indoor air quality is proposed in the end of this paper.

  9. A high-resolution tissue-specific proteome and phosphoproteome atlas of maize primary roots reveals functional gradients along the root axes.

    Science.gov (United States)

    Marcon, Caroline; Malik, Waqas Ahmed; Walley, Justin W; Shen, Zhouxin; Paschold, Anja; Smith, Laurie G; Piepho, Hans-Peter; Briggs, Steven P; Hochholdinger, Frank

    2015-05-01

    A high-resolution proteome and phosphoproteome atlas of four maize (Zea mays) primary root tissues, the cortex, stele, meristematic zone, and elongation zone, was generated. High-performance liquid chromatography coupled with tandem mass spectrometry identified 11,552 distinct nonmodified and 2,852 phosphorylated proteins across the four root tissues. Two gradients reflecting the abundance of functional protein classes along the longitudinal root axis were observed. While the classes RNA, DNA, and protein peaked in the meristematic zone, cell wall, lipid metabolism, stress, transport, and secondary metabolism culminated in the differentiation zone. Functional specialization of tissues is underscored by six of 10 cortex-specific proteins involved in flavonoid biosynthesis. Comparison of this data set with high-resolution seed and leaf proteome studies revealed 13% (1,504/11,552) root-specific proteins. While only 23% of the 1,504 root-specific proteins accumulated in all four root tissues, 61% of all 11,552 identified proteins accumulated in all four root tissues. This suggests a much higher degree of tissue-specific functionalization of root-specific proteins. In summary, these data illustrate the remarkable plasticity of the proteomic landscape of maize primary roots and thus provide a starting point for gaining a better understanding of their tissue-specific functions.

  10. Some Linguistic-based and temporal analysis on Wikipedia

    International Nuclear Information System (INIS)

    Wikipedia as a web-based, collaborative, multilingual encyclopaedia project is a very suitable field to carry out research on social dynamics and to investigate the complex concepts of conflict, collaboration, competition, dispute, etc in a large community (∼26 Million) of Wikipedia users. The other face of Wikipedia as a productive society, is its output, consisting of (∼17) Millions of articles written unsupervised by unprofessional editors in more than 270 different languages. In this talk we report some analysis performed on Wikipedia in two different approaches: temporal analysis to characterize disputes and controversies among users and linguistic-based analysis to characterize linguistic features of English texts in Wikipedia. (author)

  11. Preprocessing and Analysis of LC-MS-Based Proteomic Data.

    Science.gov (United States)

    Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W

    2016-01-01

    Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed.

  12. Fatigue analysis of steam generator cassette parts based on CAE

    International Nuclear Information System (INIS)

    Fatigue analysis has been performed for steam generator nozzle header and tube based on CAE. Three dimensional model was produced using the commercial CAD program, IDEAS and the geometry and boundary condition information have been transformed into input format of ABAQUS for thermal analysis, stress analysis, and fatigue analysis. Cassette nozzle, which has a complex geometry, has been analysed by using the three dimensional model. But steam generator tube has been analysed according to ASME procedure since it can be modelled as a two dimensional finite element model. S-N curve for the titanium alloy of the steam generator tube material was obtained from the material tests. From the analysis, it has been confirmed that these parts of the steam generator cassette satisfy the lifetime of the steam generator cassette. Three dimensional modelling strategy from the thermal analysis to fatigue analysis should be implemented into the design of reactor major components to enhance the efficiency of design procedure

  13. Agent-based analysis of organizations : formalization and simulation

    OpenAIRE

    Dignum, M.V.; Tick, C.

    2008-01-01

    Organizational effectiveness depends on many factors, including individual excellence, efficient structures, effective planning and capability to understand and match context requirements. We propose a way to model organizational performance based on a combination of formal models and agent-based simulation that supports the analysis of the congruence of different organizational structures to changing environments

  14. Differential Regulatory Analysis Based on Coexpression Network in Cancer Research.

    Science.gov (United States)

    Li, Junyi; Li, Yi-Xue; Li, Yuan-Yuan

    2016-01-01

    With rapid development of high-throughput techniques and accumulation of big transcriptomic data, plenty of computational methods and algorithms such as differential analysis and network analysis have been proposed to explore genome-wide gene expression characteristics. These efforts are aiming to transform underlying genomic information into valuable knowledges in biological and medical research fields. Recently, tremendous integrative research methods are dedicated to interpret the development and progress of neoplastic diseases, whereas differential regulatory analysis (DRA) based on gene coexpression network (GCN) increasingly plays a robust complement to regular differential expression analysis in revealing regulatory functions of cancer related genes such as evading growth suppressors and resisting cell death. Differential regulatory analysis based on GCN is prospective and shows its essential role in discovering the system properties of carcinogenesis features. Here we briefly review the paradigm of differential regulatory analysis based on GCN. We also focus on the applications of differential regulatory analysis based on GCN in cancer research and point out that DRA is necessary and extraordinary to reveal underlying molecular mechanism in large-scale carcinogenesis studies. PMID:27597964

  15. Differential Regulatory Analysis Based on Coexpression Network in Cancer Research

    Directory of Open Access Journals (Sweden)

    Junyi Li

    2016-01-01

    Full Text Available With rapid development of high-throughput techniques and accumulation of big transcriptomic data, plenty of computational methods and algorithms such as differential analysis and network analysis have been proposed to explore genome-wide gene expression characteristics. These efforts are aiming to transform underlying genomic information into valuable knowledges in biological and medical research fields. Recently, tremendous integrative research methods are dedicated to interpret the development and progress of neoplastic diseases, whereas differential regulatory analysis (DRA based on gene coexpression network (GCN increasingly plays a robust complement to regular differential expression analysis in revealing regulatory functions of cancer related genes such as evading growth suppressors and resisting cell death. Differential regulatory analysis based on GCN is prospective and shows its essential role in discovering the system properties of carcinogenesis features. Here we briefly review the paradigm of differential regulatory analysis based on GCN. We also focus on the applications of differential regulatory analysis based on GCN in cancer research and point out that DRA is necessary and extraordinary to reveal underlying molecular mechanism in large-scale carcinogenesis studies.

  16. Kernel-Based Nonlinear Discriminant Analysis for Face Recognition

    Institute of Scientific and Technical Information of China (English)

    LIU QingShan (刘青山); HUANG Rui (黄锐); LU HanQing (卢汉清); MA SongDe (马颂德)

    2003-01-01

    Linear subspace analysis methods have been successfully applied to extract features for face recognition. But they are inadequate to represent the complex and nonlinear variations of real face images, such as illumination, facial expression and pose variations, because of their linear properties. In this paper, a nonlinear subspace analysis method, Kernel-based Nonlinear Discriminant Analysis (KNDA), is presented for face recognition, which combines the nonlinear kernel trick with the linear subspace analysis method - Fisher Linear Discriminant Analysis (FLDA).First, the kernel trick is used to project the input data into an implicit feature space, then FLDA is performed in this feature space. Thus nonlinear discriminant features of the input data are yielded. In addition, in order to reduce the computational complexity, a geometry-based feature vectors selection scheme is adopted. Another similar nonlinear subspace analysis is Kernel-based Principal Component Analysis (KPCA), which combines the kernel trick with linear Principal Component Analysis (PCA). Experiments are performed with the polynomial kernel, and KNDA is compared with KPCA and FLDA. Extensive experimental results show that KNDA can give a higher recognition rate than KPCA and FLDA.

  17. Earthquake Analysis of Structure by Base Isolation Technique in SAP

    OpenAIRE

    T. Subramani; J. Jothi

    2014-01-01

    This paper presents an overview of the present state of base isolation techniques with special emphasis and a brief on other techniques developed world over for mitigating earthquake forces on the structures. The dynamic analysis procedure for isolated structures is briefly explained. The provisions of FEMA 450 for base isolated structures are highlighted. The effects of base isolation on structures located on soft soils and near active faults are given in brief. Simple case s...

  18. Computer-based modelling and analysis in engineering geology

    OpenAIRE

    Giles, David

    2014-01-01

    This body of work presents the research and publications undertaken under a general theme of computer-based modelling and analysis in engineering geology. Papers are presented on geotechnical data management, data interchange, Geographical Information Systems, surface modelling, geostatistical methods, risk-based modelling, knowledge-based systems, remote sensing in engineering geology and on the integration of computer applications into applied geoscience teaching. The work highlights my...

  19. Adaptive Fourier Decomposition Based Time-Frequency Analysis

    Institute of Scientific and Technical Information of China (English)

    Li-Ming Zhang

    2014-01-01

    The attempt to represent a signal simultaneously in time and frequency domains is full of challenges. The recently proposed adaptive Fourier decomposition (AFD) offers a practical approach to solve this problem. This paper presents the principles of the AFD based time-frequency analysis in three aspects: instantaneous frequency analysis, frequency spectrum analysis, and the spectrogram analysis. An experiment is conducted and compared with the Fourier transform in convergence rate and short-time Fourier transform in time-frequency distribution. The proposed approach performs better than both the Fourier transform and short-time Fourier transform.

  20. Abnormal traffic flow data detection based on wavelet analysis

    Directory of Open Access Journals (Sweden)

    Xiao Qian

    2016-01-01

    Full Text Available In view of the traffic flow data of non-stationary, the abnormal data detection is difficult.proposed basing on the wavelet analysis and least squares method of abnormal traffic flow data detection in this paper.First using wavelet analysis to make the traffic flow data of high frequency and low frequency component and separation, and then, combined with least square method to find abnormal points in the reconstructed signal data.Wavelet analysis and least square method, the simulation results show that using wavelet analysis of abnormal traffic flow data detection, effectively reduce the detection results of misjudgment rate and false negative rate.

  1. Noise analysis for sensitivity-based structural damage detection

    Institute of Scientific and Technical Information of China (English)

    YIN Tao; ZHU Hong-ping; YU Ling

    2007-01-01

    As vibration-based structural damage detection methods are easily affected by environmental noise, a new statistic-based noise analysis method is proposed together with the Monte Carlo technique to investigate the influence of experimental noise of modal data on sensitivity-based damage detection methods. Different from the commonly used random perturbation technique, the proposed technique is deduced directly by Moore-Penrose generalized inverse of the sensitivity matrix, which does not only make the analysis process more efficient but also can analyze the influence of noise on both frequencies and mode shapes for three commonly used sensitivity-based damage detection methods in a similar way. A one-story portal frame is adopted to evaluate the efficiency of the proposed noise analysis technique.

  2. Analysis of security protocols based on challenge-response

    Institute of Scientific and Technical Information of China (English)

    LUO JunZhou; YANG Ming

    2007-01-01

    Security protocol is specified as the procedure of challenge-response, which uses applied cryptography to confirm the existence of other principals and fulfill some data negotiation such as session keys. Most of the existing analysis methods,which either adopt theorem proving techniques such as state exploration or logic reasoning techniques such as authentication logic, face the conflicts between analysis power and operability. To solve the problem, a new efficient method is proposed that provides SSM semantics-based definition of secrecy and authentication goals and applies authentication logic as fundamental analysis techniques,in which secrecy analysis is split into two parts: Explicit-Information-Leakage and Implicit-Information-Leakage, and correspondence analysis is concluded as the analysis of the existence relationship of Strands and the agreement of Strand parameters. This new method owns both the power of the Strand Space Model and concision of authentication logic.

  3. Bayesian-network-based safety risk analysis in construction projects

    International Nuclear Information System (INIS)

    This paper presents a systemic decision support approach for safety risk analysis under uncertainty in tunnel construction. Fuzzy Bayesian Networks (FBN) is used to investigate causal relationships between tunnel-induced damage and its influential variables based upon the risk/hazard mechanism analysis. Aiming to overcome limitations on the current probability estimation, an expert confidence indicator is proposed to ensure the reliability of the surveyed data for fuzzy probability assessment of basic risk factors. A detailed fuzzy-based inference procedure is developed, which has a capacity of implementing deductive reasoning, sensitivity analysis and abductive reasoning. The “3σ criterion” is adopted to calculate the characteristic values of a triangular fuzzy number in the probability fuzzification process, and the α-weighted valuation method is adopted for defuzzification. The construction safety analysis progress is extended to the entire life cycle of risk-prone events, including the pre-accident, during-construction continuous and post-accident control. A typical hazard concerning the tunnel leakage in the construction of Wuhan Yangtze Metro Tunnel in China is presented as a case study, in order to verify the applicability of the proposed approach. The results demonstrate the feasibility of the proposed approach and its application potential. A comparison of advantages and disadvantages between FBN and fuzzy fault tree analysis (FFTA) as risk analysis tools is also conducted. The proposed approach can be used to provide guidelines for safety analysis and management in construction projects, and thus increase the likelihood of a successful project in a complex environment. - Highlights: • A systemic Bayesian network based approach for safety risk analysis is developed. • An expert confidence indicator for probability fuzzification is proposed. • Safety risk analysis progress is extended to entire life cycle of risk-prone events. • A typical

  4. A Framework for Web-Based Mechanical Design and Analysis

    Institute of Scientific and Technical Information of China (English)

    Chiaming; Yen; Wujeng; Li

    2002-01-01

    In this paper, a Web-based Mechanical Design and A na lysis Framework (WMDAF) is proposed. This WMADF allows designers to develop web -based computer aided programs in a systematic way during the collaborative mec hanical system design and analysis process. This system is based on an emerg ing web-based Content Management System (CMS) called eXtended Object Oriented P ortal System (XOOPS). Due to the Open Source Status of the XOOPS CMS, programs d eveloped with this framework can be further customized to ...

  5. Static Analysis for Event-Based XML Processing

    DEFF Research Database (Denmark)

    Møller, Anders

    2008-01-01

    Event-based processing of XML data - as exemplified by the popular SAX framework - is a powerful alternative to using W3C's DOM or similar tree-based APIs. The event-based approach is a streaming fashion with minimal memory consumption. This paper discusses challenges for creating program analyse...... for SAX applications. In particular, we consider the problem of statically guaranteeing the a given SAX program always produces only well-formed and valid XML output. We propose an analysis technique based on ecisting anglyses of Servlets, string operations, and XML graphs....

  6. Tikhonov regularization-based operational transfer path analysis

    Science.gov (United States)

    Cheng, Wei; Lu, Yingying; Zhang, Zhousuo

    2016-06-01

    To overcome ill-posed problems in operational transfer path analysis (OTPA), and improve the stability of solutions, this paper proposes a novel OTPA based on Tikhonov regularization, which considers both fitting degrees and stability of solutions. Firstly, fundamental theory of Tikhonov regularization-based OTPA is presented, and comparative studies are provided to validate the effectiveness on ill-posed problems. Secondly, transfer path analysis and source contribution evaluations for numerical cases studies on spherical radiating acoustical sources are comparatively studied. Finally, transfer path analysis and source contribution evaluations for experimental case studies on a test bed with thin shell structures are provided. This study provides more accurate transfer path analysis for mechanical systems, which can benefit for vibration reduction by structural path optimization. Furthermore, with accurate evaluation of source contributions, vibration monitoring and control by active controlling vibration sources can be effectively carried out.

  7. Matrix-based introduction to multivariate data analysis

    CERN Document Server

    Adachi, Kohei

    2016-01-01

    This book enables readers who may not be familiar with matrices to understand a variety of multivariate analysis procedures in matrix forms. Another feature of the book is that it emphasizes what model underlies a procedure and what objective function is optimized for fitting the model to data. The author believes that the matrix-based learning of such models and objective functions is the fastest way to comprehend multivariate data analysis. The text is arranged so that readers can intuitively capture the purposes for which multivariate analysis procedures are utilized: plain explanations of the purposes with numerical examples precede mathematical descriptions in almost every chapter. This volume is appropriate for undergraduate students who already have studied introductory statistics. Graduate students and researchers who are not familiar with matrix-intensive formulations of multivariate data analysis will also find the book useful, as it is based on modern matrix formulations with a special emphasis on ...

  8. Data Warehouse Requirements Analysis Framework: Business-Object Based Approach

    Directory of Open Access Journals (Sweden)

    Anirban Sarkar

    2012-01-01

    Full Text Available Detailed requirements analysis plays a key role towards the design of successful Data Warehouse (DW system. The requirements analysis specifications are used as the prime input for the construction of conceptual level multidimensional data model. This paper has proposed a Business Object based requirements analysis framework for DW system which is supported with abstraction mechanism and reuse capability. It also facilitate the stepwise mapping of requirements descriptions into high level design components of graph semantic based conceptual level object oriented multidimensional data model. The proposed framework starts with the identification of the analytical requirements using business process driven approach and finally refine the requirements in further detail to map into the conceptual level DW design model using either Demand-driven of Mixed-driven approach for DW requirements analysi

  9. NONLINEAR DATA RECONCILIATION METHOD BASED ON KERNEL PRINCIPAL COMPONENT ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In the industrial process situation, principal component analysis (PCA) is a general method in data reconciliation.However, PCA sometime is unfeasible to nonlinear feature analysis and limited in application to nonlinear industrial process.Kernel PCA (KPCA) is extension of PCA and can be used for nonlinear feature analysis.A nonlinear data reconciliation method based on KPCA is proposed.The basic idea of this method is that firstly original data are mapped to high dimensional feature space by nonlinear function, and PCA is implemented in the feature space.Then nonlinear feature analysis is implemented and data are reconstructed by using the kernel.The data reconciliation method based on KPCA is applied to ternary distillation column.Simulation results show that this method can filter the noise in measurements of nonlinear process and reconciliated data can represent the true information of nonlinear process.

  10. Intelligent Hybrid Cluster Based Classification Algorithm for Social Network Analysis

    Directory of Open Access Journals (Sweden)

    S. Muthurajkumar

    2014-05-01

    Full Text Available In this paper, we propose an hybrid clustering based classification algorithm based on mean approach to effectively classify to mine the ordered sequences (paths from weblog data in order to perform social network analysis. In the system proposed in this work for social pattern analysis, the sequences of human activities are typically analyzed by switching behaviors, which are likely to produce overlapping clusters. In this proposed system, a robust Modified Boosting algorithm is proposed to hybrid clustering based classification for clustering the data. This work is useful to provide connection between the aggregated features from the network data and traditional indices used in social network analysis. Experimental results show that the proposed algorithm improves the decision results from data clustering when combined with the proposed classification algorithm and hence it is proved that of provides better classification accuracy when tested with Weblog dataset. In addition, this algorithm improves the predictive performance especially for multiclass datasets which can increases the accuracy.

  11. UML based risk analysis - Application to a medical robot

    OpenAIRE

    Guiochet, Jérémie; Baron, Claude

    2004-01-01

    Medical robots perform complex tasks and share their working area with humans. Therefore , they belong to safety critical systems. In nowadays development process, safety is often managed by the way of dependability techniques. We propose a new global approach , based on the risk concept in order to guide designers along the safety analysis of such complex systems. Safety depends on risk management activity, which core is risk analysis. This one consists in three steps: system definition, haz...

  12. European Climate - Energy Security Nexus. A model based scenario analysis

    International Nuclear Information System (INIS)

    In this research, we have provided an overview of the climate-security nexus in the European sector through a model based scenario analysis with POLES model. The analysis underline that under stringent climate policies, Europe take advantage of a double dividend in its capacity to develop a new cleaner energy model and in lower vulnerability to potential shocks on the international energy markets. (authors)

  13. Study of engine noise based on independent component analysis

    Institute of Scientific and Technical Information of China (English)

    HAO Zhi-yong; JIN Yan; YANG Chen

    2007-01-01

    Independent component analysis was applied to analyze the acoustic signals from diesel engine. First the basic principle of independent component analysis (ICA) was reviewed. Diesel engine acoustic signal was decomposed into several independent components (Ics); Fourier transform and continuous wavelet transform (CWT) were applied to analyze the independent components. Different noise sources of the diesel engine were separated, based on the characteristics of different component in time-frequency domain.

  14. iBarcode.org: web-based molecular biodiversity analysis

    OpenAIRE

    Hajibabaei Mehrdad; Singer Gregory AC

    2009-01-01

    Abstract Background DNA sequences have become a primary source of information in biodiversity analysis. For example, short standardized species-specific genomic regions, DNA barcodes, are being used as a global standard for species identification and biodiversity studies. Most DNA barcodes are being generated by laboratories that have an expertise in DNA sequencing but not in bioinformatics data analysis. Therefore, we have developed a web-based suite of tools to help the DNA barcode research...

  15. Using the DOE Knowledge Base for Special Event Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, H.M.; Harris, J.M.; Young, C.J.

    1998-10-20

    The DOE Knowledge Base is a library of detailed information whose purpose is to support the United States National Data Center (USNDC) in its mission to monitor compliance with the Comprehensive Test Ban Treaty (CTBT). One of the important tasks which the USNDC must accomplish is to periodically perform detailed analysis of events of high interest, so-called "Special Events", to provide the national authority with information needed to make policy decisions. In this paper we investigate some possible uses of the Knowledge Base for Special Event Analysis (SEA), and make recommendations for improving Knowledge Base support for SEA. To analyze an event in detail, there are two basic types of data which must be used sensor-derived data (wave- forms, arrivals, events, etc.) and regiohalized contextual data (known sources, geological characteristics, etc.). Cur- rently there is no single package which can provide full access to both types of data, so for our study we use a separate package for each MatSeis, the Sandia Labs-developed MATLAB-based seismic analysis package, for wave- form data analysis, and ArcView, an ESRI product, for contextual data analysis. Both packages are well-suited to pro- totyping because they provide a rich set of currently available functionality and yet are also flexible and easily extensible, . Using these tools and Phase I Knowledge Base data sets, we show how the Knowledge Base can improve both the speed and the quality of SEA. Empirically-derived interpolated correction information can be accessed to improve both location estimates and associated error estimates. This information can in turn be used to identi~ any known nearby sources (e.g. mines, volcanos), which may then trigger specialized processing of the sensor data. Based on the location estimate, preferred magnitude formulas and discriminants can be retrieved, and any known blockages can be identified to prevent miscalculations. Relevant historic events can be identilled either by

  16. Segmentation of Stick Text Based on Sub Connected Area Analysis

    Institute of Scientific and Technical Information of China (English)

    高静波; 李新友; 等

    1998-01-01

    A new stick text segmentation method based on the sub connected area analysis is introduced in this paper.The foundation of this method is the sub connected area representation of text image that can represent all connected areas in an image efficiently.This method consists mainly of four steps:sub connected area classification,finding initial boundary following point,finding optimal segmentation point by boundary tracing,and text segmentaton.This method is similar to boundary analysis method but is more efficient than boundary analysis.

  17. RULE-BASED SENTIMENT ANALYSIS OF UKRAINIAN REVIEWS

    Directory of Open Access Journals (Sweden)

    Mariana Romanyshyn

    2013-07-01

    Full Text Available Last decade witnessed a lot of research in the field of sentiment analysis. Understanding the attitude and the emotions that people express in written text proved to be really important and helpful in sociology, political science, psychology, market research, and, of course, artificial intelligence. This paper demonstrates a rule-based approach to clause-level sentiment analysis of reviews in Ukrainian. The general architecture of the implemented sentiment analysis system is presented, the current stage of research is described and further work is explained. The main emphasis is made on the design of rules for computing sentiments.

  18. AN HMM BASED ANALYSIS FRAMEWORK FOR SEMANTIC VIDEO EVENTS

    Institute of Scientific and Technical Information of China (English)

    You Junyong; Liu Guizhong; Zhang Yaxin

    2007-01-01

    Semantic video analysis plays an important role in the field of machine intelligence and pattern recognition. In this paper, based on the Hidden Markov Model (HMM), a semantic recognition framework on compressed videos is proposed to analyze the video events according to six low-level features. After the detailed analysis of video events, the pattern of global motion and five features in foreground--the principal parts of videos, are employed as the observations of the Hidden Markov Model to classify events in videos. The applications of the proposed framework in some video event detections demonstrate the promising success of the proposed framework on semantic video analysis.

  19. Stability analysis of underground engineering based on multidisciplinary design optimization

    Institute of Scientific and Technical Information of China (English)

    MA Rong; ZHOU Ke-ping; GAO Feng

    2008-01-01

    Aiming at characteristics of underground engineering,analyzed the feasibility of Multidisciplinary Design Optimization (MDO) used in underground engineering,and put forward a modularization-based MDO method and the idea of MDO to resolve problems in stability analysis,proving the validity and feasibility of using MDO in underground engineering.Characteristics of uncertainty,complexity and nonlinear become bottle-neck to carry on underground engineering stability analysis by MDO.Therefore,the application of MDO in underground engineering stability analysis is still at a stage of exploration,which need some deep research.

  20. Stability analysis of underground engineering based on multidisciplinary design optimization

    Institute of Scientific and Technical Information of China (English)

    MA Rong; ZHOU Ke-ping; GAO Feng

    2008-01-01

    Aiming at characteristics of underground engineering, analyzed the feasibility of Multidisciplinary Design Optimization (MDO) used in underground engineering, and put forward a modularization-based MDO method and the idea of MDO to resolve problems in stability analysis, proving the validity and feasibility of using MDO in underground engi-neering. Characteristics of uncertainty, complexity and nonlinear become bottle-neck to carry on underground engineering stability analysis by MDO. Therefore, the application of MDO in underground engineering stability analysis is still at a stage of exploration, which need some deep research.

  1. Open access for ALICE analysis based on virtualization technology

    CERN Document Server

    Buncic, P; Schutz, Y

    2015-01-01

    Open access is one of the important leverages for long-term data preservation for a HEP experiment. To guarantee the usability of data analysis tools beyond the experiment lifetime it is crucial that third party users from the scientific community have access to the data and associated software. The ALICE Collaboration has developed a layer of lightweight components built on top of virtualization technology to hide the complexity and details of the experiment-specific software. Users can perform basic analysis tasks within CernVM, a lightweight generic virtual machine, paired with an ALICE specific contextualization. Once the virtual machine is launched, a graphical user interface is automatically started without any additional configuration. This interface allows downloading the base ALICE analysis software and running a set of ALICE analysis modules. Currently the available tools include fully documented tutorials for ALICE analysis, such as the measurement of strange particle production or the nuclear modi...

  2. Nucleonica. Web-based software tools for simulation and analysis

    International Nuclear Information System (INIS)

    The authors present a description of the Nucleonica web-based portal for simulation and analysis for a wide range of commonly encountered nuclear science applications. Advantages of a web-based approach include availability wherever there is internet access, intuitive user-friendly interface, remote access to high-power computing resources, and continual maintenance, improvement, and addition of tools and techniques common to the nuclear science industry. A description of the nuclear data resources, and some applications is given.

  3. Reliability-Based Robustness Analysis for a Croatian Sports Hall

    DEFF Research Database (Denmark)

    Čizmar, Dean; Kirkegaard, Poul Henning; Sørensen, John Dalsgaard;

    2011-01-01

    This paper presents a probabilistic approach for structural robustness assessment for a timber structure built a few years ago. The robustness analysis is based on a structural reliability based framework for robustness and a simplified mechanical system modelling of a timber truss system. A comp...... is expressed and evaluated by a robustness index. Next, the robustness is assessed using system reliability indices where the probabilistic failure model is modelled by a series system of parallel systems....

  4. Customer-Classified Algorithm Based onFuzzy Clustering Analysis

    Institute of Scientific and Technical Information of China (English)

    郭蕴华; 祖巧红; 陈定方

    2004-01-01

    A customer-classified evaluation system is described with the customization-supporting tree of evaluation indexes, in which users can determine any evaluation index independently. Based on this system, a customer-classified algorithm based on fuzzy clustering analysis is proposed to implement the customer-classified management. A numerical example is presented, which provides correct results,indicating that the algorithm can be used in the decision support system of CRM.

  5. Semiparametric theory based MIMO model and performance analysis

    Institute of Scientific and Technical Information of China (English)

    XU Fang-min; XU Xiao-dong; ZHANG Ping

    2007-01-01

    In this article, a new approach for modeling multi- input multi-output (MIMO) systems with unknown nonlinear interference is introduced. The semiparametric theory based MIMO model is established, and Kernel estimation is applied to combat the nonlinear interference. Furthermore, we derive MIMO capacity for these systems and explore the asymptotic properties of the new channel matrix via theoretical analysis. The simulation results show that the semiparametric theory based modeling and kernel estimation are valid to combat this kind of interference.

  6. Nucleonica: Web-based Software Tools for Simulations and Analysis

    OpenAIRE

    Magill, Joseph; DREHER Raymond; SOTI Zsolt; LASCHE George

    2012-01-01

    The authors present a description of a new web-based software portal for simulation and analysis for a wide range of commonly encountered nuclear science applications. Advantages of a web-based approach include availability wherever there is internet access, intuitive user-friendly interface, remote access to high-power computing resources, and continual maintenance, improvement, and addition of tools and techniques common to the nuclear science industry. A description of the nuclear data res...

  7. Lists2Networks: Integrated analysis of gene/protein lists

    Directory of Open Access Journals (Sweden)

    Ma'ayan Avi

    2010-02-01

    Full Text Available Abstract Background Systems biologists are faced with the difficultly of analyzing results from large-scale studies that profile the activity of many genes, RNAs and proteins, applied in different experiments, under different conditions, and reported in different publications. To address this challenge it is desirable to compare the results from different related studies such as mRNA expression microarrays, genome-wide ChIP-X, RNAi screens, proteomics and phosphoproteomics experiments in a coherent global framework. In addition, linking high-content multilayered experimental results with prior biological knowledge can be useful for identifying functional themes and form novel hypotheses. Results We present Lists2Networks, a web-based system that allows users to upload lists of mammalian genes/proteins onto a server-based program for integrated analysis. The system includes web-based tools to manipulate lists with different set operations, to expand lists using existing mammalian networks of protein-protein interactions, co-expression correlation, or background knowledge co-annotation correlation, as well as to apply gene-list enrichment analyses against many gene-list libraries of prior biological knowledge such as pathways, gene ontology terms, kinase-substrate, microRNA-mRAN, and protein-protein interactions, metabolites, and protein domains. Such analyses can be applied to several lists at once against many prior knowledge libraries of gene-lists associated with specific annotations. The system also contains features that allow users to export networks and share lists with other users of the system. Conclusions Lists2Networks is a user friendly web-based software system expected to significantly ease the computational analysis process for experimental systems biologists employing high-throughput experiments at multiple layers of regulation. The system is freely available at http://www.lists2networks.org.

  8. The analysis of Al-based alloys by calorimetry: quantitative analysis of reactions and reaction kinetics

    OpenAIRE

    Starink, M.J.

    2004-01-01

    Differential scanning calorimetry (DSC) and isothermal calorimetry have been applied extensively to the analysis of light metals, especially Al based alloys. Isothermal calorimetry and differential scanning calorimetry are used for analysis of solid state reactions, such as precipitation, homogenisation, devitrivication and recrystallisation; and solid–liquid reactions, such as incipient melting and solidification, are studied by differential scanning calorimetry. In producing repeatable calo...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  11. Modeling and Grid impedance Variation Analysis of Parallel Connected Grid Connected Inverter based on Impedance Based Harmonic Analysis

    DEFF Research Database (Denmark)

    Kwon, JunBum; Wang, Xiongfei; Bak, Claus Leth;

    2014-01-01

    This paper addresses the harmonic compensation error problem existing with parallel connected inverter in the same grid interface conditions by means of impedance-based analysis and modeling. Unlike the single grid connected inverter, it is found that multiple parallel connected inverters and grid...... impedance can make influence to each other if they each have a harmonic compensation function. The analysis method proposed in this paper is based on the relationship between the overall output impedance and input impedance of parallel connected inverter, where controller gain design method, which can...

  12. Situational Analysis: A Framework for Evidence-Based Practice

    Science.gov (United States)

    Annan, Jean

    2005-01-01

    Situational analysis is a framework for professional practice and research in educational psychology. The process is guided by a set of practice principles requiring that psychologists' work is evidence-based, ecological, collaborative and constructive. The framework is designed to provide direction for psychologists who wish to tailor their…

  13. Spinoza II: Conceptual Case-Based Natural Language Analysis.

    Science.gov (United States)

    Schank, Roger C.; And Others

    This paper presents the theoretical changes that have developed in Conceptual Dependency Theory and their ramifications in computer analysis of natural language. The major items of concern are: the elimination of reliance on "grammar rules" for parsing with the emphasis given to conceptual rule based parsing; the development of a conceptual case…

  14. Computer-Based Interaction Analysis with DEGREE Revisited

    Science.gov (United States)

    Barros, B.; Verdejo, M. F.

    2016-01-01

    We review our research with "DEGREE" and analyse how our work has impacted the collaborative learning community since 2000. Our research is framed within the context of computer-based interaction analysis and the development of computer-supported collaborative learning (CSCL) tools. We identify some aspects of our work which have been…

  15. Teaching of Editorial Writing Uses Claims-Based Analysis.

    Science.gov (United States)

    Porter, William C.

    1989-01-01

    Urges the use of claims-based analysis in editorial writing instruction. Explains the use of five hierarchical claim types (factual, definitional, causal, value, and policy) to teach students to analyze and formulate arguments, thus teaching editorial writing by focusing more on the process than on the product. (SR)

  16. Spironolactone use and renal toxicity: population based longitudinal analysis.

    OpenAIRE

    Wei, L; Struthers, A D; Fahey, T; Watson, A D; MacDonald, T. M.

    2010-01-01

    Objective To determine the safety of spironolactone prescribing in the setting of the UK National Health Service. Design Population based longitudinal analysis using a record linkage database. Setting Tayside, Scotland. Population All patients who received one or more dispensed prescriptions for spironolactone between 1994 and 2007. Main outcome measures Rates of prescribing for spironolactone, hospital admissions for hyperkalaemia, and hyperkalaemia and renal function without...

  17. Advancing School-Based Interventions through Economic Analysis

    Science.gov (United States)

    Olsson, Tina M.; Ferrer-Wreder, Laura; Eninger, Lilianne

    2014-01-01

    Commentators interested in school-based prevention programs point to the importance of economic issues for the future of prevention efforts. Many of the processes and aims of prevention science are dependent upon prevention resources. Although economic analysis is an essential tool for assessing resource use, the attention given economic analysis…

  18. Graph- versus Vector-Based Analysis of a Consensus Protocol

    NARCIS (Netherlands)

    Delzanno, Giorgio; Rensink, Arend; Traverso, Riccardo; Bošnački, Dragan; Edelkamp, Stefan; Lluch Lafuente, Alberto; Wijs, Anton

    2014-01-01

    The Paxos distributed consensus algorithm is a challenging case-study for standard, vector-based model checking techniques. Due to asynchronous communication, exhaustive analysis may generate very large state spaces already for small model instances. In this paper, we show the advantages of graph tr

  19. Digital Simulation-Based Training: A Meta-Analysis

    Science.gov (United States)

    Gegenfurtner, Andreas; Quesada-Pallarès, Carla; Knogler, Maximilian

    2014-01-01

    This study examines how design characteristics in digital simulation-based learning environments moderate self-efficacy and transfer of learning. Drawing on social cognitive theory and the cognitive theory of multimedia learning, the meta-analysis psychometrically cumulated k?=?15 studies of 25 years of research with a total sample size of…

  20. Likelihood-Based Confidence Intervals in Exploratory Factor Analysis

    Science.gov (United States)

    Oort, Frans J.

    2011-01-01

    In exploratory or unrestricted factor analysis, all factor loadings are free to be estimated. In oblique solutions, the correlations between common factors are free to be estimated as well. The purpose of this article is to show how likelihood-based confidence intervals can be obtained for rotated factor loadings and factor correlations, by…

  1. Automated analysis of security requirements through risk-based argumentation

    NARCIS (Netherlands)

    Yu, Yijun; Franqueira, Virginia N.L.; Tun, Thein Tan; Wieringa, Roel J.; Nuseibeh, Bashar

    2015-01-01

    Computer-based systems are increasingly being exposed to evolving security threats, which often reveal new vulnerabilities. A formal analysis of the evolving threats is difficult due to a number of practical considerations such as incomplete knowledge about the design, limited information about atta

  2. GIS based analysis of future district heating potential in Denmark

    DEFF Research Database (Denmark)

    Nielsen, Steffen; Möller, Bernd

    2013-01-01

    in Denmark have been mapped in a heat atlas which includes all buildings and their heat demands. This article focuses on developing a method for assessing the costs associated with supplying these buildings with DH. The analysis is based on the existing DH areas in Denmark. By finding the heat production...

  3. A Corpus-based Analysis of English Noun Suffixes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This paper provides a brief analysis of English suffixes. First, make a classification of the English noun suffixes etymologically; then, obtain the frequencies of each English noun suffixes in sub-corpus FR88 and WSJ88, and last draw a conclusion based on the statistics. That is from the word origins we can see its influences on English vocabulary.

  4. Project-Based Language Learning: An Activity Theory Analysis

    Science.gov (United States)

    Gibbes, Marina; Carson, Lorna

    2014-01-01

    This paper reports on an investigation of project-based language learning (PBLL) in a university language programme. Learner reflections of project work were analysed through Activity Theory, where tool-mediated activity is understood as the central unit of analysis for human interaction. Data were categorised according to the components of human…

  5. Management of Microbiologically Influenced Corrosion in Risk Based Inspection analysis

    DEFF Research Database (Denmark)

    Skovhus, Torben Lund; Hillier, Elizabeth; Andersen, Erlend S.

    2016-01-01

    Operating offshore oil and gas production facilities is often associated with high risk. In order to manage the risk, operators commonly use aids to support decision making in the establishment of a maintenance and inspection strategy. Risk Based Inspection (RBI) analysis is widely used in the of...

  6. Frailty phenotypes in the elderly based on cluster analysis

    DEFF Research Database (Denmark)

    Dato, Serena; Montesanto, Alberto; Lagani, Vincenzo;

    2012-01-01

    genetic background on the frailty status is still questioned. We investigated the applicability of a cluster analysis approach based on specific geriatric parameters, previously set up and validated in a southern Italian population, to two large longitudinal Danish samples. In both cohorts, we identified...

  7. Modal Analysis Based on the Random Decrement Technique

    DEFF Research Database (Denmark)

    Asmussen, J. C.; Brincker, Rune

    1998-01-01

    This article describes the work carried out within the project: Modal Analysis Based on the Random Decrement Technique - Application to Civil Engineering Structures. The project is part of the research programme: Dynamics of Structures sponsored by the Danish Technical Research Counsil. The planned...

  8. LES based POD analysis of Jet in Cross Flow

    DEFF Research Database (Denmark)

    Cavar, Dalibor; Meyer, Knud Erik; Jakirlic, S.;

    2010-01-01

    The paper presents results of a POD investigation of the LES based numerical simulation of the jet-in-crossflow (JICF) flowfield. LES results are firstly compared to the pointwise LDA measurements. 2D POD analysis is then used as a comparison basis for PIV measurements and LES, and finally 3D POD...

  9. System of gait analysis based on ground reaction force assessment

    Directory of Open Access Journals (Sweden)

    František Vaverka

    2015-12-01

    Full Text Available Background: Biomechanical analysis of gait employs various methods used in kinematic and kinetic analysis, EMG, and others. One of the most frequently used methods is kinetic analysis based on the assessment of the ground reaction forces (GRF recorded on two force plates. Objective: The aim of the study was to present a method of gait analysis based on the assessment of the GRF recorded during the stance phase of two steps. Methods: The GRF recorded with a force plate on one leg during stance phase has three components acting in directions: Fx - mediolateral, Fy - anteroposterior, and Fz - vertical. A custom-written MATLAB script was used for gait analysis in this study. This software displays instantaneous force data for both legs as Fx(t, Fy(t and Fz(t curves, automatically determines the extremes of functions and sets the visual markers defining the individual points of interest. Positions of these markers can be easily adjusted by the rater, which may be necessary if the GRF has an atypical pattern. The analysis is fully automated and analyzing one trial takes only 1-2 minutes. Results: The method allows quantification of temporal variables of the extremes of the Fx(t, Fy(t, Fz(t functions, durations of the braking and propulsive phase, duration of the double support phase, the magnitudes of reaction forces in extremes of measured functions, impulses of force, and indices of symmetry. The analysis results in a standardized set of 78 variables (temporal, force, indices of symmetry which can serve as a basis for further research and diagnostics. Conclusions: The resulting set of variable offers a wide choice for selecting a specific group of variables with consideration to a particular research topic. The advantage of this method is the standardization of the GRF analysis, low time requirements allowing rapid analysis of a large number of trials in a short time, and comparability of the variables obtained during different research measurements.

  10. Teaching-Learning Activity Modeling Based on Data Analysis

    Directory of Open Access Journals (Sweden)

    Kyungrog Kim

    2015-03-01

    Full Text Available Numerous studies are currently being carried out on personalized services based on data analysis to find and provide valuable information about information overload. Furthermore, the number of studies on data analysis of teaching-learning activities for personalized services in the field of teaching-learning is increasing, too. This paper proposes a learning style recency-frequency-durability (LS-RFD model for quantified analysis on the level of activities of learners, to provide the elements of teaching-learning activities according to the learning style of the learner among various parameters for personalized service. This is to measure preferences as to teaching-learning activity according to recency, frequency and durability of such activities. Based on the results, user characteristics can be classified into groups for teaching-learning activity by categorizing the level of preference and activity of the learner.

  11. Unified HMM-based layout analysis framework and algorithm

    Institute of Scientific and Technical Information of China (English)

    陈明; 丁晓青; 吴佑寿

    2003-01-01

    To manipulate the layout analysis problem for complex or irregular document image, a Unified HMM-based Layout Analysis Framework is presented in this paper. Based on the multi-resolution wavelet analysis results of the document image, we use HMM method in both inner-scale image model and trans-scale context model to classify the pixel region properties, such as text, picture or background. In each scale, a HMM direct segmentation method is used to get better inner-scale classification result. Then another HMM method is used to fuse the inner-scale result in each scale and then get better final seg- mentation result. The optimized algorithm uses a stop rule in the coarse to fine multi-scale segmentation process, so the speed is improved remarkably. Experiments prove the efficiency of proposed algorithm.

  12. Effects of Interventions Based in Behavior Analysis on Motor Skill Acquisition: A Meta-Analysis

    Science.gov (United States)

    Alstot, Andrew E.; Kang, Minsoo; Alstot, Crystal D.

    2013-01-01

    Techniques based in applied behavior analysis (ABA) have been shown to be useful across a variety of settings to improve numerous behaviors. Specifically within physical activity settings, several studies have examined the effect of interventions based in ABA on a variety of motor skills, but the overall effects of these interventions are unknown.…

  13. ODVBA: optimally-discriminative voxel-based analysis.

    Science.gov (United States)

    Zhang, Tianhao; Davatzikos, Christos

    2011-08-01

    Gaussian smoothing of images prior to applying voxel-based statistics is an important step in voxel-based analysis and statistical parametric mapping (VBA-SPM) and is used to account for registration errors, to Gaussianize the data and to integrate imaging signals from a region around each voxel. However, it has also become a limitation of VBA-SPM based methods, since it is often chosen empirically and lacks spatial adaptivity to the shape and spatial extent of the region of interest, such as a region of atrophy or functional activity. In this paper, we propose a new framework, named optimally-discriminative voxel-based analysis (ODVBA), for determining the optimal spatially adaptive smoothing of images, followed by applying voxel-based group analysis. In ODVBA, nonnegative discriminative projection is applied regionally to get the direction that best discriminates between two groups, e.g., patients and controls; this direction is equivalent to local filtering by an optimal kernel whose coefficients define the optimally discriminative direction. By considering all the neighborhoods that contain a given voxel, we then compose this information to produce the statistic for each voxel. Finally, permutation tests are used to obtain a statistical parametric map of group differences. ODVBA has been evaluated using simulated data in which the ground truth is known and with data from an Alzheimer's disease (AD) study. The experimental results have shown that the proposed ODVBA can precisely describe the shape and location of structural abnormality.

  14. Protein expression based multimarker analysis of breast cancer samples

    Directory of Open Access Journals (Sweden)

    Rajasekaran Ayyappan K

    2011-06-01

    Full Text Available Abstract Background Tissue microarray (TMA data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups. Methods We apply weighted correlation network analysis (WGCNA to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%, moderate (22% and high (50% mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-β1, and TGF β receptor II that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis. Results We find that the rule-based grouping variable (referred to as WGCNA* is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data, it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance. We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach. Conclusions We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes.

  15. Content-based analysis and indexing of sports video

    Science.gov (United States)

    Luo, Ming; Bai, Xuesheng; Xu, Guang-you

    2001-12-01

    An explosion of on-line image and video data in digital form is already well underway. With the exponential rise in interactive information exploration and dissemination through the World-Wide Web, the major inhibitors of rapid access to on-line video data are the management of capture and storage, and content-based intelligent search and indexing techniques. This paper proposes an approach for content-based analysis and event-based indexing of sports video. It includes a novel method to organize shots - classifying shots as close shots and far shots, an original idea of blur extent-based event detection, and an innovative local mutation-based algorithm for caption detection and retrieval. Results on extensive real TV programs demonstrate the applicability of our approach.

  16. Electronic Forms-Based Computing for Evidentiary Analysis

    Directory of Open Access Journals (Sweden)

    Andy Luse

    2009-09-01

    Full Text Available The paperwork associated with evidentiary collection and analysis is a highly repetitive and time-consuming process which often involves duplication of work and can frequently result in documentary errors. Electronic entry of evidencerelated information can facilitate greater accuracy and less time spent on data entry. This manuscript describes a general framework for the implementation of an electronic tablet-based system for evidentiary processing. This framework is then utilized in the design and implementation of an electronic tablet-based evidentiary input prototype system developed for use by forensic laboratories which serves as a verification of the proposed framework. The manuscript concludes with a discussion of implications and recommendations for the implementation and use of tablet-based computing for evidence analysis.

  17. Research on supplier evaluation and selection based on fuzzy hierarchy analysis and grey relational analysis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Considering the disadvantages of selecting evaluation index of supplier based on old purchase relation and in view of transformation of relation between manufacture and supplier under the dynamic, cooperative, competitive and quickly response environment, research on supplier selection evaluation was presented based on enterprise capability, cooperation degree and service level from the perspective of cooperative partnership and coordination, and the evaluation index system was established. A more objective and veracious supplier selection and evaluation method based on fuzzy analysis hierarchy process and grey relational analysis was developed, and then empirical research on electric equipment manufacturer was explored to analyze the supplier selection and evaluation.

  18. Physics-Based Simulator for NEO Exploration Analysis & Simulation

    Science.gov (United States)

    Balaram, J.; Cameron, J.; Jain, A.; Kline, H.; Lim, C.; Mazhar, H.; Myint, S.; Nayar, H.; Patton, R.; Pomerantz, M.; Quadrelli, M.; Shakkotai, P.; Tso, K.

    2011-01-01

    As part of the Space Exploration Analysis and Simulation (SEAS) task, the National Aeronautics and Space Administration (NASA) is using physics-based simulations at NASA's Jet Propulsion Laboratory (JPL) to explore potential surface and near-surface mission operations at Near Earth Objects (NEOs). The simulator is under development at JPL and can be used to provide detailed analysis of various surface and near-surface NEO robotic and human exploration concepts. In this paper we describe the SEAS simulator and provide examples of recent mission systems and operations concepts investigated using the simulation. We also present related analysis work and tools developed for both the SEAS task as well as general modeling, analysis and simulation capabilites for asteroid/small-body objects.

  19. THE ENERGY ISSUES: A CORPUS-BASED ANALYSIS

    Directory of Open Access Journals (Sweden)

    Maria-Floriana Popescu

    2015-06-01

    Full Text Available The energy-related issues have become of paramount importance in recent years due to the exhaustion of fossil fuel resources, their price variations and political dependence on nations with role of energy providers. In addition, the changing in climate conditions requires reduction emissions of greenhouse gases. Therefore, this paper proposes and assesses the novel idea of using constructions as a unit of analysis for corpus-based discourse analysis in the energy field. This article will use a standard method in linguistics to quantitatively investigate academic writings from both a lexical and a stylistic perspective. The present research paper aims to add to the knowledge about energy issues by conducting a data-driven analysis of economic academic discourse, through three periods of time and using secondary data analysis. The findings suggest the evolution of the energy sector throughout time and they gave interesting and different insights into the content of the discourses and enabled better comparison of corpora.

  20. Incorporating Semantics into Data Driven Workflows for Content Based Analysis

    Science.gov (United States)

    Argüello, M.; Fernandez-Prieto, M. J.

    Finding meaningful associations between text elements and knowledge structures within clinical narratives in a highly verbal domain, such as psychiatry, is a challenging goal. The research presented here uses a small corpus of case histories and brings into play pre-existing knowledge, and therefore, complements other approaches that use large corpus (millions of words) and no pre-existing knowledge. The paper describes a variety of experiments for content-based analysis: Linguistic Analysis using NLP-oriented approaches, Sentiment Analysis, and Semantically Meaningful Analysis. Although it is not standard practice, the paper advocates providing automatic support to annotate the functionality as well as the data for each experiment by performing semantic annotation that uses OWL and OWL-S. Lessons learnt can be transmitted to legacy clinical databases facing the conversion of clinical narratives according to prominent Electronic Health Records standards.

  1. Emergy Analysis and Sustainability Efficiency Analysis of Different Crop-Based Biodiesel in Life Cycle Perspective

    OpenAIRE

    Jingzheng Ren; Alessandro Manzardo; Anna Mazzi; Andrea Fedele; Antonio Scipioni

    2013-01-01

    Biodiesel as a promising alternative energy resource has been a hot spot in chemical engineering nowadays, but there is also an argument about the sustainability of biodiesel. In order to analyze the sustainability of biodiesel production systems and select the most sustainable scenario, various kinds of crop-based biodiesel including soybean-, rapeseed-, sunflower-, jatropha- and palm-based biodiesel production options are studied by emergy analysis; soybean-based scenario is recognized as t...

  2. Cryptographic protocol security analysis based on bounded constructing algorithm

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    An efficient approach to analyzing cryptographic protocols is to develop automatic analysis tools based on formal methods. However, the approach has encountered the high computational complexity problem due to reasons that participants of protocols are arbitrary, their message structures are complex and their executions are concurrent. We propose an efficient automatic verifying algorithm for analyzing cryptographic protocols based on the Cryptographic Protocol Algebra (CPA) model proposed recently, in which algebraic techniques are used to simplify the description of cryptographic protocols and their executions. Redundant states generated in the analysis processes are much reduced by introducing a new algebraic technique called Universal Polynomial Equation and the algorithm can be used to verify the correctness of protocols in the infinite states space. We have implemented an efficient automatic analysis tool for cryptographic protocols, called ACT-SPA, based on this algorithm, and used the tool to check more than 20 cryptographic protocols. The analysis results show that this tool is more efficient, and an attack instance not offered previously is checked by using this tool.

  3. Analysis of system trustworthiness based on information flow noninterference theory

    Institute of Scientific and Technical Information of China (English)

    Xiangying Kong; Yanhui Chen; Yi Zhuang

    2015-01-01

    The trustworthiness analysis and evaluation are the bases of the trust chain transfer. In this paper the formal method of trustworthiness analysis of a system based on the noninterfer-ence (NI) theory of the information flow is studied. Firstly, existing methods cannot analyze the impact of the system states on the trustworthiness of software during the process of trust chain trans-fer. To solve this problem, the impact of the system state on trust-worthiness of software is investigated, the run-time mutual interfer-ence behavior of software entities is described and an interference model of the access control automaton of a system is established. Secondly, based on the intransitive noninterference (INI) theory, a formal analytic method of trustworthiness for trust chain transfer is proposed, providing a theoretical basis for the analysis of dynamic trustworthiness of software during the trust chain transfer process. Thirdly, a prototype system with dynamic trustworthiness on a plat-form with dual core architecture is constructed and a verification algorithm of the system trustworthiness is provided. Final y, the monitor hypothesis is extended to the dynamic monitor hypothe-sis, a theorem of static judgment rule of system trustworthiness is provided, which is useful to prove dynamic trustworthiness of a system at the beginning of system construction. Compared with previous work in this field, this research proposes not only a formal analytic method for the determination of system trustworthiness, but also a modeling method and an analysis algorithm that are feasible for practical implementation.

  4. ROOT based Offline and Online Analysis (ROAn): An analysis framework for X-ray detector data

    International Nuclear Information System (INIS)

    The ROOT based Offline and Online Analysis (ROAn) framework was developed to perform data analysis on data from Depleted P-channel Field Effect Transistor (DePFET) detectors, a type of active pixel sensors developed at the MPI Halbleiterlabor (HLL). ROAn is highly flexible and extensible, thanks to ROOT's features like run-time type information and reflection. ROAn provides an analysis program which allows to perform configurable step-by-step analysis on arbitrary data, an associated suite of algorithms focused on DePFET data analysis, and a viewer program for displaying and processing online or offline detector data streams. The analysis program encapsulates the applied algorithms in objects called steps which produce analysis results. The dependency between results and thus the order of calculation is resolved automatically by the program. To optimize algorithms for studying detector effects, analysis parameters are often changed. Such changes of input parameters are detected in subsequent analysis runs and only the necessary recalculations are triggered. This saves time and simultaneously keeps the results consistent. The viewer program offers a configurable Graphical User Interface (GUI) and process chain, which allows the user to adapt the program to different tasks such as offline viewing of file data, online monitoring of running detector systems, or performing online data analysis (histogramming, calibration, etc.). Because of its modular design, ROAn can be extended easily, e.g. be adapted to new detector types and analysis processes

  5. ROOT based Offline and Online Analysis (ROAn): An analysis framework for X-ray detector data

    Science.gov (United States)

    Lauf, Thomas; Andritschke, Robert

    2014-10-01

    The ROOT based Offline and Online Analysis (ROAn) framework was developed to perform data analysis on data from Depleted P-channel Field Effect Transistor (DePFET) detectors, a type of active pixel sensors developed at the MPI Halbleiterlabor (HLL). ROAn is highly flexible and extensible, thanks to ROOT's features like run-time type information and reflection. ROAn provides an analysis program which allows to perform configurable step-by-step analysis on arbitrary data, an associated suite of algorithms focused on DePFET data analysis, and a viewer program for displaying and processing online or offline detector data streams. The analysis program encapsulates the applied algorithms in objects called steps which produce analysis results. The dependency between results and thus the order of calculation is resolved automatically by the program. To optimize algorithms for studying detector effects, analysis parameters are often changed. Such changes of input parameters are detected in subsequent analysis runs and only the necessary recalculations are triggered. This saves time and simultaneously keeps the results consistent. The viewer program offers a configurable Graphical User Interface (GUI) and process chain, which allows the user to adapt the program to different tasks such as offline viewing of file data, online monitoring of running detector systems, or performing online data analysis (histogramming, calibration, etc.). Because of its modular design, ROAn can be extended easily, e.g. be adapted to new detector types and analysis processes.

  6. Consistency analysis of accelerated degradation mechanism based on gray theory

    Institute of Scientific and Technical Information of China (English)

    Yunxia Chen; Hongxia Chen; Zhou Yang; Rui Kang; Yi Yang

    2014-01-01

    A fundamental premise of an accelerated testing is that the failure mechanism under elevated and normal stress levels should remain the same. Thus, verification of the consistency of failure mechanisms is essential during an accelerated testing. A new consistency analysis method based on the gray theory is pro-posed for complex products. First of al , existing consistency ana-lysis methods are reviewed with a focus on the comparison of the differences among them. Then, the proposed consistency ana-lysis method is introduced. Two effective gray prediction models, gray dynamic model and new information and equal dimensional (NIED) model, are adapted in the proposed method. The process to determine the dimension of NIED model is also discussed, and a decision rule is expanded. Based on that, the procedure of ap-plying the new consistent analysis method is developed. Final y, a case study of the consistency analysis of a reliability enhancement testing is conducted to demonstrate and validate the proposed method.

  7. Risk-based planning analysis for a single levee

    Science.gov (United States)

    Hui, Rui; Jachens, Elizabeth; Lund, Jay

    2016-04-01

    Traditional risk-based analysis for levee planning focuses primarily on overtopping failure. Although many levees fail before overtopping, few planning studies explicitly include intermediate geotechnical failures in flood risk analysis. This study develops a risk-based model for two simplified levee failure modes: overtopping failure and overall intermediate geotechnical failure from through-seepage, determined by the levee cross section represented by levee height and crown width. Overtopping failure is based only on water level and levee height, while through-seepage failure depends on many geotechnical factors as well, mathematically represented here as a function of levee crown width using levee fragility curves developed from professional judgment or analysis. These levee planning decisions are optimized to minimize the annual expected total cost, which sums expected (residual) annual flood damage and annualized construction costs. Applicability of this optimization approach to planning new levees or upgrading existing levees is demonstrated preliminarily for a levee on a small river protecting agricultural land, and a major levee on a large river protecting a more valuable urban area. Optimized results show higher likelihood of intermediate geotechnical failure than overtopping failure. The effects of uncertainty in levee fragility curves, economic damage potential, construction costs, and hydrology (changing climate) are explored. Optimal levee crown width is more sensitive to these uncertainties than height, while the derived general principles and guidelines for risk-based optimal levee planning remain the same.

  8. Barcode Server: A Visualization-Based Genome Analysis System

    Science.gov (United States)

    Mao, Fenglou; Olman, Victor; Wang, Yan; Xu, Ying

    2013-01-01

    We have previously developed a computational method for representing a genome as a barcode image, which makes various genomic features visually apparent. We have demonstrated that this visual capability has made some challenging genome analysis problems relatively easy to solve. We have applied this capability to a number of challenging problems, including (a) identification of horizontally transferred genes, (b) identification of genomic islands with special properties and (c) binning of metagenomic sequences, and achieved highly encouraging results. These application results inspired us to develop this barcode-based genome analysis server for public service, which supports the following capabilities: (a) calculation of the k-mer based barcode image for a provided DNA sequence; (b) detection of sequence fragments in a given genome with distinct barcodes from those of the majority of the genome, (c) clustering of provided DNA sequences into groups having similar barcodes; and (d) homology-based search using Blast against a genome database for any selected genomic regions deemed to have interesting barcodes. The barcode server provides a job management capability, allowing processing of a large number of analysis jobs for barcode-based comparative genome analyses. The barcode server is accessible at http://csbl1.bmb.uga.edu/Barcode. PMID:23457606

  9. Barcode server: a visualization-based genome analysis system.

    Directory of Open Access Journals (Sweden)

    Fenglou Mao

    Full Text Available We have previously developed a computational method for representing a genome as a barcode image, which makes various genomic features visually apparent. We have demonstrated that this visual capability has made some challenging genome analysis problems relatively easy to solve. We have applied this capability to a number of challenging problems, including (a identification of horizontally transferred genes, (b identification of genomic islands with special properties and (c binning of metagenomic sequences, and achieved highly encouraging results. These application results inspired us to develop this barcode-based genome analysis server for public service, which supports the following capabilities: (a calculation of the k-mer based barcode image for a provided DNA sequence; (b detection of sequence fragments in a given genome with distinct barcodes from those of the majority of the genome, (c clustering of provided DNA sequences into groups having similar barcodes; and (d homology-based search using Blast against a genome database for any selected genomic regions deemed to have interesting barcodes. The barcode server provides a job management capability, allowing processing of a large number of analysis jobs for barcode-based comparative genome analyses. The barcode server is accessible at http://csbl1.bmb.uga.edu/Barcode.

  10. Barcode server: a visualization-based genome analysis system.

    Science.gov (United States)

    Mao, Fenglou; Olman, Victor; Wang, Yan; Xu, Ying

    2013-01-01

    We have previously developed a computational method for representing a genome as a barcode image, which makes various genomic features visually apparent. We have demonstrated that this visual capability has made some challenging genome analysis problems relatively easy to solve. We have applied this capability to a number of challenging problems, including (a) identification of horizontally transferred genes, (b) identification of genomic islands with special properties and (c) binning of metagenomic sequences, and achieved highly encouraging results. These application results inspired us to develop this barcode-based genome analysis server for public service, which supports the following capabilities: (a) calculation of the k-mer based barcode image for a provided DNA sequence; (b) detection of sequence fragments in a given genome with distinct barcodes from those of the majority of the genome, (c) clustering of provided DNA sequences into groups having similar barcodes; and (d) homology-based search using Blast against a genome database for any selected genomic regions deemed to have interesting barcodes. The barcode server provides a job management capability, allowing processing of a large number of analysis jobs for barcode-based comparative genome analyses. The barcode server is accessible at http://csbl1.bmb.uga.edu/Barcode. PMID:23457606

  11. Towards Performance Measurement And Metrics Based Analysis of PLA Applications

    CERN Document Server

    Ahmed, Zeeshan

    2010-01-01

    This article is about a measurement analysis based approach to help software practitioners in managing the additional level complexities and variabilities in software product line applications. The architecture of the proposed approach i.e. ZAC is designed and implemented to perform preprocessesed source code analysis, calculate traditional and product line metrics and visualize results in two and three dimensional diagrams. Experiments using real time data sets are performed which concluded with the results that the ZAC can be very helpful for the software practitioners in understanding the overall structure and complexity of product line applications. Moreover the obtained results prove strong positive correlation between calculated traditional and product line measures.

  12. FARO base case post-test analysis by COMETA code

    Energy Technology Data Exchange (ETDEWEB)

    Annunziato, A.; Addabbo, C. [Joint Research Centre, Ispra (Italy)

    1995-09-01

    The paper analyzes the COMETA (Core Melt Thermal-Hydraulic Analysis) post test calculations of FARO Test L-11, the so-called Base Case Test. The FARO Facility, located at JRC Ispra, is used to simulate the consequences of Severe Accidents in Nuclear Power Plants under a variety of conditions. The COMETA Code has a 6 equations two phase flow field and a 3 phases corium field: the jet, the droplets and the fused-debris bed. The analysis shown that the code is able to pick-up all the major phenomena occurring during the fuel-coolant interaction pre-mixing phase.

  13. APL-based flexibility analysis of manufacturing grid

    Institute of Scientific and Technical Information of China (English)

    LIU Li-lan; SUN Xue-hua; CAI Hong-xia; CHAI Jian-fei

    2009-01-01

    With the characteristics of diversity, randomness, concurrency and decomposability, tasks in manufacturing field are very complicated, and so manufacturing grid(MG)should have considerable flexibility to deal with this problem. With the definition of node and arc, MG structure is converted into a small-world network. Given construction cost constraint, the problem of shortest task waiting time is transformed into the constrained optimization problem, and a corresponding flexibility analysis model based on average path length(APL)is proposed, and the premise of arc-length and node-distance are defined. The results of application example show that the analysis model is effective.

  14. Logistics Enterprise Evaluation Model Based On Fuzzy Clustering Analysis

    Science.gov (United States)

    Fu, Pei-hua; Yin, Hong-bo

    In this thesis, we introduced an evaluation model based on fuzzy cluster algorithm of logistics enterprises. First of all,we present the evaluation index system which contains basic information, management level, technical strength, transport capacity,informatization level, market competition and customer service. We decided the index weight according to the grades, and evaluated integrate ability of the logistics enterprises using fuzzy cluster analysis method. In this thesis, we introduced the system evaluation module and cluster analysis module in detail and described how we achieved these two modules. At last, we gave the result of the system.

  15. Dynamic Chest Image Analysis: Model-Based Perfusion Analysis in Dynamic Pulmonary Imaging

    Science.gov (United States)

    Liang, Jianming; Järvi, Timo; Kiuru, Aaro; Kormano, Martti; Svedström, Erkki

    2003-12-01

    The "Dynamic Chest Image Analysis" project aims to develop model-based computer analysis and visualization methods for showing focal and general abnormalities of lung ventilation and perfusion based on a sequence of digital chest fluoroscopy frames collected with the dynamic pulmonary imaging technique. We have proposed and evaluated a multiresolutional method with an explicit ventilation model for ventilation analysis. This paper presents a new model-based method for pulmonary perfusion analysis. According to perfusion properties, we first devise a novel mathematical function to form a perfusion model. A simple yet accurate approach is further introduced to extract cardiac systolic and diastolic phases from the heart, so that this cardiac information may be utilized to accelerate the perfusion analysis and improve its sensitivity in detecting pulmonary perfusion abnormalities. This makes perfusion analysis not only fast but also robust in computation; consequently, perfusion analysis becomes computationally feasible without using contrast media. Our clinical case studies with 52 patients show that this technique is effective for pulmonary embolism even without using contrast media, demonstrating consistent correlations with computed tomography (CT) and nuclear medicine (NM) studies. This fluoroscopical examination takes only about 2 seconds for perfusion study with only low radiation dose to patient, involving no preparation, no radioactive isotopes, and no contrast media.

  16. Dynamic Chest Image Analysis: Model-Based Perfusion Analysis in Dynamic Pulmonary Imaging

    Directory of Open Access Journals (Sweden)

    Kiuru Aaro

    2003-01-01

    Full Text Available The "Dynamic Chest Image Analysis" project aims to develop model-based computer analysis and visualization methods for showing focal and general abnormalities of lung ventilation and perfusion based on a sequence of digital chest fluoroscopy frames collected with the dynamic pulmonary imaging technique. We have proposed and evaluated a multiresolutional method with an explicit ventilation model for ventilation analysis. This paper presents a new model-based method for pulmonary perfusion analysis. According to perfusion properties, we first devise a novel mathematical function to form a perfusion model. A simple yet accurate approach is further introduced to extract cardiac systolic and diastolic phases from the heart, so that this cardiac information may be utilized to accelerate the perfusion analysis and improve its sensitivity in detecting pulmonary perfusion abnormalities. This makes perfusion analysis not only fast but also robust in computation; consequently, perfusion analysis becomes computationally feasible without using contrast media. Our clinical case studies with 52 patients show that this technique is effective for pulmonary embolism even without using contrast media, demonstrating consistent correlations with computed tomography (CT and nuclear medicine (NM studies. This fluoroscopical examination takes only about 2 seconds for perfusion study with only low radiation dose to patient, involving no preparation, no radioactive isotopes, and no contrast media.

  17. Open access for ALICE analysis based on virtualization technology

    Science.gov (United States)

    Buncic, P.; Gheata, M.; Schutz, Y.

    2015-12-01

    Open access is one of the important leverages for long-term data preservation for a HEP experiment. To guarantee the usability of data analysis tools beyond the experiment lifetime it is crucial that third party users from the scientific community have access to the data and associated software. The ALICE Collaboration has developed a layer of lightweight components built on top of virtualization technology to hide the complexity and details of the experiment-specific software. Users can perform basic analysis tasks within CernVM, a lightweight generic virtual machine, paired with an ALICE specific contextualization. Once the virtual machine is launched, a graphical user interface is automatically started without any additional configuration. This interface allows downloading the base ALICE analysis software and running a set of ALICE analysis modules. Currently the available tools include fully documented tutorials for ALICE analysis, such as the measurement of strange particle production or the nuclear modification factor in Pb-Pb collisions. The interface can be easily extended to include an arbitrary number of additional analysis modules. We present the current status of the tools used by ALICE through the CERN open access portal, and the plans for future extensions of this system.

  18. Job optimization in ATLAS TAG-based distributed analysis

    International Nuclear Information System (INIS)

    The ATLAS experiment is projected to collect over one billion events/year during the first few years of operation. The efficient selection of events for various physics analyses across all appropriate samples presents a significant technical challenge. ATLAS computing infrastructure leverages the Grid to tackle the analysis across large samples by organizing data into a hierarchical structure and exploiting distributed computing to churn through the computations. This includes events at different stages of processing: RAW, ESD (Event Summary Data), AOD (Analysis Object Data), DPD (Derived Physics Data). Event Level Metadata Tags (TAGs) contain information about each event stored using multiple technologies accessible by POOL and various web services. This allows users to apply selection cuts on quantities of interest across the entire sample to compile a subset of events that are appropriate for their analysis. This paper describes new methods for organizing jobs using the TAGs criteria to analyze ATLAS data. It further compares different access patterns to the event data and explores ways to partition the workload for event selection and analysis. Here analysis is defined as a broader set of event processing tasks including event selection and reduction operations ('skimming', 'slimming' and 'thinning') as well as DPD making. Specifically it compares analysis with direct access to the events (AOD and ESD data) to access mediated by different TAG-based event selections. We then compare different ways of splitting the processing to maximize performance.

  19. Transit Traffic Analysis Zone Delineating Method Based on Thiessen Polygon

    Directory of Open Access Journals (Sweden)

    Shuwei Wang

    2014-04-01

    Full Text Available A green transportation system composed of transit, busses and bicycles could be a significant in alleviating traffic congestion. However, the inaccuracy of current transit ridership forecasting methods is imposing a negative impact on the development of urban transit systems. Traffic Analysis Zone (TAZ delineating is a fundamental and essential step in ridership forecasting, existing delineating method in four-step models have some problems in reflecting the travel characteristics of urban transit. This paper aims to come up with a Transit Traffic Analysis Zone delineation method as supplement of traditional TAZs in transit service analysis. The deficiencies of current TAZ delineating methods were analyzed, and the requirements of Transit Traffic Analysis Zone (TTAZ were summarized. Considering these requirements, Thiessen Polygon was introduced into TTAZ delineating. In order to validate its feasibility, Beijing was then taken as an example to delineate TTAZs, followed by a spatial analysis of office buildings within a TTAZ and transit station departure passengers. Analysis result shows that the TTAZs based on Thiessen polygon could reflect the transit travel characteristic and is of in-depth research value.

  20. Model based process-product design and analysis

    DEFF Research Database (Denmark)

    Gani, Rafiqul

    This paper gives a perspective on modelling and the important role it has within product-process design and analysis. Different modelling issues related to development and application of systematic model-based solution approaches for product-process design is discussed and the need for a hybrid...... model-based framework is highlighted. This framework should be able to manage knowledge-data, models, and associated methods and tools integrated with design work-flows and data-flows for specific product-process design problems. In particular, the framework needs to manage models of different types...

  1. Coverage analysis for sensor networks based on Clifford algebra

    Institute of Scientific and Technical Information of China (English)

    XIE WeiXin; CAO WenMing; MENG Shan

    2008-01-01

    The coverage performance is the foundation of information acquisition in distrib-uted sensor networks. The previously proposed coverage work was mostly based on unit disk coverage model or ball coverage model in 2D or 3D space, respectively. However, most methods cannot give a homogeneous coverage model for targets with hybrid types. This paper presents a coverage analysis approach for sensor networks based on Clifford algebra and establishes a homogeneous coverage model for sensor networks with hybrid types of targets. The effectiveness of the approach is demonstrated with examples.

  2. Image Analysis of Fabric Pilling Based on Light Projection

    Institute of Scientific and Technical Information of China (English)

    陈霞; 黄秀宝

    2003-01-01

    The objective assessment of fabric pilling based on light projection and image analysis has been exploited recently.The device for capturing the cross-sectional images of the pilled fabrics with light projection is elaborated.The detection of the profile line and integration of the sequential cross-sectional pilled image are discussed.The threshold based on Gaussian model is recommended for pill segmentation.The results show that the installed system is capable of eliminating the interference with pill information from the fabric color and pattern.

  3. Image registration based on matrix perturbation analysis using spectral graph

    Institute of Scientific and Technical Information of China (English)

    Chengcai Leng; Zheng Tian; Jing Li; Mingtao Ding

    2009-01-01

    @@ We present a novel perspective on characterizing the spectral correspondence between nodes of the weighted graph with application to image registration.It is based on matrix perturbation analysis on the spectral graph.The contribution may be divided into three parts.Firstly, the perturbation matrix is obtained by perturbing the matrix of graph model.Secondly, an orthogonal matrix is obtained based on an optimal parameter, which can better capture correspondence features.Thirdly, the optimal matching matrix is proposed by adjusting signs of orthogonal matrix for image registration.Experiments on both synthetic images and real-world images demonstrate the effectiveness and accuracy of the proposed method.

  4. Plug-in Based Analysis Framework for LHC Post-Mortem Analysis

    CERN Document Server

    Gorbonosov, R; Zerlauth, M; Baggiolini, V

    2014-01-01

    Plug-in based software architectures [1] are extensible, enforce modularity and allow several teams to work in parallel. But they have certain technical and organizational challenges, which we discuss in this paper. We gained our experience when developing the Post-Mortem Analysis (PMA) system, which is a mission critical system for the Large Hadron Collider (LHC). We used a plugin-based architecture with a general-purpose analysis engine, for which physicists and equipment experts code plugins containing the analysis algorithms. We have over 45 analysis plugins developed by a dozen of domain experts. This paper focuses on the design challenges we faced in order to mitigate the risks of executing third-party code: assurance that even a badly written plugin doesn't perturb the work of the overall application; plugin execution control which allows to detect plugin misbehaviour and react; robust communication mechanism between plugins, diagnostics facilitation in case of plugin failure; testing of the plugins be...

  5. Physics-based stability analysis of MOS transistors

    Science.gov (United States)

    Ferrara, A.; Steeneken, P. G.; Boksteen, B. K.; Heringa, A.; Scholten, A. J.; Schmitz, J.; Hueting, R. J. E.

    2015-11-01

    In this work, a physics-based model is derived based on a linearization procedure for investigating the electrical, thermal and electro-thermal instability of power metal-oxide-semiconductor (MOS) transistors. The proposed model can be easily interfaced with a circuit or device simulator to perform a failure analysis, making it particularly useful for power transistors. Furthermore, it allows mapping the failure points on a three-dimensional (3D) space defined by the gate-width normalized drain current, drain voltage and junction temperature. This leads to the definition of the Safe Operating Volume (SOV), a powerful frame work for making failure predictions and determining the main root of instability (electrical, thermal or electro-thermal) in different bias and operating conditions. A comparison between the modeled and the measured SOV of silicon-on-insulator (SOI) LDMOS transistors is reported to support the validity of the proposed stability analysis.

  6. Image-Analysis Based on Seed Phenomics in Sesame

    Directory of Open Access Journals (Sweden)

    Prasad R.

    2014-10-01

    Full Text Available The seed coat (testa structure of twenty-three cultivated (Sesamum indicum L. and six wild sesame (s. occidentale Regel & Heer., S. mulayanum Nair, S. prostratum Retz., S. radiatum Schumach. & Thonn., S. angustifolium (Oliv. Engl. and S. schinzianum Asch germplasm was analyzed from digital and Scanning Electron Microscopy (SEM images with dedicated software using the descriptors for computer based seed image analysis to understand the diversity of seed morphometric traits, which later on can be extended to screen and evaluate improved genotypes of sesame. Seeds of wild sesame species could conveniently be distinguished from cultivated varieties based on shape and architectural analysis. Results indicated discrete ‘cut off values to identify definite shape and contour of seed for a desirable sesame genotype along with the con-ventional practice of selecting lighter colored testa.

  7. GNSS Spoofing Detection Based on Signal Power Measurements: Statistical Analysis

    Directory of Open Access Journals (Sweden)

    V. Dehghanian

    2012-01-01

    Full Text Available A threat to GNSS receivers is posed by a spoofing transmitter that emulates authentic signals but with randomized code phase and Doppler values over a small range. Such spoofing signals can result in large navigational solution errors that are passed onto the unsuspecting user with potentially dire consequences. An effective spoofing detection technique is developed in this paper, based on signal power measurements and that can be readily applied to present consumer grade GNSS receivers with minimal firmware changes. An extensive statistical analysis is carried out based on formulating a multihypothesis detection problem. Expressions are developed to devise a set of thresholds required for signal detection and identification. The detection processing methods developed are further manipulated to exploit incidental antenna motion arising from user interaction with a GNSS handheld receiver to further enhance the detection performance of the proposed algorithm. The statistical analysis supports the effectiveness of the proposed spoofing detection technique under various multipath conditions.

  8. Estimating Driving Performance Based on EEG Spectrum Analysis

    Directory of Open Access Journals (Sweden)

    Jung Tzyy-Ping

    2005-01-01

    Full Text Available The growing number of traffic accidents in recent years has become a serious concern to society. Accidents caused by driver's drowsiness behind the steering wheel have a high fatality rate because of the marked decline in the driver's abilities of perception, recognition, and vehicle control abilities while sleepy. Preventing such accidents caused by drowsiness is highly desirable but requires techniques for continuously detecting, estimating, and predicting the level of alertness of drivers and delivering effective feedbacks to maintain their maximum performance. This paper proposes an EEG-based drowsiness estimation system that combines electroencephalogram (EEG log subband power spectrum, correlation analysis, principal component analysis, and linear regression models to indirectly estimate driver's drowsiness level in a virtual-reality-based driving simulator. Our results demonstrated that it is feasible to accurately estimate quantitatively driving performance, expressed as deviation between the center of the vehicle and the center of the cruising lane, in a realistic driving simulator.

  9. Empirical Analysis of Signature-Based Sign Language Recognition

    Directory of Open Access Journals (Sweden)

    Sumaira Kausar

    2014-10-01

    Full Text Available The significance of automated SLR (Sign Language Recognition proved not only in the deaf community but in various other spheres of life. The automated SLR are mainly based on the machine learning methods.PSL (Pakistani Sign Languageis an emerging area in order to benefit a big community in this region of the world. This paper presents recognition of PSL using machine learning methods. We propose an efficient and invariant method of classification of signs of PSL. This paper also presents a thorough empirical analysis of signature-based classification methods. Six different signatures are analyzed for two distinct groups of signs having total of forty five signs. Signs of PSL are close enough in terms of inter-sign similarity distancetherefore, it is a challenge to make the classification. Methodical empirical analysis proves that proposed method deals with these challenges adequately and effectively

  10. CORBA-Based Analysis of Multi Agent Behavior

    Institute of Scientific and Technical Information of China (English)

    Swapan Bhattacharya; Anirban Banerjee; Shibdas Bandyopadhyay

    2005-01-01

    An agent is a computer software that is capable of taking independent action on behalf of its user or owner. It is an entity with goals, actions and domain knowledge, situated in an environment. Multiagent systems comprises of multiple autonomous, interacting computer software, or agents. These systems can successfully emulate the entities active in a distributed environment. The analysis of multiagent behavior has been studied in this paper based on a specific board game problem similar to the famous problem of GO. In this paper a framework is developed to define the states of the multiagent entities and measure the convergence metrics for this problem. An analysis of the changes of states leading to the goal state is also made. We support our study of multiagent behavior by simulations based on a CORBA framework in order to substantiate our findings.

  11. Protein analysis based on molecular beacon probes and biofunctionalized nanoparticles

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    With the completion of the human genome-sequencing project, there has been a resulting change in the focus of studies from genomics to proteomics. By utilizing the inherent advantages of molecular beacon probes and biofunctionalized nanoparticles, a series of novel principles, methods and techniques have been exploited for bioanalytical and biomedical studies. This review mainly discusses the applications of molecular beacon probes and biofunctionalized nanoparticles-based technologies for realtime, in-situ, highly sensitive and highly selective protein analysis, including the nonspecific or specific protein detection and separation, protein/DNA interaction studies, cell surface protein recognition, and antigen-antibody binding process-based bacteria assays. The introduction of molecular beacon probes and biofunctionalized nanoparticles into the protein analysis area would necessarily advance the proteomics research.

  12. Gold Nanoparticles-Based Barcode Analysis for Detection of Norepinephrine.

    Science.gov (United States)

    An, Jeung Hee; Lee, Kwon-Jai; Choi, Jeong-Woo

    2016-02-01

    Nanotechnology-based bio-barcode amplification analysis offers an innovative approach for detecting neurotransmitters. We evaluated the efficacy of this method for detecting norepinephrine in normal and oxidative-stress damaged dopaminergic cells. Our approach use a combination of DNA barcodes and bead-based immunoassays for detecting neurotransmitters with surface-enhanced Raman spectroscopy (SERS), and provides polymerase chain reaction (PCR)-like sensitivity. This method relies on magnetic Dynabeads containing antibodies and nanoparticles that are loaded both with DNA barcords and with antibodies that can sandwich the target protein captured by the Dynabead-bound antibodies. The aggregate sandwich structures are magnetically separated from the solution and treated to remove the conjugated barcode DNA. The DNA barcodes are then identified by SERS and PCR analysis. The concentration of norepinephrine in dopaminergic cells can be readily detected using the bio-barcode assay, which is a rapid, high-throughput screening tool for detecting neurotransmitters. PMID:27305769

  13. Classification analysis of microarray data based on ontological engineering

    Institute of Scientific and Technical Information of China (English)

    LI Guo-qi; SHENG Huan-ye

    2007-01-01

    Background knowledge is important for data mining, especially in complicated situation. Ontological engineering is the successor of knowledge engineering. The sharable knowledge bases built on ontology can be used to provide background knowledge to direct the process of data mining. This paper gives a common introduction to the method and presents a practical analysis example using SVM (support vector machine) as the classifier. Gene Ontology and the accompanying annotations compose a big knowledge base, on which many researches have been carried out. Microarray dataset is the output of DNA chip.With the help of Gene Ontology we present a more elaborate analysis on microarray data than former researchers. The method can also be used in other fields with similar scenario.

  14. Student Engagement: A Principle-Based Concept Analysis.

    Science.gov (United States)

    Bernard, Jean S

    2015-08-04

    A principle-based concept analysis of student engagement was used to examine the state of the science across disciplines. Four major perspectives of philosophy of science guided analysis and provided a framework for study of interrelationships and integration of conceptual components which then resulted in formulation of a theoretical definition. Findings revealed student engagement as a dynamic reiterative process marked by positive behavioral, cognitive, and affective elements exhibited in pursuit of deep learning. This process is influenced by a broader sociocultural environment bound by contextual preconditions of self-investment, motivation, and a valuing of learning. Outcomes of student engagement include satisfaction, sense of well-being, and personal development. Findings of this analysis prove relevant to nursing education as faculty transition from traditional teaching paradigms, incorporate learner-centered strategies, and adopt innovative pedagogical methodologies. It lends support for curricula reform, development of more accurate evaluative measures, and creation of meaningful teaching-learning environments within the discipline.

  15. Error Analysis of English Writing Based on Interlanguage Theory

    Institute of Scientific and Technical Information of China (English)

    李玲

    2014-01-01

    Language learning process has been hunted by learner’s errors,which is an unavoidable phenomenon.In the 1950s and 1960s,Contractive Analysis (CA) based on behaviorism and structuralism was generally employed in analyzing learners’ errors. CA soon lost its popularity.Error Analysis (EA),a branch of applied linguistics,has made great contributions to the study of second language learning and throws some light on the process of second language learning.Careful study of the errors reveals the common problems shared by the language learners.Writing is important in language learning process.Under Chinese context,English writing is always a difficult question for Chinese teachers and students,so errors in students’ written works are unavoidable.In this thesis,the author studies on error analysis of English writing with the interlanguage theory as its theoretical guidance.

  16. Error Analysis of English Writing Based on Interlanguage Theory

    Institute of Scientific and Technical Information of China (English)

    李玲

    2014-01-01

    Language learning process has been hunted by learner’s errors,which is an unavoidable phenomenon.In the 1950 s and 1960 s,Contractive Analysis(CA) based on behaviorism and structuralism was generally employed in analyzing learners’ errors.CA soon lost its popularity.Error Analysis(EA),a branch of applied linguistics,has made great contributions to the study of second language learning and throws some light on the process of second language learning.Careful study of the errors reveals the common problems shared by the language learners.Writing is important in language learning process.Under Chinese context,English writing is always a difficult question for Chinese teachers and students,so errors in students’ written works are unavoidable.In this thesis,the author studies on error analysis of English writing with the interlanguage theory as its theoretical guidance.

  17. Facial Affect Recognition Using Regularized Discriminant Analysis-Based Algorithms

    Directory of Open Access Journals (Sweden)

    Cheng-Yuan Shih

    2010-01-01

    Full Text Available This paper presents a novel and effective method for facial expression recognition including happiness, disgust, fear, anger, sadness, surprise, and neutral state. The proposed method utilizes a regularized discriminant analysis-based boosting algorithm (RDAB with effective Gabor features to recognize the facial expressions. Entropy criterion is applied to select the effective Gabor feature which is a subset of informative and nonredundant Gabor features. The proposed RDAB algorithm uses RDA as a learner in the boosting algorithm. The RDA combines strengths of linear discriminant analysis (LDA and quadratic discriminant analysis (QDA. It solves the small sample size and ill-posed problems suffered from QDA and LDA through a regularization technique. Additionally, this study uses the particle swarm optimization (PSO algorithm to estimate optimal parameters in RDA. Experiment results demonstrate that our approach can accurately and robustly recognize facial expressions.

  18. GOMA: functional enrichment analysis tool based on GO modules

    Institute of Scientific and Technical Information of China (English)

    Qiang Huang; Ling-Yun Wu; Yong Wang; Xiang-Sun Zhang

    2013-01-01

    Analyzing the function of gene sets is a critical step in interpreting the results of high-throughput experiments in systems biology.A variety of enrichment analysis tools have been developed in recent years,but most output a long list of significantly enriched terms that are often redundant,making it difficult to extract the most meaningful functions.In this paper,we present GOMA,a novel enrichment analysis method based on the new concept of enriched functional Gene Ontology (GO) modules.With this method,we systematically revealed functional GO modules,i.e.,groups of functionally similar GO terms,via an optimization model and then ranked them by enrichment scores.Our new method simplifies enrichment analysis results by reducing redundancy,thereby preventing inconsistent enrichment results among functionally similar terms and providing more biologically meaningful results.

  19. Error Analysis of Robotic Assembly System Based on Screw Theory

    Institute of Scientific and Technical Information of China (English)

    韩卫军; 费燕琼; 赵锡芳

    2003-01-01

    Assembly errors have great influence on assembly quality in robotic assembly systems. Error analysis is directed to the propagations and accumula-tions of various errors and their effect on assembly success.Using the screw coordinates, assembly errors are represented as "error twist", the extremely compact expression. According to the law of screw composition, relative position and orientation errors of mating parts are computed and the necessary condition of assembly success is concluded. A new simple method for measuring assembly errors is also proposed based on the transformation law of a screw.Because of the compact representation of error, the model presented for error analysis can be applied to various part- mating types and especially useful for error analysis of complexity assembly.

  20. Student Engagement: A Principle-Based Concept Analysis.

    Science.gov (United States)

    Bernard, Jean S

    2015-01-01

    A principle-based concept analysis of student engagement was used to examine the state of the science across disciplines. Four major perspectives of philosophy of science guided analysis and provided a framework for study of interrelationships and integration of conceptual components which then resulted in formulation of a theoretical definition. Findings revealed student engagement as a dynamic reiterative process marked by positive behavioral, cognitive, and affective elements exhibited in pursuit of deep learning. This process is influenced by a broader sociocultural environment bound by contextual preconditions of self-investment, motivation, and a valuing of learning. Outcomes of student engagement include satisfaction, sense of well-being, and personal development. Findings of this analysis prove relevant to nursing education as faculty transition from traditional teaching paradigms, incorporate learner-centered strategies, and adopt innovative pedagogical methodologies. It lends support for curricula reform, development of more accurate evaluative measures, and creation of meaningful teaching-learning environments within the discipline. PMID:26234950