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Sample records for quantitative phosphoproteome analysis

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

    PubMed

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

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

  2. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    SciTech Connect

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

    2012-11-11

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-11

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

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

    PubMed

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

    2017-03-20

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

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

    PubMed

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

    2015-05-01

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

  11. SILAC for global phosphoproteomic analysis.

    PubMed

    Pimienta, Genaro; Chaerkady, Raghothama; Pandey, Akhilesh

    2009-01-01

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

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

    PubMed

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

    2017-02-07

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

  13. Insights Regarding Fungal Phosphoproteomic Analysis.

    PubMed

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

    2017-03-10

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    SciTech Connect

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

    2008-10-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-12-02

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-04

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2011-05-15

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

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

    PubMed

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

    2015-09-04

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2016-03-17

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2017-02-21

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2017-02-10

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

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

    PubMed

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

    2015-08-07

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Phosphoproteomics in Cancer

    PubMed Central

    Harsha, H. C.; Pandey, Akhilesh

    2010-01-01

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

  1. Phosphoproteomic analysis of Her2/neu signaling and inhibition

    PubMed Central

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

    2006-01-01

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

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

    SciTech Connect

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

    2012-04-17

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

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

    PubMed

    Guo, Mingquan; Huang, Bill X

    2013-02-01

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

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

    PubMed

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

    2016-02-02

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2014-02-01

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

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

    PubMed

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

    2015-06-05

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2011-03-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-05

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-08-25

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

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

    PubMed

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

    2014-10-03

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2016-02-09

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

  5. Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

    PubMed Central

    Rinschen, Markus M.; Wu, Xiongwu; König, Tim; Pisitkun, Trairak; Hagmann, Henning; Pahmeyer, Caroline; Lamkemeyer, Tobias; Kohli, Priyanka; Schnell, Nicole; Schermer, Bernhard; Dryer, Stuart; Brooks, Bernard R.; Beltrao, Pedro; Krueger, Marcus

    2014-01-01

    Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry–based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains. PMID:24511133

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

    PubMed

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

    2016-02-05

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. Phosphoproteomics in cereals.

    PubMed

    Yang, Pingfang

    2015-01-01

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

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

    PubMed

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

    2014-12-05

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

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

    PubMed

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

    2010-12-21

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-04-13

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

  15. Phosphoproteomic profiling of the myocyte.

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    2012-06-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-04-04

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

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

    PubMed

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

    2012-07-17

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

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2013-05-01

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

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

    PubMed Central

    Domanski, Dominik; Helbing, Caren C

    2007-01-01

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

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

    PubMed

    Cesaro, Luca; Salvi, Mauro

    2010-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2017-03-01

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

  20. Databases for plant phosphoproteomics.

    PubMed

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

    2015-01-01

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

  1. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

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

  2. Multivariate Quantitative Chemical Analysis

    NASA Technical Reports Server (NTRS)

    Kinchen, David G.; Capezza, Mary

    1995-01-01

    Technique of multivariate quantitative chemical analysis devised for use in determining relative proportions of two components mixed and sprayed together onto object to form thermally insulating foam. Potentially adaptable to other materials, especially in process-monitoring applications in which necessary to know and control critical properties of products via quantitative chemical analyses of products. In addition to chemical composition, also used to determine such physical properties as densities and strengths.

  3. Quantitative Hydrocarbon Surface Analysis

    NASA Technical Reports Server (NTRS)

    Douglas, Vonnie M.

    2000-01-01

    The elimination of ozone depleting substances, such as carbon tetrachloride, has resulted in the use of new analytical techniques for cleanliness verification and contamination sampling. The last remaining application at Rocketdyne which required a replacement technique was the quantitative analysis of hydrocarbons by infrared spectrometry. This application, which previously utilized carbon tetrachloride, was successfully modified using the SOC-400, a compact portable FTIR manufactured by Surface Optics Corporation. This instrument can quantitatively measure and identify hydrocarbons from solvent flush of hardware as well as directly analyze the surface of metallic components without the use of ozone depleting chemicals. Several sampling accessories are utilized to perform analysis for various applications.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-10-22

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

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

    PubMed Central

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

    2012-01-01

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

  7. Quantitative environmental risk analysis

    SciTech Connect

    Klovning, J.; Nilsen, E.F.

    1995-12-31

    According to regulations relating to implementation and rise of risk analysis in the petroleum activities issued by the Norwegian Petroleum Directorate, it is mandatory for an operator on the Norwegian Continental Shelf to establish acceptance criteria for environmental risk in the activities and carry out environmental risk analysis. This paper presents a {open_quotes}new{close_quotes} method for environmental risk analysis developed by the company. The objective has been to assist the company to meet rules and regulations and to assess and describe the environmental risk in a systematic manner. In the environmental risk analysis the most sensitive biological resource in the affected area is used to assess the environmental damage. The analytical method is based on the methodology for quantitative risk analysis related to loss of life. In addition it incorporates the effect of seasonal fluctuations in the environmental risk evaluations. The paper is describing the function of the main analytical sequences exemplified through an analysis of environmental risk related to exploration drilling in an environmental sensitive area on the Norwegian Continental Shelf.

  8. Characterization of the Phosphoproteome in SLE Patients

    PubMed Central

    Huang, Jianrong; Dai, Yong

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-01-01

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

  11. Quantitative Techniques in Volumetric Analysis

    NASA Astrophysics Data System (ADS)

    Zimmerman, John; Jacobsen, Jerrold J.

    1996-12-01

    Quantitative Techniques in Volumetric Analysis is a visual library of techniques used in making volumetric measurements. This 40-minute VHS videotape is designed as a resource for introducing students to proper volumetric methods and procedures. The entire tape, or relevant segments of the tape, can also be used to review procedures used in subsequent experiments that rely on the traditional art of quantitative analysis laboratory practice. The techniques included are: Quantitative transfer of a solid with a weighing spoon Quantitative transfer of a solid with a finger held weighing bottle Quantitative transfer of a solid with a paper strap held bottle Quantitative transfer of a solid with a spatula Examples of common quantitative weighing errors Quantitative transfer of a solid from dish to beaker to volumetric flask Quantitative transfer of a solid from dish to volumetric flask Volumetric transfer pipet A complete acid-base titration Hand technique variations The conventional view of contemporary quantitative chemical measurement tends to focus on instrumental systems, computers, and robotics. In this view, the analyst is relegated to placing standards and samples on a tray. A robotic arm delivers a sample to the analysis center, while a computer controls the analysis conditions and records the results. In spite of this, it is rare to find an analysis process that does not rely on some aspect of more traditional quantitative analysis techniques, such as careful dilution to the mark of a volumetric flask. Figure 2. Transfer of a solid with a spatula. Clearly, errors in a classical step will affect the quality of the final analysis. Because of this, it is still important for students to master the key elements of the traditional art of quantitative chemical analysis laboratory practice. Some aspects of chemical analysis, like careful rinsing to insure quantitative transfer, are often an automated part of an instrumental process that must be understood by the

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. Quantitative analysis of PET studies.

    PubMed

    Weber, Wolfgang A

    2010-09-01

    Quantitative analysis can be included relatively easily in clinical PET-imaging protocols, but in order to obtain meaningful quantitative results one needs to follow a standardized protocol for image acquisition and data analysis. Important factors to consider are the calibration of the PET scanner, the radiotracer uptake time and the approach for definition of regions of interests. Using such standardized acquisition protocols quantitative parameters of tumor metabolism or receptor status can be derived from tracer kinetic analysis and simplified approaches such as calculation of standardized uptake values (SUVs).

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

    PubMed Central

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

    2016-01-01

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

  17. Quantitative analysis in megageomorphology

    NASA Technical Reports Server (NTRS)

    Mayer, L.

    1985-01-01

    Megageomorphology is the study of regional topographic features and their relations to independent geomorphic variables that operate at the regional scale. These independent variables can be classified as either tectonic or climatic in nature. Quantitative megageomorphology stresses the causal relations between plate tectonic factors and landscape features or correlations between climatic factors and geomorphic processes. In addition, the cumulative effects of tectonics and climate on landscape evolution that simultaneously operate in a complex system of energy transfer is of interst. Regional topographic differentiation, say between continents and ocean floors, is largely the result of the different densities and density contrasts within the oceanic and continental lithosphere and their isostatic consequences. Regional tectonic processes that alter these lithospheric characteristics include rifting, collision, subduction, transpression and transtension.

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. Functional phosphoproteomic mass spectrometry-based approaches

    PubMed Central

    2012-01-01

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

  1. Software for quantitative trait analysis.

    PubMed

    Almasy, Laura; Warren, Diane M

    2005-09-01

    This paper provides a brief overview of software currently available for the genetic analysis of quantitative traits in humans. Programs that implement variance components, Markov Chain Monte Carlo (MCMC), Haseman-Elston (H-E) and penetrance model-based linkage analyses are discussed, as are programs for measured genotype association analyses and quantitative trait transmission disequilibrium tests. The software compared includes LINKAGE, FASTLINK, PAP, SOLAR, SEGPATH, ACT, Mx, MERLIN, GENEHUNTER, Loki, Mendel, SAGE, QTDT and FBAT. Where possible, the paper provides URLs for acquiring these programs through the internet, details of the platforms for which the software is available and the types of analyses performed.

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

    PubMed Central

    2015-01-01

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

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

    PubMed Central

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Phosphoproteomics for the masses

    PubMed Central

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

    2010-01-01

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

  6. Quantitative analysis of glycated proteins.

    PubMed

    Priego-Capote, Feliciano; Ramírez-Boo, María; Finamore, Francesco; Gluck, Florent; Sanchez, Jean-Charles

    2014-02-07

    The proposed protocol presents a comprehensive approach for large-scale qualitative and quantitative analysis of glycated proteins (GP) in complex biological samples including biological fluids and cell lysates such as plasma and red blood cells. The method, named glycation isotopic labeling (GIL), is based on the differential labeling of proteins with isotopic [(13)C6]-glucose, which supports quantitation of the resulting glycated peptides after enzymatic digestion with endoproteinase Glu-C. The key principle of the GIL approach is the detection of doublet signals for each glycated peptide in MS precursor scanning (glycated peptide with in vivo [(12)C6]- and in vitro [(13)C6]-glucose). The mass shift of the doublet signals is +6, +3 or +2 Da depending on the peptide charge state and the number of glycation sites. The intensity ratio between doublet signals generates quantitative information of glycated proteins that can be related to the glycemic state of the studied samples. Tandem mass spectrometry with high-energy collisional dissociation (HCD-MS2) and data-dependent methods with collision-induced dissociation (CID-MS3 neutral loss scan) are used for qualitative analysis.

  7. Bioimaging for quantitative phenotype analysis.

    PubMed

    Chen, Weiyang; Xia, Xian; Huang, Yi; Chen, Xingwei; Han, Jing-Dong J

    2016-06-01

    With the development of bio-imaging techniques, an increasing number of studies apply these techniques to generate a myriad of image data. Its applications range from quantification of cellular, tissue, organismal and behavioral phenotypes of model organisms, to human facial phenotypes. The bio-imaging approaches to automatically detect, quantify, and profile phenotypic changes related to specific biological questions open new doors to studying phenotype-genotype associations and to precisely evaluating molecular changes associated with quantitative phenotypes. Here, we review major applications of bioimage-based quantitative phenotype analysis. Specifically, we describe the biological questions and experimental needs addressable by these analyses, computational techniques and tools that are available in these contexts, and the new perspectives on phenotype-genotype association uncovered by such analyses.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  10. Phosphoproteome Discovery in Human Biological Fluids

    PubMed Central

    Giorgianni, Francesco; Beranova-Giorgianni, Sarka

    2016-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-04-01

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

  13. Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells.

    PubMed

    Popova, Taissia G; Turell, Michael J; Espina, Virginia; Kehn-Hall, Kylene; Kidd, Jessica; Narayanan, Aarthi; Liotta, Lance; Petricoin, Emanuel F; Kashanchi, Fatah; Bailey, Charles; Popov, Serguei G

    2010-11-03

    Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK) and downstream transcriptional factors [STAT1 (Y701), ATF2 (T69/71), MSK1 (S360) and CREB (S133)]. NF-κB phosphorylation was also increased. Activation of p53 (S15, S46) correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473), along with phosphorylation of FOX 01/03 (T24/31) which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication.

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

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

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

  15. Quantitative analysis of retinal OCT.

    PubMed

    Sonka, Milan; Abràmoff, Michael D

    2016-10-01

    Clinical acceptance of 3-D OCT retinal imaging brought rapid development of quantitative 3-D analysis of retinal layers, vasculature, retinal lesions as well as facilitated new research in retinal diseases. One of the cornerstones of many such analyses is segmentation and thickness quantification of retinal layers and the choroid, with an inherently 3-D simultaneous multi-layer LOGISMOS (Layered Optimal Graph Image Segmentation for Multiple Objects and Surfaces) segmentation approach being extremely well suited for the task. Once retinal layers are segmented, regional thickness, brightness, or texture-based indices of individual layers can be easily determined and thus contribute to our understanding of retinal or optic nerve head (ONH) disease processes and can be employed for determination of disease status, treatment responses, visual function, etc. Out of many applications, examples provided in this paper focus on image-guided therapy and outcome prediction in age-related macular degeneration and on assessing visual function from retinal layer structure in glaucoma.

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

    PubMed

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

    2008-11-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2017-03-21

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

  20. Quantitative analysis of endogenous compounds.

    PubMed

    Thakare, Rhishikesh; Chhonker, Yashpal S; Gautam, Nagsen; Alamoudi, Jawaher Abdullah; Alnouti, Yazen

    2016-09-05

    Accurate quantitative analysis of endogenous analytes is essential for several clinical and non-clinical applications. LC-MS/MS is the technique of choice for quantitative analyses. Absolute quantification by LC/MS requires preparing standard curves in the same matrix as the study samples so that the matrix effect and the extraction efficiency for analytes are the same in both the standard and study samples. However, by definition, analyte-free biological matrices do not exist for endogenous compounds. To address the lack of blank matrices for the quantification of endogenous compounds by LC-MS/MS, four approaches are used including the standard addition, the background subtraction, the surrogate matrix, and the surrogate analyte methods. This review article presents an overview these approaches, cite and summarize their applications, and compare their advantages and disadvantages. In addition, we discuss in details, validation requirements and compatibility with FDA guidelines to ensure method reliability in quantifying endogenous compounds. The standard addition, background subtraction, and the surrogate analyte approaches allow the use of the same matrix for the calibration curve as the one to be analyzed in the test samples. However, in the surrogate matrix approach, various matrices such as artificial, stripped, and neat matrices are used as surrogate matrices for the actual matrix of study samples. For the surrogate analyte approach, it is required to demonstrate similarity in matrix effect and recovery between surrogate and authentic endogenous analytes. Similarly, for the surrogate matrix approach, it is required to demonstrate similar matrix effect and extraction recovery in both the surrogate and original matrices. All these methods represent indirect approaches to quantify endogenous compounds and regardless of what approach is followed, it has to be shown that none of the validation criteria have been compromised due to the indirect analyses.

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

    PubMed

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

    2017-03-06

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

  2. Versatile nanocomposites in phosphoproteomics: a review.

    PubMed

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

    2012-10-17

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

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

    PubMed

    Salih, Erdjan

    2005-01-01

    The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass

  4. Identifying differentially regulated subnetworks from phosphoproteomic data

    PubMed Central

    2010-01-01

    Background Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. Results Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. Conclusions SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data. PMID:20584295

  5. Quantitative analysis of glycoprotein glycans.

    PubMed

    Orlando, Ron

    2013-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-10-01

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

  8. Quantitative analysis of qualitative images

    NASA Astrophysics Data System (ADS)

    Hockney, David; Falco, Charles M.

    2005-03-01

    We show optical evidence that demonstrates artists as early as Jan van Eyck and Robert Campin (c1425) used optical projections as aids for producing their paintings. We also have found optical evidence within works by later artists, including Bermejo (c1475), Lotto (c1525), Caravaggio (c1600), de la Tour (c1650), Chardin (c1750) and Ingres (c1825), demonstrating a continuum in the use of optical projections by artists, along with an evolution in the sophistication of that use. However, even for paintings where we have been able to extract unambiguous, quantitative evidence of the direct use of optical projections for producing certain of the features, this does not mean that paintings are effectively photographs. Because the hand and mind of the artist are intimately involved in the creation process, understanding these complex images requires more than can be obtained from only applying the equations of geometrical optics.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Quantitative histogram analysis of images

    NASA Astrophysics Data System (ADS)

    Holub, Oliver; Ferreira, Sérgio T.

    2006-11-01

    A routine for histogram analysis of images has been written in the object-oriented, graphical development environment LabVIEW. The program converts an RGB bitmap image into an intensity-linear greyscale image according to selectable conversion coefficients. This greyscale image is subsequently analysed by plots of the intensity histogram and probability distribution of brightness, and by calculation of various parameters, including average brightness, standard deviation, variance, minimal and maximal brightness, mode, skewness and kurtosis of the histogram and the median of the probability distribution. The program allows interactive selection of specific regions of interest (ROI) in the image and definition of lower and upper threshold levels (e.g., to permit the removal of a constant background signal). The results of the analysis of multiple images can be conveniently saved and exported for plotting in other programs, which allows fast analysis of relatively large sets of image data. The program file accompanies this manuscript together with a detailed description of two application examples: The analysis of fluorescence microscopy images, specifically of tau-immunofluorescence in primary cultures of rat cortical and hippocampal neurons, and the quantification of protein bands by Western-blot. The possibilities and limitations of this kind of analysis are discussed. Program summaryTitle of program: HAWGC Catalogue identifier: ADXG_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADXG_v1_0 Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Computers: Mobile Intel Pentium III, AMD Duron Installations: No installation necessary—Executable file together with necessary files for LabVIEW Run-time engine Operating systems or monitors under which the program has been tested: WindowsME/2000/XP Programming language used: LabVIEW 7.0 Memory required to execute with typical data:˜16MB for starting and ˜160MB used for

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

    PubMed Central

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

    2016-01-01

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

  13. Mobile app-based quantitative scanometric analysis.

    PubMed

    Wong, Jessica X H; Liu, Frank S F; Yu, Hua-Zhong

    2014-12-16

    The feasibility of using smartphones and other mobile devices as the detection platform for quantitative scanometric assays is demonstrated. The different scanning modes (color, grayscale, black/white) and grayscale converting protocols (average, weighted average/luminosity, and software specific) have been compared in determining the optical darkness ratio (ODR) values, a conventional quantitation measure for scanometric assays. A mobile app was developed to image and analyze scanometric assays, as demonstrated by paper-printed tests and a biotin-streptavidin assay on a plastic substrate. Primarily for ODR analysis, the app has been shown to perform as well as a traditional desktop scanner, augmenting that smartphones (and other mobile devices) promise to be a practical platform for accurate, quantitative chemical analysis and medical diagnostics.

  14. Quantitative WDS analysis using electron probe microanalyzer

    SciTech Connect

    Ul-Hamid, Anwar . E-mail: anwar@kfupm.edu.sa; Tawancy, Hani M.; Mohammed, Abdul-Rashid I.; Al-Jaroudi, Said S.; Abbas, Nureddin M.

    2006-04-15

    In this paper, the procedure for conducting quantitative elemental analysis by ZAF correction method using wavelength dispersive X-ray spectroscopy (WDS) in an electron probe microanalyzer (EPMA) is elaborated. Analysis of a thermal barrier coating (TBC) system formed on a Ni-based single crystal superalloy is presented as an example to illustrate the analysis of samples consisting of a large number of major and minor elements. The analysis was performed by known standards and measured peak-to-background intensity ratios. The procedure for using separate set of acquisition conditions for major and minor element analysis is explained and its importance is stressed.

  15. Seniors' Online Communities: A Quantitative Content Analysis

    ERIC Educational Resources Information Center

    Nimrod, Galit

    2010-01-01

    Purpose: To examine the contents and characteristics of seniors' online communities and to explore their potential benefits to older adults. Design and Methods: Quantitative content analysis of a full year's data from 14 leading online communities using a novel computerized system. The overall database included 686,283 messages. Results: There was…

  16. A quantitative approach to scar analysis.

    PubMed

    Khorasani, Hooman; Zheng, Zhong; Nguyen, Calvin; Zara, Janette; Zhang, Xinli; Wang, Joyce; Ting, Kang; Soo, Chia

    2011-02-01

    Analysis of collagen architecture is essential to wound healing research. However, to date no consistent methodologies exist for quantitatively assessing dermal collagen architecture in scars. In this study, we developed a standardized approach for quantitative analysis of scar collagen morphology by confocal microscopy using fractal dimension and lacunarity analysis. Full-thickness wounds were created on adult mice, closed by primary intention, and harvested at 14 days after wounding for morphometrics and standard Fourier transform-based scar analysis as well as fractal dimension and lacunarity analysis. In addition, transmission electron microscopy was used to evaluate collagen ultrastructure. We demonstrated that fractal dimension and lacunarity analysis were superior to Fourier transform analysis in discriminating scar versus unwounded tissue in a wild-type mouse model. To fully test the robustness of this scar analysis approach, a fibromodulin-null mouse model that heals with increased scar was also used. Fractal dimension and lacunarity analysis effectively discriminated unwounded fibromodulin-null versus wild-type skin as well as healing fibromodulin-null versus wild-type wounds, whereas Fourier transform analysis failed to do so. Furthermore, fractal dimension and lacunarity data also correlated well with transmission electron microscopy collagen ultrastructure analysis, adding to their validity. These results demonstrate that fractal dimension and lacunarity are more sensitive than Fourier transform analysis for quantification of scar morphology.

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

    PubMed

    Hudson, Claire A; López Bernal, Andrés

    2017-01-22

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

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

    PubMed

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

    2016-06-01

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

  19. Quantitative ADF STEM: acquisition, analysis and interpretation

    NASA Astrophysics Data System (ADS)

    Jones, L.

    2016-01-01

    Quantitative annular dark-field in the scanning transmission electron microscope (ADF STEM), where image intensities are used to provide composition and thickness measurements, has enjoyed a renaissance during the last decade. Now in a post aberration-correction era many aspects of the technique are being revisited. Here the recent progress and emerging best-practice for such aberration corrected quantitative ADF STEM is discussed including issues relating to proper acquisition of experimental data and its calibration, approaches for data analysis, the utility of such data, its interpretation and limitations.

  20. Method and apparatus for chromatographic quantitative analysis

    DOEpatents

    Fritz, James S.; Gjerde, Douglas T.; Schmuckler, Gabriella

    1981-06-09

    An improved apparatus and method for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single eluent and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

  1. Quantitative analysis of blood vessel geometry

    NASA Astrophysics Data System (ADS)

    Fuhrman, Michael G.; Abdul-Karim, Othman; Shah, Sujal; Gilbert, Steven G.; Van Bibber, Richard

    2001-07-01

    Re-narrowing or restenosis of a human coronary artery occurs within six months in one third of balloon angioplasty procedures. Accurate and repeatable quantitative analysis of vessel shape is important to characterize the progression and type of restenosis, and to evaluate effects new therapies might have. A combination of complicated geometry and image variability, and the need for high resolution and large image size makes visual/manual analysis slow, difficult, and prone to error. The image processing and analysis described here was developed to automate feature extraction of the lumen, internal elastic lamina, neointima, external elastic lamina, and tunica adventitia and to enable an objective, quantitative definition of blood vessel geometry. The quantitative geometrical analysis enables the measurement of several features including perimeter, area, and other metrics of vessel damage. Automation of feature extraction creates a high throughput capability that enables analysis of serial sections for more accurate measurement of restenosis dimensions. Measurement results are input into a relational database where they can be statistically analyzed compared across studies. As part of the integrated process, results are also imprinted on the images themselves to facilitate auditing of the results. The analysis is fast, repeatable and accurate while allowing the pathologist to control the measurement process.

  2. Comprehensive quantitative analysis on privacy leak behavior.

    PubMed

    Fan, Lejun; Wang, Yuanzhuo; Jin, Xiaolong; Li, Jingyuan; Cheng, Xueqi; Jin, Shuyuan

    2013-01-01

    Privacy information is prone to be leaked by illegal software providers with various motivations. Privacy leak behavior has thus become an important research issue of cyber security. However, existing approaches can only qualitatively analyze privacy leak behavior of software applications. No quantitative approach, to the best of our knowledge, has been developed in the open literature. To fill this gap, in this paper we propose for the first time four quantitative metrics, namely, possibility, severity, crypticity, and manipulability, for privacy leak behavior analysis based on Privacy Petri Net (PPN). In order to compare the privacy leak behavior among different software, we further propose a comprehensive metric, namely, overall leak degree, based on these four metrics. Finally, we validate the effectiveness of the proposed approach using real-world software applications. The experimental results demonstrate that our approach can quantitatively analyze the privacy leak behaviors of various software types and reveal their characteristics from different aspects.

  3. Comprehensive Quantitative Analysis on Privacy Leak Behavior

    PubMed Central

    Fan, Lejun; Wang, Yuanzhuo; Jin, Xiaolong; Li, Jingyuan; Cheng, Xueqi; Jin, Shuyuan

    2013-01-01

    Privacy information is prone to be leaked by illegal software providers with various motivations. Privacy leak behavior has thus become an important research issue of cyber security. However, existing approaches can only qualitatively analyze privacy leak behavior of software applications. No quantitative approach, to the best of our knowledge, has been developed in the open literature. To fill this gap, in this paper we propose for the first time four quantitative metrics, namely, possibility, severity, crypticity, and manipulability, for privacy leak behavior analysis based on Privacy Petri Net (PPN). In order to compare the privacy leak behavior among different software, we further propose a comprehensive metric, namely, overall leak degree, based on these four metrics. Finally, we validate the effectiveness of the proposed approach using real-world software applications. The experimental results demonstrate that our approach can quantitatively analyze the privacy leak behaviors of various software types and reveal their characteristics from different aspects. PMID:24066046

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

    PubMed Central

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

    2015-01-01

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

  5. Good practices for quantitative bias analysis.

    PubMed

    Lash, Timothy L; Fox, Matthew P; MacLehose, Richard F; Maldonado, George; McCandless, Lawrence C; Greenland, Sander

    2014-12-01

    Quantitative bias analysis serves several objectives in epidemiological research. First, it provides a quantitative estimate of the direction, magnitude and uncertainty arising from systematic errors. Second, the acts of identifying sources of systematic error, writing down models to quantify them, assigning values to the bias parameters and interpreting the results combat the human tendency towards overconfidence in research results, syntheses and critiques and the inferences that rest upon them. Finally, by suggesting aspects that dominate uncertainty in a particular research result or topic area, bias analysis can guide efficient allocation of sparse research resources. The fundamental methods of bias analyses have been known for decades, and there have been calls for more widespread use for nearly as long. There was a time when some believed that bias analyses were rarely undertaken because the methods were not widely known and because automated computing tools were not readily available to implement the methods. These shortcomings have been largely resolved. We must, therefore, contemplate other barriers to implementation. One possibility is that practitioners avoid the analyses because they lack confidence in the practice of bias analysis. The purpose of this paper is therefore to describe what we view as good practices for applying quantitative bias analysis to epidemiological data, directed towards those familiar with the methods. We focus on answering questions often posed to those of us who advocate incorporation of bias analysis methods into teaching and research. These include the following. When is bias analysis practical and productive? How does one select the biases that ought to be addressed? How does one select a method to model biases? How does one assign values to the parameters of a bias model? How does one present and interpret a bias analysis?. We hope that our guide to good practices for conducting and presenting bias analyses will encourage

  6. Phosphoproteomics technologies and applications in plant biology research

    PubMed Central

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

    2015-01-01

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

  7. Quantitative image analysis of celiac disease.

    PubMed

    Ciaccio, Edward J; Bhagat, Govind; Lewis, Suzanne K; Green, Peter H

    2015-03-07

    We outline the use of quantitative techniques that are currently used for analysis of celiac disease. Image processing techniques can be useful to statistically analyze the pixular data of endoscopic images that is acquired with standard or videocapsule endoscopy. It is shown how current techniques have evolved to become more useful for gastroenterologists who seek to understand celiac disease and to screen for it in suspected patients. New directions for focus in the development of methodology for diagnosis and treatment of this disease are suggested. It is evident that there are yet broad areas where there is potential to expand the use of quantitative techniques for improved analysis in suspected or known celiac disease patients.

  8. Quantitative analysis to guide orphan drug development.

    PubMed

    Lesko, L J

    2012-08-01

    The development of orphan drugs for rare diseases has made impressive strides in the past 10 years. There has been a surge in orphan drug designations, but new drug approvals have not kept up. This article presents a three-pronged hierarchical strategy for quantitative analysis of data at the descriptive, mechanistic, and systems levels of the biological system that could represent a standardized and rational approach to orphan drug development. Examples are provided to illustrate the concept.

  9. Using Qualitative Hazard Analysis to Guide Quantitative Safety Analysis

    NASA Technical Reports Server (NTRS)

    Shortle, J. F.; Allocco, M.

    2005-01-01

    Quantitative methods can be beneficial in many types of safety investigations. However, there are many difficulties in using quantitative m ethods. Far example, there may be little relevant data available. This paper proposes a framework for using quantitative hazard analysis to prioritize hazard scenarios most suitable for quantitative mziysis. The framework first categorizes hazard scenarios by severity and likelihood. We then propose another metric "modeling difficulty" that desc ribes the complexity in modeling a given hazard scenario quantitatively. The combined metrics of severity, likelihood, and modeling difficu lty help to prioritize hazard scenarios for which quantitative analys is should be applied. We have applied this methodology to proposed concepts of operations for reduced wake separation for airplane operatio ns at closely spaced parallel runways.

  10. Influence analysis in quantitative trait loci detection

    PubMed Central

    Dou, Xiaoling; Kuriki, Satoshi; Maeno, Akiteru; Takada, Toyoyuki; Shiroishi, Toshihiko

    2014-01-01

    This paper presents systematic methods for the detection of influential individuals that affect the log odds (LOD) score curve. We derive general formulas of influence functions for profile likelihoods and introduce them into two standard quantitative trait locus detection methods—the interval mapping method and single marker analysis. Besides influence analysis on specific LOD scores, we also develop influence analysis methods on the shape of the LOD score curves. A simulation-based method is proposed to assess the significance of the influence of the individuals. These methods are shown useful in the influence analysis of a real dataset of an experimental population from an F2 mouse cross. By receiver operating characteristic analysis, we confirm that the proposed methods show better performance than existing diagnostics. PMID:24740424

  11. Quantitative resilience analysis through control design.

    SciTech Connect

    Sunderland, Daniel; Vugrin, Eric D.; Camphouse, Russell Chris

    2009-09-01

    Critical infrastructure resilience has become a national priority for the U. S. Department of Homeland Security. System resilience has been studied for several decades in many different disciplines, but no standards or unifying methods exist for critical infrastructure resilience analysis. Few quantitative resilience methods exist, and those existing approaches tend to be rather simplistic and, hence, not capable of sufficiently assessing all aspects of critical infrastructure resilience. This report documents the results of a late-start Laboratory Directed Research and Development (LDRD) project that investigated the development of quantitative resilience through application of control design methods. Specifically, we conducted a survey of infrastructure models to assess what types of control design might be applicable for critical infrastructure resilience assessment. As a result of this survey, we developed a decision process that directs the resilience analyst to the control method that is most likely applicable to the system under consideration. Furthermore, we developed optimal control strategies for two sets of representative infrastructure systems to demonstrate how control methods could be used to assess the resilience of the systems to catastrophic disruptions. We present recommendations for future work to continue the development of quantitative resilience analysis methods.

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

    PubMed

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

    2016-06-01

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

  13. Quantitative analysis of non-Hodgkin's lymphoma.

    PubMed Central

    Abbott, C R; Blewitt, R W; Bird, C C

    1982-01-01

    A preliminary attempt has been made to characterise a small series of non-Hodgkin's lymphomas (NHL) by morphometric means using the Quantimet 720 Kontron MOP/AMO3 image analysis systems. In most cases it was found that the distribution of nuclear area and correlation between mean nuclear area and frequency per unit field, corresponded closely with tumour classification determined by light microscopy. These results suggest that it may be possible to devise an objective and reproducible grading system for NHL using quantitative morphometric techniques. PMID:7040479

  14. Quantitative NIR Raman analysis in liquid mixtures.

    PubMed

    Sato-Berrú, R Ysacc; Medina-Valtierra, Jorge; Medina-Gutiérrez, Cirilo; Frausto-Reyes, Claudio

    2004-08-01

    The capability to obtain quantitative information of a simple way from Raman spectra is a subject of considerable interest. In this work, this is demonstrated for mixtures of ethanol with water and rhodamine-6G (R-6G) with methanol, which were analyzed directly in glass vessel. The Raman intensities and a simple mathematical model have been used and applied for the analysis of liquid samples. It is starting point to generate a general expression, from the experimental spectra, as the sum of the particular expression for each pure compound allow us to obtain an expression for the mixtures which can be used for determining concentrations, from the Raman spectrum, of the mixture.

  15. Cross-Species PTM Mapping from Phosphoproteomic Data.

    PubMed

    Chaudhuri, Rima; Yang, Jean Yee Hwa

    2017-01-01

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

  16. Quantitative analysis of retinal changes in hypertension

    NASA Astrophysics Data System (ADS)

    Giansanti, Roberto; Boemi, Massimo; Fumelli, Paolo; Passerini, Giorgio; Zingaretti, Primo

    1995-05-01

    Arterial hypertension is a high prevalence disease in Western countries and it is associated with increased risk for cardiovascular accidents. Retinal vessel changes are common findings in patients suffering from long-standing hypertensive disease. Morphological evaluations of the fundus oculi represent a fundamental tool for the clinical approach to the patient with hypertension. A qualitative analysis of the retinal lesions is usually performed and this implies severe limitations both in the classification of the different degrees of the pathology and in the follow-up of the disease. A diagnostic system based on a quantitative analysis of the retinal changes could overcome these problems. Our computerized approach was intended for this scope. The paper concentrates on the results and the implications of a computerized approach to the automatic extraction of numerical indexes describing morphological details of the fundus oculi. A previously developed image processing and recognition system, documented elsewhere and briefly described here, was successfully tested in pre-clinical experiments and applied in the evaluation of normal as well as of pathological fundus. The software system was developed to extract indexes such as caliber and path of vessels, local tortuosity of arteries and arterioles, positions and angles of crossings between two vessels. The reliability of the results, justified by their low variability, makes feasible the standardization of quantitative parameters to be used both in the diagnosis and in the prognosis of hypertension, and also allows prospective studies based upon them.

  17. Quantitative interactome analysis reveals a chemoresistant edgotype

    PubMed Central

    Chavez, Juan D.; Schweppe, Devin K.; Eng, Jimmy K.; Zheng, Chunxiang; Taipale, Alex; Zhang, Yiyi; Takara, Kohji; Bruce, James E.

    2015-01-01

    Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for ‘edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype. PMID:26235782

  18. Large-Scale Proteomics and Phosphoproteomics of Urinary Exosomes

    PubMed Central

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

    2009-01-01

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

  19. Automated quantitative image analysis of nanoparticle assembly

    NASA Astrophysics Data System (ADS)

    Murthy, Chaitanya R.; Gao, Bo; Tao, Andrea R.; Arya, Gaurav

    2015-05-01

    The ability to characterize higher-order structures formed by nanoparticle (NP) assembly is critical for predicting and engineering the properties of advanced nanocomposite materials. Here we develop a quantitative image analysis software to characterize key structural properties of NP clusters from experimental images of nanocomposites. This analysis can be carried out on images captured at intermittent times during assembly to monitor the time evolution of NP clusters in a highly automated manner. The software outputs averages and distributions in the size, radius of gyration, fractal dimension, backbone length, end-to-end distance, anisotropic ratio, and aspect ratio of NP clusters as a function of time along with bootstrapped error bounds for all calculated properties. The polydispersity in the NP building blocks and biases in the sampling of NP clusters are accounted for through the use of probabilistic weights. This software, named Particle Image Characterization Tool (PICT), has been made publicly available and could be an invaluable resource for researchers studying NP assembly. To demonstrate its practical utility, we used PICT to analyze scanning electron microscopy images taken during the assembly of surface-functionalized metal NPs of differing shapes and sizes within a polymer matrix. PICT is used to characterize and analyze the morphology of NP clusters, providing quantitative information that can be used to elucidate the physical mechanisms governing NP assembly.The ability to characterize higher-order structures formed by nanoparticle (NP) assembly is critical for predicting and engineering the properties of advanced nanocomposite materials. Here we develop a quantitative image analysis software to characterize key structural properties of NP clusters from experimental images of nanocomposites. This analysis can be carried out on images captured at intermittent times during assembly to monitor the time evolution of NP clusters in a highly automated

  20. Materials characterization through quantitative digital image analysis

    SciTech Connect

    J. Philliber; B. Antoun; B. Somerday; N. Yang

    2000-07-01

    A digital image analysis system has been developed to allow advanced quantitative measurement of microstructural features. This capability is maintained as part of the microscopy facility at Sandia, Livermore. The system records images digitally, eliminating the use of film. Images obtained from other sources may also be imported into the system. Subsequent digital image processing enhances image appearance through the contrast and brightness adjustments. The system measures a variety of user-defined microstructural features--including area fraction, particle size and spatial distributions, grain sizes and orientations of elongated particles. These measurements are made in a semi-automatic mode through the use of macro programs and a computer controlled translation stage. A routine has been developed to create large montages of 50+ separate images. Individual image frames are matched to the nearest pixel to create seamless montages. Results from three different studies are presented to illustrate the capabilities of the system.

  1. Near Real Time Quantitative Gas Analysis Techniques

    NASA Astrophysics Data System (ADS)

    Herget, William F.; Tromp, Marianne L.; Anderson, Charles R.

    1985-12-01

    A Fourier transform infrared (FT-IR) - based system has been developed and is undergoing evaluation for near real time multicomponent quantitative analysis of undiluted gaseous automotive exhaust emissions. The total system includes: (1) a gas conditioning system (GCS) for tracer gas injection, gas mixing, and temperature stabilization; and (2) an exhaust gas analyzer (EGA) consisting of a sample cell, an FT-IR system, and a computerized data processing system. Tests have shown that the system can monitor about 20 individual species (concentrations down to the 1-20 ppm range) with a time resolution of one second. Tests have been conducted on a chassis dynamometer system utilizing different autos, different fuels, and different driving cycles. Results were compared with those obtained using a standard constant volume sampling (CVS) system.

  2. Nonlinear dynamics and quantitative EEG analysis.

    PubMed

    Jansen, B H

    1996-01-01

    Quantitative, computerized electroencephalogram (EEG) analysis appears to be based on a phenomenological approach to EEG interpretation, and is primarily rooted in linear systems theory. A fundamentally different approach to computerized EEG analysis, however, is making its way into the laboratories. The basic idea, inspired by recent advances in the area of nonlinear dynamics and chaos theory, is to view an EEG as the output of a deterministic system of relatively simple complexity, but containing nonlinearities. This suggests that studying the geometrical dynamics of EEGs, and the development of neurophysiologically realistic models of EEG generation may produce more successful automated EEG analysis techniques than the classical, stochastic methods. A review of the fundamentals of chaos theory is provided. Evidence supporting the nonlinear dynamics paradigm to EEG interpretation is presented, and the kind of new information that can be extracted from the EEG is discussed. A case is made that a nonlinear dynamic systems viewpoint to EEG generation will profoundly affect the way EEG interpretation is currently done.

  3. Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

    PubMed

    Parte, Priyanka P; Rao, Parimala; Redij, Shweta; Lobo, Vivian; D'Souza, Serena J; Gajbhiye, Rahul; Kulkarni, Vijay

    2012-10-22

    Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.

  4. Quantitative analysis of protein turnover in plants.

    PubMed

    Nelson, Clark J; Li, Lei; Millar, A Harvey

    2014-03-01

    Proteins are constantly being synthesised and degraded as plant cells age and as plants grow, develop and adapt the proteome. Given that plants develop through a series of events from germination to fruiting and even undertake whole organ senescence, an understanding of protein turnover as a fundamental part of this process in plants is essential. Both synthesis and degradation processes are spatially separated in a cell across its compartmented structure. The majority of protein synthesis occurs in the cytosol, while synthesis of specific components occurs inside plastids and mitochondria. Degradation of proteins occurs in both the cytosol, through the action of the plant proteasome, and in organelles and lytic structures through different protease classes. Tracking the specific synthesis and degradation rate of individual proteins can be undertaken using stable isotope feeding and the ability of peptide MS to track labelled peptide fractions over time. Mathematical modelling can be used to follow the isotope signature of newly synthesised protein as it accumulates and natural abundance proteins as they are lost through degradation. Different technical and biological constraints govern the potential for the use of (13)C, (15)N, (2)H and (18)O for these experiments in complete labelling and partial labelling strategies. Future development of quantitative protein turnover analysis will involve analysis of protein populations in complexes and subcellular compartments, assessing the effect of PTMs and integrating turnover studies into wider system biology study of plants.

  5. Quantitative Analysis of Triple Mutant Genetic Interactions

    PubMed Central

    Braberg, Hannes; Alexander, Richard; Shales, Michael; Xu, Jiewei; Franks-Skiba, Kathleen E.; Wu, Qiuqin; Haber, James E.; Krogan, Nevan J.

    2014-01-01

    The quantitative analysis of genetic interactions between pairs of gene mutations has proven effective for characterizing cellular functions but can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed Triple Mutant Analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, that is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principle actors are deleted. TMA has also uncovered double mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete, and measures interactions for up to 30 double mutants against a library of 1536 single mutants. PMID:25010907

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

    PubMed

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

    2016-03-01

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

  7. Applying Knowledge of Quantitative Design and Analysis

    ERIC Educational Resources Information Center

    Baskas, Richard S.

    2011-01-01

    This study compared and contrasted two quantitative scholarly articles in relation to their research designs. Their designs were analyzed by the comparison of research references and research specific vocabulary to describe how various research methods were used. When researching and analyzing quantitative scholarly articles, it is imperative to…

  8. Phosphoproteome Analysis of Drosophila melanogaster Embryos

    PubMed Central

    Zhai, Bo; Villén, Judit; Beausoleil, Sean A.; Mintseris, Julian; Gygi, Steven P.

    2011-01-01

    Protein phosphorylation is a key regulatory event in most cellular processes and development. Mass spectrometry-based proteomics provides a framework for the large-scale identification and characterization of phosphorylation sites. Here, we used a well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange chromatography, immobilized metal affinity chromatography, and high-accuracy mass spectrometry instrumentation to study phosphorylation in developing Drosophila embryos. In total, 13 720 different phosphorylation sites were discovered from 2702 proteins with an estimated false-discovery rate (FDR) of 0.63% at the peptide level. Because of the large size of the data set, both novel and known phosphorylation motifs were extracted using the Motif-X algorithm, including those representative of potential ordered phosphorylation events. PMID:18327897

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

    PubMed Central

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

    2016-01-01

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

  10. Quantitative Analysis of Hypoperfusion in Acute Stroke

    PubMed Central

    Nael, Kambiz; Meshksar, Arash; Liebeskind, David S.; Coull, Bruce M.; Krupinski, Elizabeth A.; Villablanca, J. Pablo

    2014-01-01

    Background and Purpose This study compares the concordance between arterial spin labeling (ASL) and dynamic susceptibility contrast (DSC) for the identification of regional hypoperfusion and diffusion-perfusion mismatch tissue classification using a quantitative method. Methods The inclusion criteria for this retrospective study were as follows: patients with acute ischemic syndrome with symptom onset <24 hours and acquisition of both ASL and DSC MR perfusion. The volumes of infarction and hypoperfused lesions were calculated on ASL and DSC multi-parametric maps. Patients were classified into reperfused, matched, or mismatch groups using time to maximum >6 sec as the reference. In a subset of patients who were successfully recanalized, the identical analysis was performed and the infarction and hypoperfused lesion volumes were used for paired pre- and posttreatment comparisons. Results Forty-one patients met our inclusion criteria. Twenty patients underwent successful endovascular revascularization (TICI>2a), resulting in a total of 61 ASL-DSC data pairs for comparison. The hypoperfusion volume on ASL-cerebral blood flow best approximated the DSC-time to peak volume (r=0.83) in pretreatment group and time to maximum (r=0.46) after recanalization. Both ASL-cerebral blood flow and DSC-TTP overestimated the hypoperfusion volume compared with time to maximum volume in pretreatment (F=27.41, P<0.0001) and recanalized patients (F=8.78, P<0.0001). Conclusions ASL-cerebral blood flow overestimates the DSC time to maximum hypoperfusion volume and mismatch classification in patients with acute ischemic syndrome. Continued overestimation of hypoperfused volume after recanalization suggests flow pattern and velocity changes in addition to arterial transit delay can affects the performance of ASL. PMID:23988646

  11. Quantitative Auger analysis of Nb-Ge superconducting alloys

    SciTech Connect

    Buitrago, R.H.

    1980-01-01

    The feasibility of using Auger electron analysis for quantitative analysis was investigated by studying Nb/sub 3/Ge thin-film Auger data with different approaches. A method base on elemental standards gave consistent quantitative values with reported Nb-Ge data. Alloy sputter yields were also calculated and results were consistent with those for pure elements.

  12. Quantitative Analysis of Radar Returns from Insects

    NASA Technical Reports Server (NTRS)

    Riley, J. R.

    1979-01-01

    When a number of flying insects is low enough to permit their resolution as individual radar targets, quantitative estimates of their aerial density are developed. Accurate measurements of heading distribution using a rotating polarization radar to enhance the wingbeat frequency method of identification are presented.

  13. Some Epistemological Considerations Concerning Quantitative Analysis

    ERIC Educational Resources Information Center

    Dobrescu, Emilian

    2008-01-01

    This article presents the author's address at the 2007 "Journal of Applied Quantitative Methods" ("JAQM") prize awarding festivity. The festivity was included in the opening of the 4th International Conference on Applied Statistics, November 22, 2008, Bucharest, Romania. In the address, the author reflects on three theses that…

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

    SciTech Connect

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

    2015-01-15

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

  15. Quantitative analysis of planetary reflectance spectra with principal components analysis

    NASA Technical Reports Server (NTRS)

    Johnson, P. E.; Smith, M. O.; Adams, J. B.

    1985-01-01

    A technique is presented for quantitative analysis of planetary reflectance spectra as mixtures of particles on microscopic and macroscopic scales using principal components analysis. This technique allows for determination of the endmembers being mixed, their abundance, and the scale of mixing, as well as other physical parameters. Eighteen lunar telescopic reflectance spectra of the Copernicus crater region, from 600 nm to 1800 nm in wavelength, are modeled in terms of five likely endmembers: mare basalt, mature mare soil, anorthosite, mature highland soil, and clinopyroxene. These endmembers were chosen from a similar analysis of 92 lunar soil and rock samples. The models fit the data to within 2 percent rms. It is found that the goodness of fit is marginally better for intimate mixing over macroscopic mixing.

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

    PubMed

    McNulty, Dean E; Annan, Roland S

    2008-05-01

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

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

    PubMed

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

    2010-01-01

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

  18. Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2012-12-01

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

  1. Energy Dispersive Spectrometry and Quantitative Analysis Short Course. Introduction to X-ray Energy Dispersive Spectrometry and Quantitative Analysis

    NASA Technical Reports Server (NTRS)

    Carpenter, Paul; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    This course will cover practical applications of the energy-dispersive spectrometer (EDS) to x-ray microanalysis. Topics covered will include detector technology, advances in pulse processing, resolution and performance monitoring, detector modeling, peak deconvolution and fitting, qualitative and quantitative analysis, compositional mapping, and standards. An emphasis will be placed on use of the EDS for quantitative analysis, with discussion of typical problems encountered in the analysis of a wide range of materials and sample geometries.

  2. Structural and quantitative analysis of Equisetum alkaloids.

    PubMed

    Cramer, Luise; Ernst, Ludger; Lubienski, Marcus; Papke, Uli; Schiebel, Hans-Martin; Jerz, Gerold; Beuerle, Till

    2015-08-01

    Equisetum palustre L. is known for its toxicity for livestock. Several studies in the past addressed the isolation and identification of the responsible alkaloids. So far, palustrine (1) and N(5)-formylpalustrine (2) are known alkaloids of E. palustre. A HPLC-ESI-MS/MS method in combination with simple sample work-up was developed to identify and quantitate Equisetum alkaloids. Besides the two known alkaloids six related alkaloids were detected in different Equisetum samples. The structure of the alkaloid palustridiene (3) was derived by comprehensive 1D and 2D NMR experiments. N(5)-Acetylpalustrine (4) was also thoroughly characterized by NMR for the first time. The structure of N(5)-formylpalustridiene (5) is proposed based on mass spectrometry results. Twenty-two E. palustre samples were screened by a HPLC-ESI-MS/MS method after development of a simple sample work-up and in most cases the set of all eight alkaloids were detected in all parts of the plant. A high variability of the alkaloid content and distribution was found depending on plant organ, plant origin and season ranging from 88 to 597mg/kg dried weight. However, palustrine (1) and the alkaloid palustridiene (3) always represented the main alkaloids. For the first time, a comprehensive identification, quantitation and distribution of Equisetum alkaloids was achieved.

  3. Joint association analysis of bivariate quantitative and qualitative traits.

    PubMed

    Yuan, Mengdie; Diao, Guoqing

    2011-11-29

    Univariate genome-wide association analysis of quantitative and qualitative traits has been investigated extensively in the literature. In the presence of correlated phenotypes, it is more intuitive to analyze all phenotypes simultaneously. We describe an efficient likelihood-based approach for the joint association analysis of quantitative and qualitative traits in unrelated individuals. We assume a probit model for the qualitative trait, under which an unobserved latent variable and a prespecified threshold determine the value of the qualitative trait. To jointly model the quantitative and qualitative traits, we assume that the quantitative trait and the latent variable follow a bivariate normal distribution. The latent variable is allowed to be correlated with the quantitative phenotype. Simultaneous modeling of the quantitative and qualitative traits allows us to make more precise inference on the pleiotropic genetic effects. We derive likelihood ratio tests for the testing of genetic effects. An application to the Genetic Analysis Workshop 17 data is provided. The new method yields reasonable power and meaningful results for the joint association analysis of the quantitative trait Q1 and the qualitative trait disease status at SNPs with not too small MAF.

  4. Qualitative and quantitative analysis of endocytic recycling.

    PubMed

    Reineke, James B; Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.

  5. Impact of TGF-b on breast cancer from a quantitative proteomic analysis.

    PubMed

    Ahn, Jaegyoon; Yoon, Youngmi; Yeu, Yunku; Lee, Hookuen; Park, Sanghyun

    2013-12-01

    There has been much active research in bioinformatics to support our understanding of oncogenesis and tumor progression. Most research relies on mRNA gene expression data to identify marker genes or cancer specific gene networks. However, considering that proteins are functional molecules that carry out the biological tasks of genes, they can be direct markers of biological functions. Protein abundance data on a genome scale have not been investigated in depth due to the limited availability of high throughput protein assays. This hindrance is chiefly caused by a lack of robust techniques such as RT-PCR (real-time polymerase chain reaction). In this study, we quantified phospho-proteomes of breast cancer cell lines treated with TGF-beta (transforming growth factor beta). To discover biomarkers and observe changes in the signaling pathways related to breast cancer, we applied a protein network-based approach to generate a classifier of subnet markers. The accuracy of that classifier outperformed other network-based classification algorithms, and current feature selection and classification algorithms. Moreover, many cancer-related proteins were identified in those sub-networks. Each sub-network provides functional insights and can serve as a potential marker for TGF-beta treatments. After interpreting the roles of proteins in sub-networks with various signaling pathways, we found strong candidate proteins and various related interactions that are expected to affect breast cancer outcomes. These results demonstrate the high quality of the quantified phospho-proteomes data and show that our network construction and classification method is appropriate for an analysis of this type of data.

  6. Quantitative infrared analysis of hydrogen fluoride

    SciTech Connect

    Manuta, D.M.

    1997-04-01

    This work was performed at the Portsmouth Gaseous Diffusion Plant where hydrogen fluoride is produced upon the hydrolysis of UF{sub 6}. This poses a problem for in this setting and a method for determining the mole percent concentration was desired. HF has been considered to be a non-ideal gas for many years. D. F. Smith utilized complex equations in his HF studies in the 1950s. We have evaluated HF behavior as a function of pressure from three different perspectives. (1) Absorbance at 3877 cm{sup -1} as a function of pressure for 100% HF. (2) Absorbance at 3877 cm{sup -1} as a function of increasing partial pressure HF. Total pressure = 300 mm HgA maintained with nitrogen. (3) Absorbance at 3877 cm{sup -1} for constant partial pressure HF. Total pressure is increased to greater than 800 mm HgA with nitrogen. These experiments have shown that at partial pressures up to 35mm HgA, HIF follows the ideal gas law. The absorbance at 3877 cm{sup -1} can be quantitatively analyzed via infrared methods.

  7. Quantitative Analysis of HIV-1 Preintegration Complexes

    PubMed Central

    Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

    2009-01-01

    Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

  8. Quantitative multi-modal NDT data analysis

    SciTech Connect

    Heideklang, René; Shokouhi, Parisa

    2014-02-18

    A single NDT technique is often not adequate to provide assessments about the integrity of test objects with the required coverage or accuracy. In such situations, it is often resorted to multi-modal testing, where complementary and overlapping information from different NDT techniques are combined for a more comprehensive evaluation. Multi-modal material and defect characterization is an interesting task which involves several diverse fields of research, including signal and image processing, statistics and data mining. The fusion of different modalities may improve quantitative nondestructive evaluation by effectively exploiting the augmented set of multi-sensor information about the material. It is the redundant information in particular, whose quantification is expected to lead to increased reliability and robustness of the inspection results. There are different systematic approaches to data fusion, each with its specific advantages and drawbacks. In our contribution, these will be discussed in the context of nondestructive materials testing. A practical study adopting a high-level scheme for the fusion of Eddy Current, GMR and Thermography measurements on a reference metallic specimen with built-in grooves will be presented. Results show that fusion is able to outperform the best single sensor regarding detection specificity, while retaining the same level of sensitivity.

  9. The quantitative failure of human reliability analysis

    SciTech Connect

    Bennett, C.T.

    1995-07-01

    This philosophical treatise argues the merits of Human Reliability Analysis (HRA) in the context of the nuclear power industry. Actually, the author attacks historic and current HRA as having failed in informing policy makers who make decisions based on risk that humans contribute to systems performance. He argues for an HRA based on Bayesian (fact-based) inferential statistics, which advocates a systems analysis process that employs cogent heuristics when using opinion, and tempers itself with a rational debate over the weight given subjective and empirical probabilities.

  10. Quantitive and Sociological Analysis of Blog Networks

    NASA Astrophysics Data System (ADS)

    Bachnik, W.; Szymczyk, S.; Leszczynski, S.; Podsiadlo, R.; Rymszewicz, E.; Kurylo, L.; Makowiec, D.; Bykowska, B.

    2005-10-01

    This paper examines the emerging phenomenon of blogging, using three different Polish blogging services as the base of the research. Authors show that blog networks are sharing their characteristics with complex networks (gamma coefficients, small worlds, cliques, etc.). Elements of sociometric analysis were used to prove existence of some social structures in the blog networks.

  11. A Comparative Assessment of Greek Universities' Efficiency Using Quantitative Analysis

    ERIC Educational Resources Information Center

    Katharaki, Maria; Katharakis, George

    2010-01-01

    In part due to the increased demand for higher education, typical evaluation frameworks for universities often address the key issue of available resource utilisation. This study seeks to estimate the efficiency of 20 public universities in Greece through quantitative analysis (including performance indicators, data envelopment analysis (DEA) and…

  12. Influence of corrosion layers on quantitative analysis

    NASA Astrophysics Data System (ADS)

    Denker, A.; Bohne, W.; Opitz-Coutureau, J.; Rauschenberg, J.; Röhrich, J.; Strub, E.

    2005-09-01

    Art historians and restorers in charge of ancient metal objects are often reluctant to remove the corrosion layer evolved over time, as this would change the appearance of the artefact dramatically. Therefore, when an elemental analysis of the objects is required, this has to be done by penetrating the corrosion layer. In this work the influence of corrosion was studied on Chinese and Roman coins, where removal of oxidized material was possible. Measurements on spots with and without corrosion are presented and the results discussed.

  13. Segmentation and Quantitative Analysis of Epithelial Tissues.

    PubMed

    Aigouy, Benoit; Umetsu, Daiki; Eaton, Suzanne

    2016-01-01

    Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

  14. Quantitative Analysis of Seismicity in Iran

    NASA Astrophysics Data System (ADS)

    Raeesi, Mohammad; Zarifi, Zoya; Nilfouroushan, Faramarz; Boroujeni, Samar Amini; Tiampo, Kristy

    2017-03-01

    We use historical and recent major earthquakes and GPS geodetic data to compute seismic strain rate, geodetic slip deficit, static stress drop, the parameters of the magnitude-frequency distribution and geodetic strain rate in the Iranian Plateau to identify seismically mature fault segments and regions. Our analysis suggests that 11 fault segments are in the mature stage of the earthquake cycle, with the possibility of generating major earthquakes. These faults primarily are located in the north and the east of Iran. Four seismically mature regions in southern Iran with the potential for damaging strong earthquakes are also identified. We also delineate four additional fault segments in Iran that can generate major earthquakes without robust clues to their maturity.The most important fault segment in this study is the strike-slip system near the capital city of Tehran, with the potential to cause more than one million fatalities.

  15. Quantitative Analysis of Seismicity in Iran

    NASA Astrophysics Data System (ADS)

    Raeesi, Mohammad; Zarifi, Zoya; Nilfouroushan, Faramarz; Boroujeni, Samar Amini; Tiampo, Kristy

    2016-12-01

    We use historical and recent major earthquakes and GPS geodetic data to compute seismic strain rate, geodetic slip deficit, static stress drop, the parameters of the magnitude-frequency distribution and geodetic strain rate in the Iranian Plateau to identify seismically mature fault segments and regions. Our analysis suggests that 11 fault segments are in the mature stage of the earthquake cycle, with the possibility of generating major earthquakes. These faults primarily are located in the north and the east of Iran. Four seismically mature regions in southern Iran with the potential for damaging strong earthquakes are also identified. We also delineate four additional fault segments in Iran that can generate major earthquakes without robust clues to their maturity.The most important fault segment in this study is the strike-slip system near the capital city of Tehran, with the potential to cause more than one million fatalities.

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

    PubMed Central

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

    2017-01-01

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

  17. Uncertainty of quantitative microbiological methods of pharmaceutical analysis.

    PubMed

    Gunar, O V; Sakhno, N G

    2015-12-30

    The total uncertainty of quantitative microbiological methods, used in pharmaceutical analysis, consists of several components. The analysis of the most important sources of the quantitative microbiological methods variability demonstrated no effect of culture media and plate-count techniques in the estimation of microbial count while the highly significant effect of other factors (type of microorganism, pharmaceutical product and individual reading and interpreting errors) was established. The most appropriate method of statistical analysis of such data was ANOVA which enabled not only the effect of individual factors to be estimated but also their interactions. Considering all the elements of uncertainty and combining them mathematically the combined relative uncertainty of the test results was estimated both for method of quantitative examination of non-sterile pharmaceuticals and microbial count technique without any product. These data did not exceed 35%, appropriated for a traditional plate count methods.

  18. Quantitative flow cytometric analysis of membrane antigen expression.

    PubMed

    D'hautcourt, Jean-Luc

    2002-11-01

    Immunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen under- or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.

  19. Control of separation and quantitative analysis by GC-FTIR

    NASA Astrophysics Data System (ADS)

    Semmoud, A.; Huvenne, Jean P.; Legrand, P.

    1992-03-01

    Software for 3-D representations of the 'Absorbance-Wavenumber-Retention time' is used to control the quality of the GC separation. Spectral information given by the FTIR detection allows the user to be sure that a chromatographic peak is 'pure.' The analysis of peppermint essential oil is presented as an example. This assurance is absolutely required for quantitative applications. In these conditions, we have worked out a quantitative analysis of caffeine. Correlation coefficients between integrated absorbance measurements and concentration of caffeine are discussed at two steps of the data treatment.

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

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2013-10-04

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

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

    PubMed

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

    2010-02-05

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

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

    SciTech Connect

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

    2013-06-05

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

  4. A quantitative analysis of the F18 flight control system

    NASA Technical Reports Server (NTRS)

    Doyle, Stacy A.; Dugan, Joanne B.; Patterson-Hine, Ann

    1993-01-01

    This paper presents an informal quantitative analysis of the F18 flight control system (FCS). The analysis technique combines a coverage model with a fault tree model. To demonstrate the method's extensive capabilities, we replace the fault tree with a digraph model of the F18 FCS, the only model available to us. The substitution shows that while digraphs have primarily been used for qualitative analysis, they can also be used for quantitative analysis. Based on our assumptions and the particular failure rates assigned to the F18 FCS components, we show that coverage does have a significant effect on the system's reliability and thus it is important to include coverage in the reliability analysis.

  5. Issues in Quantitative Analysis of Ultraviolet Imager (UV) Data: Airglow

    NASA Technical Reports Server (NTRS)

    Germany, G. A.; Richards, P. G.; Spann, J. F.; Brittnacher, M. J.; Parks, G. K.

    1999-01-01

    The GGS Ultraviolet Imager (UVI) has proven to be especially valuable in correlative substorm, auroral morphology, and extended statistical studies of the auroral regions. Such studies are based on knowledge of the location, spatial, and temporal behavior of auroral emissions. More quantitative studies, based on absolute radiometric intensities from UVI images, require a more intimate knowledge of the instrument behavior and data processing requirements and are inherently more difficult than studies based on relative knowledge of the oval location. In this study, UVI airglow observations are analyzed and compared with model predictions to illustrate issues that arise in quantitative analysis of UVI images. These issues include instrument calibration, long term changes in sensitivity, and imager flat field response as well as proper background correction. Airglow emissions are chosen for this study because of their relatively straightforward modeling requirements and because of their implications for thermospheric compositional studies. The analysis issues discussed here, however, are identical to those faced in quantitative auroral studies.

  6. CUMULATIVE RISK ASSESSMENT: GETTING FROM TOXICOLOGY TO QUANTITATIVE ANALYSIS

    EPA Science Inventory

    INTRODUCTION: GETTING FROM TOXICOLOGY TO QUANTITATIVE ANALYSIS FOR CUMULATIVE RISK

    Hugh A. Barton1 and Carey N. Pope2
    1US EPA, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC
    2Department of...

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

    PubMed Central

    2014-01-01

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

  8. Quantitating the subtleties of microglial morphology with fractal analysis

    PubMed Central

    Karperien, Audrey; Ahammer, Helmut; Jelinek, Herbert F.

    2013-01-01

    It is well established that microglial form and function are inextricably linked. In recent years, the traditional view that microglial form ranges between “ramified resting” and “activated amoeboid” has been emphasized through advancing imaging techniques that point to microglial form being highly dynamic even within the currently accepted morphological categories. Moreover, microglia adopt meaningful intermediate forms between categories, with considerable crossover in function and varying morphologies as they cycle, migrate, wave, phagocytose, and extend and retract fine and gross processes. From a quantitative perspective, it is problematic to measure such variability using traditional methods, but one way of quantitating such detail is through fractal analysis. The techniques of fractal analysis have been used for quantitating microglial morphology, to categorize gross differences but also to differentiate subtle differences (e.g., amongst ramified cells). Multifractal analysis in particular is one technique of fractal analysis that may be useful for identifying intermediate forms. Here we review current trends and methods of fractal analysis, focusing on box counting analysis, including lacunarity and multifractal analysis, as applied to microglial morphology. PMID:23386810

  9. Quantitative analysis of regional myocardial performance in coronary artery disease

    NASA Technical Reports Server (NTRS)

    Stewart, D. K.; Dodge, H. T.; Frimer, M.

    1975-01-01

    Findings from a group of subjects with significant coronary artery stenosis are given. A group of controls determined by use of a quantitative method for the study of regional myocardial performance based on the frame-by-frame analysis of biplane left ventricular angiograms are presented. Particular emphasis was placed upon the analysis of wall motion in terms of normalized segment dimensions, timing and velocity of contraction. The results were compared with the method of subjective assessment used clinically.

  10. Quantitative analysis of culture using millions of digitized books.

    PubMed

    Michel, Jean-Baptiste; Shen, Yuan Kui; Aiden, Aviva Presser; Veres, Adrian; Gray, Matthew K; Pickett, Joseph P; Hoiberg, Dale; Clancy, Dan; Norvig, Peter; Orwant, Jon; Pinker, Steven; Nowak, Martin A; Aiden, Erez Lieberman

    2011-01-14

    We constructed a corpus of digitized texts containing about 4% of all books ever printed. Analysis of this corpus enables us to investigate cultural trends quantitatively. We survey the vast terrain of 'culturomics,' focusing on linguistic and cultural phenomena that were reflected in the English language between 1800 and 2000. We show how this approach can provide insights about fields as diverse as lexicography, the evolution of grammar, collective memory, the adoption of technology, the pursuit of fame, censorship, and historical epidemiology. Culturomics extends the boundaries of rigorous quantitative inquiry to a wide array of new phenomena spanning the social sciences and the humanities.

  11. Quantitative analysis of culture using millions of digitized books

    PubMed Central

    Michel, Jean-Baptiste; Shen, Yuan Kui; Aiden, Aviva P.; Veres, Adrian; Gray, Matthew K.; Pickett, Joseph P.; Hoiberg, Dale; Clancy, Dan; Norvig, Peter; Orwant, Jon; Pinker, Steven; Nowak, Martin A.; Aiden, Erez Lieberman

    2011-01-01

    We constructed a corpus of digitized texts containing about 4% of all books ever printed. Analysis of this corpus enables us to investigate cultural trends quantitatively. We survey the vast terrain of ‘culturomics’, focusing on linguistic and cultural phenomena that were reflected in the English language between 1800 and 2000. We show how this approach can provide insights about fields as diverse as lexicography, the evolution of grammar, collective memory, the adoption of technology, the pursuit of fame, censorship, and historical epidemiology. ‘Culturomics’ extends the boundaries of rigorous quantitative inquiry to a wide array of new phenomena spanning the social sciences and the humanities. PMID:21163965

  12. Quantitative numerical analysis of transient IR-experiments on buildings

    NASA Astrophysics Data System (ADS)

    Maierhofer, Ch.; Wiggenhauser, H.; Brink, A.; Röllig, M.

    2004-12-01

    Impulse-thermography has been established as a fast and reliable tool in many areas of non-destructive testing. In recent years several investigations have been done to apply active thermography to civil engineering. For quantitative investigations in this area of application, finite difference calculations have been performed for systematic studies on the influence of environmental conditions, heating power and time, defect depth and size and thermal properties of the bulk material (concrete). The comparison of simulated and experimental data enables the quantitative analysis of defects.

  13. Improved method and apparatus for chromatographic quantitative analysis

    DOEpatents

    Fritz, J.S.; Gjerde, D.T.; Schmuckler, G.

    An improved apparatus and method are described for the quantitative analysis of a solution containing a plurality of anion species by ion exchange chromatography which utilizes a single element and a single ion exchange bed which does not require periodic regeneration. The solution containing the anions is added to an anion exchange resin bed which is a low capacity macroreticular polystyrene-divinylbenzene resin containing quarternary ammonium functional groups, and is eluted therefrom with a dilute solution of a low electrical conductance organic acid salt. As each anion species is eluted from the bed, it is quantitatively sensed by conventional detection means such as a conductivity cell.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  17. Markov chain Monte Carlo linkage analysis of complex quantitative phenotypes.

    PubMed

    Hinrichs, A; Reich, T

    2001-01-01

    We report a Markov chain Monte Carlo analysis of the five simulated quantitative traits in Genetic Analysis Workshop 12 using the Loki software. Our objectives were to determine the efficacy of the Markov chain Monte Carlo method and to test a new scoring technique. Our initial blind analysis, on replicate 42 (the "best replicate") successfully detected four out of the five disease loci and found no false positives. A power analysis shows that the software could usually detect 4 of the 10 trait/gene combinations at an empirical point-wise p-value of 1.5 x 10(-4).

  18. Spotsizer: High-throughput quantitative analysis of microbial growth

    PubMed Central

    Jeffares, Daniel C.; Arzhaeva, Yulia; Bähler, Jürg

    2017-01-01

    Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license. PMID:27712582

  19. Spotsizer: High-throughput quantitative analysis of microbial growth.

    PubMed

    Bischof, Leanne; Převorovský, Martin; Rallis, Charalampos; Jeffares, Daniel C; Arzhaeva, Yulia; Bähler, Jürg

    2016-10-01

    Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license.

  20. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

    PubMed

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies.

  1. Some selected quantitative methods of thermal image analysis in Matlab.

    PubMed

    Koprowski, Robert

    2016-05-01

    The paper presents a new algorithm based on some selected automatic quantitative methods for analysing thermal images. It shows the practical implementation of these image analysis methods in Matlab. It enables to perform fully automated and reproducible measurements of selected parameters in thermal images. The paper also shows two examples of the use of the proposed image analysis methods for the area of ​​the skin of a human foot and face. The full source code of the developed application is also provided as an attachment. The main window of the program during dynamic analysis of the foot thermal image.

  2. Quantitative risk analysis of oil storage facilities in seismic areas.

    PubMed

    Fabbrocino, Giovanni; Iervolino, Iunio; Orlando, Francesca; Salzano, Ernesto

    2005-08-31

    Quantitative risk analysis (QRA) of industrial facilities has to take into account multiple hazards threatening critical equipment. Nevertheless, engineering procedures able to evaluate quantitatively the effect of seismic action are not well established. Indeed, relevant industrial accidents may be triggered by loss of containment following ground shaking or other relevant natural hazards, either directly or through cascade effects ('domino effects'). The issue of integrating structural seismic risk into quantitative probabilistic seismic risk analysis (QpsRA) is addressed in this paper by a representative study case regarding an oil storage plant with a number of atmospheric steel tanks containing flammable substances. Empirical seismic fragility curves and probit functions, properly defined both for building-like and non building-like industrial components, have been crossed with outcomes of probabilistic seismic hazard analysis (PSHA) for a test site located in south Italy. Once the seismic failure probabilities have been quantified, consequence analysis has been performed for those events which may be triggered by the loss of containment following seismic action. Results are combined by means of a specific developed code in terms of local risk contour plots, i.e. the contour line for the probability of fatal injures at any point (x, y) in the analysed area. Finally, a comparison with QRA obtained by considering only process-related top events is reported for reference.

  3. An improved quantitative analysis method for plant cortical microtubules.

    PubMed

    Lu, Yi; Huang, Chenyang; Wang, Jia; Shang, Peng

    2014-01-01

    The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies.

  4. Data from quantitative label free proteomics analysis of rat spleen.

    PubMed

    Dudekula, Khadar; Le Bihan, Thierry

    2016-09-01

    The dataset presented in this work has been obtained using a label-free quantitative proteomic analysis of rat spleen. A robust method for extraction of proteins from rat spleen tissue and LC-MS-MS analysis was developed using a urea and SDS-based buffer. Different fractionation methods were compared. A total of 3484 different proteins were identified from the pool of all experiments run in this study (a total of 2460 proteins with at least two peptides). A total of 1822 proteins were identified from nine non-fractionated pulse gels, 2288 proteins and 2864 proteins were identified by SDS-PAGE fractionation into three and five fractions respectively. The proteomics data are deposited in ProteomeXchange Consortium via PRIDE PXD003520, Progenesis and Maxquant output are presented in the supported information. The generated list of proteins under different regimes of fractionation allow assessing the nature of the identified proteins; variability in the quantitative analysis associated with the different sampling strategy and allow defining a proper number of replicates for future quantitative analysis.

  5. Qualitative and quantitative stability analysis of penta-rhythmic circuits

    NASA Astrophysics Data System (ADS)

    Schwabedal, Justus T. C.; Knapper, Drake E.; Shilnikov, Andrey L.

    2016-12-01

    Inhibitory circuits of relaxation oscillators are often-used models for dynamics of biological networks. We present a qualitative and quantitative stability analysis of such a circuit constituted by three generic oscillators (of a Fitzhugh-Nagumo type) as its nodes coupled reciprocally. Depending on inhibitory strengths, and parameters of individual oscillators, the circuit exhibits polyrhythmicity of up to five simultaneously stable rhythms. With methods of bifurcation analysis and phase reduction, we investigate qualitative changes in stability of these circuit rhythms for a wide range of parameters. Furthermore, we quantify robustness of the rhythms maintained under random perturbations by monitoring phase diffusion in the circuit. Our findings allow us to describe how circuit dynamics relate to dynamics of individual nodes. We also find that quantitative and qualitative stability properties of polyrhythmicity do not always align.

  6. Implementing a Quantitative Analysis Design Tool for Future Generation Interfaces

    DTIC Science & Technology

    2012-03-01

    future MAC-enabled systems. A human-computer interaction ( HCI ) Index, originally applied to multi-function displays was applied to the prototype Vigilant...Spirit interface. A modified version of the HCI Index was successfully applied to perform a quantitative analysis of the baseline VSCS interface and...two modified interface designs. The modified HCI Index incorporates the Hick-Hyman decision time, Fitts’ Law time, and the physical actions

  7. Quantitative NMR Analysis of Partially Substituted Biodiesel Glycerols

    SciTech Connect

    Nagy, M.; Alleman, T. L.; Dyer, T.; Ragauskas, A. J.

    2009-01-01

    Phosphitylation of hydroxyl groups in biodiesel samples with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane followed by 31P-NMR analysis provides a rapid quantitative analytical technique for the determination of substitution patterns on partially esterified glycerols. The unique 31P-NMR chemical shift data was established with a series mono and di-substituted fatty acid esters of glycerol and then utilized to characterize an industrial sample of partially processed biodiesel.

  8. Comprehensive Quantitative Analysis of SQ Injection Using Multiple Chromatographic Technologies.

    PubMed

    Chau, Siu-Leung; Huang, Zhi-Bing; Song, Yan-Gang; Yue, Rui-Qi; Ho, Alan; Lin, Chao-Zhan; Huang, Wen-Hua; Han, Quan-Bin

    2016-08-19

    Quality control of Chinese medicine injections remains a challenge due to our poor knowledge of their complex chemical profile. This study aims to investigate the chemical composition of one of the best-selling injections, Shenqi Fuzheng (SQ) injection (SQI), via a full component quantitative analysis. A total of 15 representative small molecular components of SQI were simultaneously determined using ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole tandem time-of-flight mass spectrometry (Q-TOF-MS); saccharide composition of SQI was also quantitatively determined by high performance liquid chromatography (HPLC) with evaporative light scattering detector (ELSD) on an amino column before and after acid hydrolysis. The existence of polysaccharides was also examined on a gel permeation chromatography column. The method was well validated in terms of linearity, sensitivity, precision, accuracy and stability, and was successfully applied to analyze 13 SQI samples. The results demonstrate that up to 94.69% (w/w) of this injection product are quantitatively determined, in which small molecules and monosaccharide/sucrose account for 0.18%-0.21%, and 53.49%-58.2%, respectively. The quantitative information contributes to accumulating scientific evidence to better understand the therapy efficacy and safety of complex Chinese medicine injections.

  9. Quantitative Proteomic Approaches for Analysis of Protein S-Nitrosylation.

    PubMed

    Qu, Zhe; Greenlief, C Michael; Gu, Zezong

    2016-01-04

    S-Nitrosylation is a redox-based post-translational modification of a protein in response to nitric oxide (NO) signaling, and it participates in a variety of processes in diverse biological systems. The significance of this type of protein modification in health and diseases is increasingly recognized. In the central nervous system, aberrant S-nitrosylation, due to excessive NO production, is known to cause protein misfolding, mitochondrial dysfunction, transcriptional dysregulation, and neuronal death. This leads to an altered physiological state and consequently contributes to pathogenesis of neurodegenerative disorders. To date, much effort has been made to understand the mechanisms underlying protein S-nitrosylation, and several approaches have been developed to unveil S-nitrosylated proteins from different organisms. Interest in determining the dynamic changes of protein S-nitrosylation under different physiological and pathophysiological conditions has underscored the need for the development of quantitative proteomic approaches. Currently, both gel-based and gel-free mass spectrometry-based quantitative methods are widely used, and they each have advantages and disadvantages but may also be used together to produce complementary data. This review evaluates current available quantitative proteomic techniques for the analysis of protein S-nitrosylation and highlights recent advances, with emphasis on applications in neurodegenerative diseases. An important goal is to provide a comprehensive guide of feasible quantitative proteomic methodologies for examining protein S-nitrosylation in research to yield insights into disease mechanisms, diagnostic biomarkers, and drug discovery.

  10. Quantitative analysis of the heterogeneous population of endocytic vesicles.

    PubMed

    Kozlov, Konstantin; Kosheverova, Vera; Kamentseva, Rimma; Kharchenko, Marianna; Sokolkova, Alena; Kornilova, Elena; Samsonova, Maria

    2017-03-07

    The quantitative characterization of endocytic vesicles in images acquired with microscope is critically important for deciphering of endocytosis mechanisms. Image segmentation is the most important step of quantitative image analysis. In spite of availability of many segmentation methods, the accurate segmentation is challenging when the images are heterogeneous with respect to object shapes and signal intensities what is typical for images of endocytic vesicles. We present a Morphological reconstruction and Contrast mapping segmentation method (MrComas) for the segmentation of the endocytic vesicle population that copes with the heterogeneity in their shape and intensity. The method uses morphological opening and closing by reconstruction in the vicinity of local minima and maxima respectively thus creating the strong contrast between their basins of attraction. As a consequence, the intensity is flattened within the objects and their edges are enhanced. The method accurately recovered quantitative characteristics of synthetic images that preserve characteristic features of the endocytic vesicle population. In benchmarks and quantitative comparisons with two other popular segmentation methods, namely manual thresholding and Squash plugin, MrComas shows the best segmentation results on real biological images of EGFR (Epidermal Growth Factor Receptor) endocytosis. As a proof of feasibility, the method was applied to quantify the dynamical behavior of Early Endosomal Autoantigen 1 (EEA1)-positive endosome subpopulations during EGF-stimulated endocytosis.

  11. Mini-Column Ion-Exchange Separation and Atomic Absorption Quantitation of Nickel, Cobalt, and Iron: An Undergraduate Quantitative Analysis Experiment.

    ERIC Educational Resources Information Center

    Anderson, James L.; And Others

    1980-01-01

    Presents an undergraduate quantitative analysis experiment, describing an atomic absorption quantitation scheme that is fast, sensitive and comparatively simple relative to other titration experiments. (CS)

  12. A Quantitative Method for Microtubule Analysis in Fluorescence Images.

    PubMed

    Lan, Xiaodong; Li, Lingfei; Hu, Jiongyu; Zhang, Qiong; Dang, Yongming; Huang, Yuesheng

    2015-12-01

    Microtubule analysis is of significant value for a better understanding of normal and pathological cellular processes. Although immunofluorescence microscopic techniques have proven useful in the study of microtubules, comparative results commonly rely on a descriptive and subjective visual analysis. We developed an objective and quantitative method based on image processing and analysis of fluorescently labeled microtubular patterns in cultured cells. We used a multi-parameter approach by analyzing four quantifiable characteristics to compose our quantitative feature set. Then we interpreted specific changes in the parameters and revealed the contribution of each feature set using principal component analysis. In addition, we verified that different treatment groups could be clearly discriminated using principal components of the multi-parameter model. High predictive accuracy of four commonly used multi-classification methods confirmed our method. These results demonstrated the effectiveness and efficiency of our method in the analysis of microtubules in fluorescence images. Application of the analytical methods presented here provides information concerning the organization and modification of microtubules, and could aid in the further understanding of structural and functional aspects of microtubules under normal and pathological conditions.

  13. Cell poking: quantitative analysis of indentation of thick viscoelastic layers.

    PubMed

    Duszyk, M; Schwab, B; Zahalak, G I; Qian, H; Elson, E L

    1989-04-01

    A recently introduced device, the cell poker, measures the force required to indent the exposed surface of a cell adherent to a rigid substratum. The cell poker has provided phenomenological information about the viscoelastic properties of several different types of cells, about mechanical changes triggered by external stimuli, and about the role of the cytoskeleton in these mechanical functions. Except in special cases, however, it has not been possible to extract quantitative estimates of viscosity and elasticity moduli from cell poker measurements. This paper presents cell poker measurements of well characterized viscoelastic polymeric materials, polydimethylsiloxanes of different degrees of polymerization, in a simple shape, a flat, thick layer, which for our purposes can be treated as a half space. Analysis of the measurements in terms of a linear viscoelasticity theory yields viscosity values for three polymer samples in agreement with those determined by measurements on a macroscopic scale. Theoretical analysis further indicates that the measured limiting static elasticity of the layers may result from the tension generated at the interface between the polymer and water. This work demonstrates the possibility of obtaining quantitative viscoelastic material properties from cell poker measurements and represents the first step in extending these quantitative studies to more complicated structures including cells.

  14. Cell poking: quantitative analysis of indentation of thick viscoelastic layers.

    PubMed Central

    Duszyk, M; Schwab, B; Zahalak, G I; Qian, H; Elson, E L

    1989-01-01

    A recently introduced device, the cell poker, measures the force required to indent the exposed surface of a cell adherent to a rigid substratum. The cell poker has provided phenomenological information about the viscoelastic properties of several different types of cells, about mechanical changes triggered by external stimuli, and about the role of the cytoskeleton in these mechanical functions. Except in special cases, however, it has not been possible to extract quantitative estimates of viscosity and elasticity moduli from cell poker measurements. This paper presents cell poker measurements of well characterized viscoelastic polymeric materials, polydimethylsiloxanes of different degrees of polymerization, in a simple shape, a flat, thick layer, which for our purposes can be treated as a half space. Analysis of the measurements in terms of a linear viscoelasticity theory yields viscosity values for three polymer samples in agreement with those determined by measurements on a macroscopic scale. Theoretical analysis further indicates that the measured limiting static elasticity of the layers may result from the tension generated at the interface between the polymer and water. This work demonstrates the possibility of obtaining quantitative viscoelastic material properties from cell poker measurements and represents the first step in extending these quantitative studies to more complicated structures including cells. PMID:2720066

  15. Quantitative multivariate analysis of dynamic multicellular morphogenic trajectories.

    PubMed

    White, Douglas E; Sylvester, Jonathan B; Levario, Thomas J; Lu, Hang; Streelman, J Todd; McDevitt, Todd C; Kemp, Melissa L

    2015-07-01

    Interrogating fundamental cell biology principles that govern tissue morphogenesis is critical to better understanding of developmental biology and engineering novel multicellular systems. Recently, functional micro-tissues derived from pluripotent embryonic stem cell (ESC) aggregates have provided novel platforms for experimental investigation; however elucidating the factors directing emergent spatial phenotypic patterns remains a significant challenge. Computational modelling techniques offer a unique complementary approach to probe mechanisms regulating morphogenic processes and provide a wealth of spatio-temporal data, but quantitative analysis of simulations and comparison to experimental data is extremely difficult. Quantitative descriptions of spatial phenomena across multiple systems and scales would enable unprecedented comparisons of computational simulations with experimental systems, thereby leveraging the inherent power of computational methods to interrogate the mechanisms governing emergent properties of multicellular biology. To address these challenges, we developed a portable pattern recognition pipeline consisting of: the conversion of cellular images into networks, extraction of novel features via network analysis, and generation of morphogenic trajectories. This novel methodology enabled the quantitative description of morphogenic pattern trajectories that could be compared across diverse systems: computational modelling of multicellular structures, differentiation of stem cell aggregates, and gastrulation of cichlid fish. Moreover, this method identified novel spatio-temporal features associated with different stages of embryo gastrulation, and elucidated a complex paracrine mechanism capable of explaining spatiotemporal pattern kinetic differences in ESC aggregates of different sizes.

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

    PubMed

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

    2013-09-15

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-02

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

  19. Quantitative analysis of endocytosis with cytoplasmic pHluorin chimeras.

    PubMed

    Prosser, Derek C; Whitworth, Karen; Wendland, Beverly

    2010-09-01

    The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking. In contrast, cytoplasmic fusions are well tolerated, allowing for the quantitative assessment of trafficking of virtually any transmembrane protein. Quenching of degradation-resistant pHluorin in the acidic vacuole permits quantification of extravacuolar cargo proteins at steady-state levels and is compatible with kinetic analysis of endocytosis in live cells.

  20. [Simultaneous quantitative analysis of four lignanoids in Schisandra chinensis by quantitative analysis of multi-components by single marker].

    PubMed

    He, Feng-Cheng; Li, Shou-Xin; Zhao, Zhi-Quan; Dong, Jin-Ping; Liu, Wu-Zhan; Su, Rui-Qiang

    2012-07-01

    The aim of the study is to establish a new method of quality evaluation and validate its feasibilities by the simultaneous quantitative assay of four lignanoids in Schisandra chinensis. A new quality evaluation method, quantitative analysis of multi-components by single marker (QAMS), was established and validated with Schisandra chinensis. Four main lignanoids, schisandrin, schisantherin A, deoxyschizandrin and gamma-schizandrin, were selected as analytes and schisandrin as internal reference substance to evaluate the quality. Their contents in 13 different batches of samples, collected from different bathes, were determined by both external standard method and QAMS. The method was evaluated by comparison of the quantitative results between external standard method and QAMS. No significant differences were found in the quantitative results of four lignanoids in 13 batches of S. chinensis determined by external standard method and QAMS. QAMS is feasible for determination of four lignanoids simultaneously when some authentic standard substances were unavailable, and the developed method can be used for quality control of S. chinensis.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2015-12-01

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

  4. Quantitative analysis of in vivo confocal microscopy images: a review.

    PubMed

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

    In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

  5. A quantitative analysis of IRAS maps of molecular clouds

    NASA Technical Reports Server (NTRS)

    Wiseman, Jennifer J.; Adams, Fred C.

    1994-01-01

    We present an analysis of IRAS maps of five molecular clouds: Orion, Ophiuchus, Perseus, Taurus, and Lupus. For the classification and description of these astrophysical maps, we use a newly developed technique which considers all maps of a given type to be elements of a pseudometric space. For each physical characteristic of interest, this formal system assigns a distance function (a pseudometric) to the space of all maps: this procedure allows us to measure quantitatively the difference between any two maps and to order the space of all maps. We thus obtain a quantitative classification scheme for molecular clouds. In this present study we use the IRAS continuum maps at 100 and 60 micrometer(s) to produce column density (or optical depth) maps for the five molecular cloud regions given above. For this sample of clouds, we compute the 'output' functions which measure the distribution of density, the distribution of topological components, the self-gravity, and the filamentary nature of the clouds. The results of this work provide a quantitative description of the structure in these molecular cloud regions. We then order the clouds according to the overall environmental 'complexity' of these star-forming regions. Finally, we compare our results with the observed populations of young stellar objects in these clouds and discuss the possible environmental effects on the star-formation process. Our results are consistent with the recently stated conjecture that more massive stars tend to form in more 'complex' environments.

  6. Simulating realistic predator signatures in quantitative fatty acid signature analysis

    USGS Publications Warehouse

    Bromaghin, Jeffrey F.

    2015-01-01

    Diet estimation is an important field within quantitative ecology, providing critical insights into many aspects of ecology and community dynamics. Quantitative fatty acid signature analysis (QFASA) is a prominent method of diet estimation, particularly for marine mammal and bird species. Investigators using QFASA commonly use computer simulation to evaluate statistical characteristics of diet estimators for the populations they study. Similar computer simulations have been used to explore and compare the performance of different variations of the original QFASA diet estimator. In both cases, computer simulations involve bootstrap sampling prey signature data to construct pseudo-predator signatures with known properties. However, bootstrap sample sizes have been selected arbitrarily and pseudo-predator signatures therefore may not have realistic properties. I develop an algorithm to objectively establish bootstrap sample sizes that generates pseudo-predator signatures with realistic properties, thereby enhancing the utility of computer simulation for assessing QFASA estimator performance. The algorithm also appears to be computationally efficient, resulting in bootstrap sample sizes that are smaller than those commonly used. I illustrate the algorithm with an example using data from Chukchi Sea polar bears (Ursus maritimus) and their marine mammal prey. The concepts underlying the approach may have value in other areas of quantitative ecology in which bootstrap samples are post-processed prior to their use.

  7. Quantitative analysis of motion control in long term microgravity.

    PubMed

    Baroni, G; Ferrigno, G; Anolli, A; Andreoni, G; Pedotti, A

    1998-01-01

    In the frame of the 179-days EUROMIR '95 space mission, two in-flight experiments have foreseen quantitative three-dimensional human movement analysis in microgravity. For this aim, a space qualified opto-electronic motion analyser based on passive markers has been installed onboard the Russian Space Station MIR and 8 in flight sessions have been performed. Techhology and method for the collection of kinematics data are described, evaluating the accuracy in three-dimensional marker localisation. Results confirm the suitability of opto-electronic technology for quantitative human motion analysis on orbital modules and raise a set of "lessons learned", leading to the improvement of motion analyser performance with a contemporary swiftness of the on-board operations. Among the experimental program of T4, results of three voluntary posture perturbation protocols are described. The analysis suggests that a short term reinterpretation of proprioceptive information and re-calibration of sensorimotor mechanisms seem to end within the first weeks of flight, while a continuous long term adaptation process allows the refinement of motor performance, in the frame of never abandoned terrestrial strategies.

  8. QUANTITATIVE MASS SPECTROMETRIC ANALYSIS OF GLYCOPROTEINS COMBINED WITH ENRICHMENT METHODS

    PubMed Central

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:148–165, 2015. PMID:24889823

  9. Flow quantitation by radio frequency analysis of contrast echocardiography.

    PubMed

    Rovai, D; Lombardi, M; Mazzarisi, A; Landini, L; Taddei, L; Distante, A; Benassi, A; L'Abbate, A

    1993-03-01

    Contrast echocardiography has the potential for measuring cardiac output and regional blood flow. However, accurate quantitation is limited both by the use of non-standard contrast agents and by the electronic signal distortion inherent to the echocardiographic instruments. Thus, the aim of this study is to quantify flow by combining a stable contrast agent and a modified echo equipment, able to sample the radio frequency (RF) signal from a region of interest (ROI) in the echo image. The contrast agent SHU-454 (0.8 ml) was bolus injected into an in vitro calf vein, at 23 flow rates (ranging from 376 to 3620 ml/min) but constant volume and pressure. The ROI was placed in the centre of the vein, the RF signal was processed in real time and transferred to a personal computer to generate time-intensity curves. In the absence of recirculation, contrast washout slope and mean transit time (MTT) of curves (1.11-8.52 seconds) yielded excellent correlations with flow: r = 0.93 and 0.95, respectively. To compare the accuracy of RF analysis with that of conventional image processing as to flow quantitation, conventional images were collected in the same flow model by two different scanners: a) the mechanical sector scanner used for RF analysis, and b) a conventional electronic sector scanner. These images were digitized off-line, mean videodensity inside an identical ROI was measured and time-intensity curves were built. MTT by RF was shorter than by videodensitometric analysis of the images generated by the same scanner (p < 0.001). In contrast, MTT by RF was longer than by the conventional scanner (p < 0.001). Significant differences in MTT were also found with changes in the gain setting controls of the conventional scanner. To study the stability of the contrast effect, 6 contrast injections (20 ml) were performed at a constant flow rate during recirculation: the spontaneous decay in RF signal intensity (t1/2 = 64 +/- 8 seconds) was too long to affect MTT significantly

  10. Computer compensation for NMR quantitative analysis of trace components

    SciTech Connect

    Nakayama, T.; Fujiwara, Y.

    1981-07-22

    A computer program has been written that determines trace components and separates overlapping components in multicomponent NMR spectra. This program uses the Lorentzian curve as a theoretical curve of NMR spectra. The coefficients of the Lorentzian are determined by the method of least squares. Systematic errors such as baseline/phase distortion are compensated and random errors are smoothed by taking moving averages, so that there processes contribute substantially to decreasing the accumulation time of spectral data. The accuracy of quantitative analysis of trace components has been improved by two significant figures. This program was applied to determining the abundance of 13C and the saponification degree of PVA.

  11. Quantitative analysis of sideband coupling in photoinduced force microscopy

    NASA Astrophysics Data System (ADS)

    Jahng, Junghoon; Kim, Bongsu; Lee, Eun Seong; Potma, Eric Olaf

    2016-11-01

    We present a theoretical and experimental analysis of the cantilever motions detected in photoinduced force microscopy (PiFM) using the sideband coupling detection scheme. In sideband coupling, the cantilever dynamics are probed at a combination frequency of a fundamental mechanical eigenmode and the modulation frequency of the laser beam. Using this detection mode, we develop a method for reconstructing the modulated photoinduced force gradient from experimental parameters in a quantitative manner. We show evidence, both theoretically and experimentally, that the sideband coupling detection mode provides PiFM images with superior contrast compared to images obtained when detecting the cantilever motions directly at the laser modulation frequency.

  12. Lipid biomarker analysis for the quantitative analysis of airborne microorganisms

    SciTech Connect

    Macnaughton, S.J.; Jenkins, T.L.; Cormier, M.R.

    1997-08-01

    There is an ever increasing concern regarding the presence of airborne microbial contaminants within indoor air environments. Exposure to such biocontaminants can give rise to large numbers of different health effects including infectious diseases, allergenic responses and respiratory problems, Biocontaminants typically round in indoor air environments include bacteria, fungi, algae, protozoa and dust mites. Mycotoxins, endotoxins, pollens and residues of organisms are also known to cause adverse health effects. A quantitative detection/identification technique independent of culturability that assays both culturable and non culturable biomass including endotoxin is critical in defining risks from indoor air biocontamination. Traditionally, methods employed for the monitoring of microorganism numbers in indoor air environments involve classical culture based techniques and/or direct microscopic counting. It has been repeatedly documented that viable microorganism counts only account for between 0.1-10% of the total community detectable by direct counting. The classic viable microbiologic approach doe`s not provide accurate estimates of microbial fragments or other indoor air components that can act as antigens and induce or potentiate allergic responses. Although bioaerosol samplers are designed to damage the microbes as little as possible, microbial stress has been shown to result from air sampling, aerosolization and microbial collection. Higher collection efficiency results in greater cell damage while less cell damage often results in lower collection efficiency. Filtration can collect particulates at almost 100% efficiency, but captured microorganisms may become dehydrated and damaged resulting in non-culturability, however, the lipid biomarker assays described herein do not rely on cell culture. Lipids are components that are universally distributed throughout cells providing a means to assess independent of culturability.

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

    PubMed

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

    2015-01-01

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

  14. Functional Regression Models for Epistasis Analysis of Multiple Quantitative Traits.

    PubMed

    Zhang, Futao; Xie, Dan; Liang, Meimei; Xiong, Momiao

    2016-04-01

    To date, most genetic analyses of phenotypes have focused on analyzing single traits or analyzing each phenotype independently. However, joint epistasis analysis of multiple complementary traits will increase statistical power and improve our understanding of the complicated genetic structure of the complex diseases. Despite their importance in uncovering the genetic structure of complex traits, the statistical methods for identifying epistasis in multiple phenotypes remains fundamentally unexplored. To fill this gap, we formulate a test for interaction between two genes in multiple quantitative trait analysis as a multiple functional regression (MFRG) in which the genotype functions (genetic variant profiles) are defined as a function of the genomic position of the genetic variants. We use large-scale simulations to calculate Type I error rates for testing interaction between two genes with multiple phenotypes and to compare the power with multivariate pairwise interaction analysis and single trait interaction analysis by a single variate functional regression model. To further evaluate performance, the MFRG for epistasis analysis is applied to five phenotypes of exome sequence data from the NHLBI's Exome Sequencing Project (ESP) to detect pleiotropic epistasis. A total of 267 pairs of genes that formed a genetic interaction network showed significant evidence of epistasis influencing five traits. The results demonstrate that the joint interaction analysis of multiple phenotypes has a much higher power to detect interaction than the interaction analysis of a single trait and may open a new direction to fully uncovering the genetic structure of multiple phenotypes.

  15. Quantitative chemical analysis of ocular melanosomes in the TEM.

    PubMed

    Eibl, O; Schultheiss, S; Blitgen-Heinecke, P; Schraermeyer, U

    2006-01-01

    Melanosomes in retinal tissues of a human, monkey and rat were analyzed by EDX in the TEM. Samples were prepared by ultramicrotomy at different thicknesses. The material was mounted on Al grids and samples were analyzed in a Zeiss 912 TEM equipped with an Omega filter and EDX detector with ultrathin window. Melanosomes consist of C and O as main components, mole fractions are about 90 and 3-10 at.%, respectively, and small mole fraction ratios, between 2 and 0.1 at.%, of Na, Mg, K, Si, P, S, Cl, Ca. All elements were measured quantitatively by standardless EDX with high precision. Mole fractions of transition metals Fe, Cu and Zn were also measured. For Fe a mole fraction ratio of less than 0.1at.% was found and gives the melanin its paramagnetic properties. Its mole fraction is however close to or below the minimum detectable mass fraction of the used equipment. Only in the human eye and only in the retinal pigment epitelium (rpe) the mole fractions of Zn (0.1 at.% or 5000 microg/g) and Cu were clearly beyond the minimum detectable mass fraction. In the rat and monkey eye the mole fraction of Zn was at or below the minimum detectable mass fraction and could not be measured quantitatively. The obtained results yielded the chemical composition of the melanosomes in the choroidal tissue and the retinal pigment epitelium (rpe) of the three different species. The results of the chemical analysis are discussed by mole fraction correlation diagrams. Similarities and differences between the different species are outlined. Correlation behavior was found to hold over species, e.g. the Ca-O correlation. It indicates that Ca is bound to oxygen rich sites in the melanin. These are the first quantitative analyses of melanosomes by EDX reported so far. The quantitative chemical analysis should open a deeper understanding of the metabolic processes in the eye that are of central importance for the understanding of a large number of eye-related diseases. The chemical analysis also

  16. Quantitative analysis of intermolecular interactions in orthorhombic rubrene

    SciTech Connect

    Hathwar, Venkatesha R.; Sist, Mattia; Jørgensen, Mads R. V.; Mamakhel, Aref H.; Wang, Xiaoping; Hoffmann, Christina M.; Sugimoto, Kunihisa; Overgaard, Jacob; Iversen, Bo Brummerstedt

    2015-08-14

    Rubrene is one of the most studied organic semiconductors to date due to its high charge carrier mobility which makes it a potentially applicable compound in modern electronic devices. Previous electronic device characterizations and first principles theoretical calculations assigned the semiconducting properties of rubrene to the presence of a large overlap of the extended π-conjugated core between molecules. We present here the electron density distribution in rubrene at 20 K and at 100 K obtained using a combination of high-resolution X-ray and neutron diffraction data. The topology of the electron density and energies of intermolecular interactions are studied quantitatively. Specifically, the presence of Cπ...Cπinteractions between neighbouring tetracene backbones of the rubrene molecules is experimentally confirmed from a topological analysis of the electron density, Non-Covalent Interaction (NCI) analysis and the calculated interaction energy of molecular dimers. A significant contribution to the lattice energy of the crystal is provided by H—H interactions. The electron density features of H—H bonding, and the interaction energy of molecular dimers connected by H—H interaction clearly demonstrate an importance of these weak interactions in the stabilization of the crystal structure. Finally, the quantitative nature of the intermolecular interactions is virtually unchanged between 20 K and 100 K suggesting that any changes in carrier transport at these low temperatures would have a different origin. The obtained experimental results are further supported by theoretical calculations.

  17. Quantitative analysis of Caenorhabditis elegans chemotaxis using a microfluidic device.

    PubMed

    Hu, Liang; Ye, Jinjuan; Tan, Haowei; Ge, Anle; Tang, Lichun; Feng, Xiaojun; Du, Wei; Liu, Bi-Feng

    2015-08-05

    Caenorhabditis elegans, one of the widely studied model organisms, sense external chemical cues and perform relative chemotaxis behaviors through its simple chemosensory neuronal system. To study the mechanism underlying chemosensory behavior, a rapid and reliable method for quantitatively analyzing the worms' behaviors is essential. In this work, we demonstrated a microfluidic approach for investigating chemotaxis responses of worms to chemical gradients. The flow-based microfluidic chip was consisted of circular tree-like microchannels, which was able to generate eight flow streams containing stepwise chemical concentrations without the difference in flow velocity. Worms' upstream swimming into microchannels with various concentrations was monitored for quantitative analysis of the chemotaxis behavior. By using this microfluidic chip, the attractive and repellent responses of C. elegans to NaCl were successfully quantified within several minutes. The results demonstrated the wild type-like repellent responses and severely impaired attractive responses in grk-2 mutant animals with defects in calcium influx. In addition, the chemotaxis analysis of the third stage larvae revealed that its gustatory response was different from that in the adult stage. Thus, our microfluidic method provided a useful platform for studying the chemosensory behaviors of C. elegans and screening of chemosensation-related chemical drugs.

  18. Quantitative analysis in outcome assessment of instrumented lumbosacral arthrodesis.

    PubMed

    Champain, Sabina; Mazel, Christian; Mitulescu, Anca; Skalli, Wafa

    2007-08-01

    The outcome assessment in instrumented lumbosacral fusion mostly focuses on clinical criteria, complications and scores, with a high variability of imaging means, methods of fusion grading and parameters describing degenerative changes, making comparisons between studies difficult. The aim of this retrospective evaluation was to evaluate the interest of quantified radiographic analysis of lumbar spine in global outcome assessment and to highlight the key biomechanical factors involved. Clinical data and Beaujon-Lassale scores were collected for 49 patients who underwent lumbosacral arthrodesis after prior lumbar discectomy (mean follow-up: 5 years). Sagittal standing and lumbar flexion-extension X-ray films allowed quantifying vertebral, lumbar, pelvic and kinematic parameters of the lumbar spine, which were compared to reference values. Statistics were performed to assess evolution for all variables. At long-term follow-up, 90% of patients presented satisfactory clinical outcomes, associated to normal sagittal alignment; vertebral parameters objectified adjacent level degeneration in four cases (8%). Clinical outcome was correlated (r = 0.8) with fusion that was confirmed in 80% of cases, doubtful in 16% and pseudarthrosis seemed to occur in 4% (2) of cases. In addition to clinical data (outcomes comparable to the literature), quantitative analysis accurately described lumbar spine geometry and kinematics, highlighting parameters related to adjacent level's degeneration and a significant correlation between clinical outcome and fusion. Furthermore, criteria proposed to quantitatively evaluate fusion from lumbar dynamic radiographs seem to be appropriate and in agreement with surgeon's qualitative grading in 87% of cases.

  19. Multivariate calibration applied to the quantitative analysis of infrared spectra

    SciTech Connect

    Haaland, D.M.

    1991-01-01

    Multivariate calibration methods are very useful for improving the precision, accuracy, and reliability of quantitative spectral analyses. Spectroscopists can more effectively use these sophisticated statistical tools if they have a qualitative understanding of the techniques involved. A qualitative picture of the factor analysis multivariate calibration methods of partial least squares (PLS) and principal component regression (PCR) is presented using infrared calibrations based upon spectra of phosphosilicate glass thin films on silicon wafers. Comparisons of the relative prediction abilities of four different multivariate calibration methods are given based on Monte Carlo simulations of spectral calibration and prediction data. The success of multivariate spectral calibrations is demonstrated for several quantitative infrared studies. The infrared absorption and emission spectra of thin-film dielectrics used in the manufacture of microelectronic devices demonstrate rapid, nondestructive at-line and in-situ analyses using PLS calibrations. Finally, the application of multivariate spectral calibrations to reagentless analysis of blood is presented. We have found that the determination of glucose in whole blood taken from diabetics can be precisely monitored from the PLS calibration of either mind- or near-infrared spectra of the blood. Progress toward the non-invasive determination of glucose levels in diabetics is an ultimate goal of this research. 13 refs., 4 figs.

  20. Quantitative analysis of live cells using digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Lewis, Tan Rongwei; Qu, Weijuan; Chee, Oi Choo; Singh, Vijay Raj; Asundi, Anand

    2010-03-01

    During the life time of a cell, it goes through changes to the plasma membrane as well as its internal structures especially distinctive during processes like cell division and death. Different types of microscope are used to fulfill the observation of the cell's variation. In our experiment, Vero cells have been investigated by using phase contrast microscopy and digital holographic microscopy (DHM). A comparison of the images obtained for cell division is presented here. The conventional phase contrast microscope provided a good imaging method in the real time analysis of cell division. The off-axis digital hologram recorded by the DHM system can be reconstructed to obtain both the intensity image and phase contrast image of the test object. These can be used for live cell imaging to provide multiple results from a single equipment setup. The DHM system, besides being a qualitative tool, is able to provide quantitative results and 3D images of the cell division process. The ability of DHM to provide quantitative analysis makes it an ideal tool for life science applications.

  1. Quantitative analysis of live cells using digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Lewis, Tan Rongwei; Qu, Weijuan; Chee, Oi Choo; Singh, Vijay Raj; Asundi, Anand

    2009-12-01

    During the life time of a cell, it goes through changes to the plasma membrane as well as its internal structures especially distinctive during processes like cell division and death. Different types of microscope are used to fulfill the observation of the cell's variation. In our experiment, Vero cells have been investigated by using phase contrast microscopy and digital holographic microscopy (DHM). A comparison of the images obtained for cell division is presented here. The conventional phase contrast microscope provided a good imaging method in the real time analysis of cell division. The off-axis digital hologram recorded by the DHM system can be reconstructed to obtain both the intensity image and phase contrast image of the test object. These can be used for live cell imaging to provide multiple results from a single equipment setup. The DHM system, besides being a qualitative tool, is able to provide quantitative results and 3D images of the cell division process. The ability of DHM to provide quantitative analysis makes it an ideal tool for life science applications.

  2. Quantitative analysis of intermolecular interactions in orthorhombic rubrene

    PubMed Central

    Hathwar, Venkatesha R.; Sist, Mattia; Jørgensen, Mads R. V.; Mamakhel, Aref H.; Wang, Xiaoping; Hoffmann, Christina M.; Sugimoto, Kunihisa; Overgaard, Jacob; Iversen, Bo Brummerstedt

    2015-01-01

    Rubrene is one of the most studied organic semiconductors to date due to its high charge carrier mobility which makes it a potentially applicable compound in modern electronic devices. Previous electronic device characterizations and first principles theoretical calculations assigned the semiconducting properties of rubrene to the presence of a large overlap of the extended π-conjugated core between molecules. We present here the electron density distribution in rubrene at 20 K and at 100 K obtained using a combination of high-resolution X-ray and neutron diffraction data. The topology of the electron density and energies of intermolecular interactions are studied quantitatively. Specifically, the presence of Cπ⋯Cπ interactions between neighbouring tetracene backbones of the rubrene molecules is experimentally confirmed from a topological analysis of the electron density, Non-Covalent Interaction (NCI) analysis and the calculated interaction energy of molecular dimers. A significant contribution to the lattice energy of the crystal is provided by H—H interactions. The electron density features of H—H bonding, and the interaction energy of molecular dimers connected by H—H interaction clearly demonstrate an importance of these weak interactions in the stabilization of the crystal structure. The quantitative nature of the intermolecular interactions is virtually unchanged between 20 K and 100 K suggesting that any changes in carrier transport at these low temperatures would have a different origin. The obtained experimental results are further supported by theoretical calculations. PMID:26306198

  3. Quantitative analysis of intermolecular interactions in orthorhombic rubrene

    DOE PAGES

    Hathwar, Venkatesha R.; Sist, Mattia; Jørgensen, Mads R. V.; ...

    2015-08-14

    Rubrene is one of the most studied organic semiconductors to date due to its high charge carrier mobility which makes it a potentially applicable compound in modern electronic devices. Previous electronic device characterizations and first principles theoretical calculations assigned the semiconducting properties of rubrene to the presence of a large overlap of the extended π-conjugated core between molecules. We present here the electron density distribution in rubrene at 20 K and at 100 K obtained using a combination of high-resolution X-ray and neutron diffraction data. The topology of the electron density and energies of intermolecular interactions are studied quantitatively. Specifically,more » the presence of Cπ...Cπinteractions between neighbouring tetracene backbones of the rubrene molecules is experimentally confirmed from a topological analysis of the electron density, Non-Covalent Interaction (NCI) analysis and the calculated interaction energy of molecular dimers. A significant contribution to the lattice energy of the crystal is provided by H—H interactions. The electron density features of H—H bonding, and the interaction energy of molecular dimers connected by H—H interaction clearly demonstrate an importance of these weak interactions in the stabilization of the crystal structure. Finally, the quantitative nature of the intermolecular interactions is virtually unchanged between 20 K and 100 K suggesting that any changes in carrier transport at these low temperatures would have a different origin. The obtained experimental results are further supported by theoretical calculations.« less

  4. Quantitative Analysis of the Interdisciplinarity of Applied Mathematics.

    PubMed

    Xie, Zheng; Duan, Xiaojun; Ouyang, Zhenzheng; Zhang, Pengyuan

    2015-01-01

    The increasing use of mathematical techniques in scientific research leads to the interdisciplinarity of applied mathematics. This viewpoint is validated quantitatively here by statistical and network analysis on the corpus PNAS 1999-2013. A network describing the interdisciplinary relationships between disciplines in a panoramic view is built based on the corpus. Specific network indicators show the hub role of applied mathematics in interdisciplinary research. The statistical analysis on the corpus content finds that algorithms, a primary topic of applied mathematics, positively correlates, increasingly co-occurs, and has an equilibrium relationship in the long-run with certain typical research paradigms and methodologies. The finding can be understood as an intrinsic cause of the interdisciplinarity of applied mathematics.

  5. [Quantitative analysis of transformer oil dissolved gases using FTIR].

    PubMed

    Zhao, An-xin; Tang, Xiao-jun; Wang, Er-zhen; Zhang, Zhong-hua; Liu, Jun-hua

    2013-09-01

    For the defects of requiring carrier gas and regular calibration, and low safety using chromatography to on line monitor transformer dissolved gases, it was attempted to establish a dissolved gas analysis system based on Fourier transform infrared spectroscopy. Taking into account the small amount of characteristic gases, many components, detection limit and safety requirements and the difficulty of degasser to put an end to the presence of interference gas, the quantitative analysis model was established based on sparse partial least squares, piecewise section correction and feature variable extraction algorithm using improvement TR regularization. With the characteristic gas of CH4, C2H6, C2H6, and CO2, the results show that using FTIR meets DGA requirements with the spectrum wave number resolution of 1 cm(-1) and optical path of 10 cm.

  6. Quantitative Analysis of the Interdisciplinarity of Applied Mathematics

    PubMed Central

    Zhang, Pengyuan

    2015-01-01

    The increasing use of mathematical techniques in scientific research leads to the interdisciplinarity of applied mathematics. This viewpoint is validated quantitatively here by statistical and network analysis on the corpus PNAS 1999–2013. A network describing the interdisciplinary relationships between disciplines in a panoramic view is built based on the corpus. Specific network indicators show the hub role of applied mathematics in interdisciplinary research. The statistical analysis on the corpus content finds that algorithms, a primary topic of applied mathematics, positively correlates, increasingly co-occurs, and has an equilibrium relationship in the long-run with certain typical research paradigms and methodologies. The finding can be understood as an intrinsic cause of the interdisciplinarity of applied mathematics. PMID:26352604

  7. Fusing Quantitative Requirements Analysis with Model-based Systems Engineering

    NASA Technical Reports Server (NTRS)

    Cornford, Steven L.; Feather, Martin S.; Heron, Vance A.; Jenkins, J. Steven

    2006-01-01

    A vision is presented for fusing quantitative requirements analysis with model-based systems engineering. This vision draws upon and combines emergent themes in the engineering milieu. "Requirements engineering" provides means to explicitly represent requirements (both functional and non-functional) as constraints and preferences on acceptable solutions, and emphasizes early-lifecycle review, analysis and verification of design and development plans. "Design by shopping" emphasizes revealing the space of options available from which to choose (without presuming that all selection criteria have previously been elicited), and provides means to make understandable the range of choices and their ramifications. "Model-based engineering" emphasizes the goal of utilizing a formal representation of all aspects of system design, from development through operations, and provides powerful tool suites that support the practical application of these principles. A first step prototype towards this vision is described, embodying the key capabilities. Illustrations, implications, further challenges and opportunities are outlined.

  8. Quantitative Phase Analysis by the Rietveld Method for Forensic Science.

    PubMed

    Deng, Fei; Lin, Xiaodong; He, Yonghong; Li, Shu; Zi, Run; Lai, Shijun

    2015-07-01

    Quantitative phase analysis (QPA) is helpful to determine the type attribute of the object because it could present the content of the constituents. QPA by Rietveld method requires neither measurement of calibration data nor the use of an internal standard; however, the approximate crystal structure of each phase in a mixture is necessary. In this study, 8 synthetic mixtures composed of potassium nitrate and sulfur were analyzed by Rietveld QPA method. The Rietveld refinement was accomplished with a material analysis using diffraction program and evaluated by three agreement indices. Results showed that Rietveld QPA yielded precise results, with errors generally less than 2.0% absolute. In addition, a criminal case which was broken successfully with the help of Rietveld QPA method was also introduced. This method will allow forensic investigators to acquire detailed information of the material evidence, which could point out the direction for case detection and court proceedings.

  9. 3D quantitative analysis of brain SPECT images

    NASA Astrophysics Data System (ADS)

    Loncaric, Sven; Ceskovic, Ivan; Petrovic, Ratimir; Loncaric, Srecko

    2001-07-01

    The main purpose of this work is to develop a computer-based technique for quantitative analysis of 3-D brain images obtained by single photon emission computed tomography (SPECT). In particular, the volume and location of ischemic lesion and penumbra is important for early diagnosis and treatment of infracted regions of the brain. SPECT imaging is typically used as diagnostic tool to assess the size and location of the ischemic lesion. The segmentation method presented in this paper utilizes a 3-D deformable model in order to determine size and location of the regions of interest. The evolution of the model is computed using a level-set implementation of the algorithm. In addition to 3-D deformable model the method utilizes edge detection and region growing for realization of a pre-processing. Initial experimental results have shown that the method is useful for SPECT image analysis.

  10. Quantitative morphometric analysis for the tectonic characterisation of northern Tunisia.

    NASA Astrophysics Data System (ADS)

    Camafort, Miquel; Pérez-Peña, José Vicente; Booth-Rea, Guillermo; Ranero, César R.; Gràcia, Eulàlia; Azañón, José Miguel; Melki, Fetheddine; Ouadday, Mohamed

    2016-04-01

    Northern Tunisia is characterized by low deformation rates and low to moderate seismicity. Although instrumental seismicity reaches maximum magnitudes of Mw 5.5, some historical earthquakes have occurred with catastrophic consequences in this region. Aiming to improve our knowledge of active tectonics in Tunisia, we carried out both a quantitative morphometric analysis and field study in the north-western region. We applied different morphometric tools, like river profiles, knickpoint analysis, hypsometric curves and integrals and drainage pattern anomalies in order to differentiate between zones with high or low recent tectonic activity. This analysis helps identifying uplift and subsidence zones, which we relate to fault activity. Several active faults in a sparse distribution were identified. A selected sector was studied with a field campaign to test the results obtained with the quantitative analysis. During the fieldwork we identified geological evidence of recent activity and a considerable seismogenic potential along El Alia-Teboursouk (ETF) and Dkhila (DF) faults. The ETF fault could be responsible of one of the most devastating historical earthquakes in northern Tunisia that destroyed Utique in 412 A.D. Geological evidence include fluvial terraces folded by faults, striated and cracked pebbles, clastic dikes, sand volcanoes, coseismic cracks, etc. Although not reflected in the instrumental seismicity, our results support an important seismic hazard, evidenced by the several active tectonic structures identified and the two seismogenic faults described. After obtaining the current active tectonic framework of Tunisia we discuss our results within the western Mediterranean trying to contribute to the understanding of the western Mediterranean tectonic context. With our results, we suggest that the main reason explaining the sparse and scarce seismicity of the area in contrast with the adjacent parts of the Nubia-Eurasia boundary is due to its extended

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

    SciTech Connect

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

    2010-09-17

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

  12. Quantitative analysis of cyclic beta-turn models.

    PubMed Central

    Perczel, A.; Fasman, G. D.

    1992-01-01

    The beta-turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the alpha-helix and beta-pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of beta-turns. Recently, it has been reported (Hollósi, M., Kövér, K.E., Holly, S., Radics, L., & Fasman, G.D., 1987, Biopolymers 26, 1527-1572; Perczel, A., Hollósi, M., Foxman, B.M., & Fasman, G.D., 1991a, J. Am. Chem. Soc. 113, 9772-9784) that some pseudohexapeptides (e.g., the cyclo[(delta)Ava-Gly-Pro-Aaa-Gly] where Aaa = Ser, Ser(OtBu), or Gly) in many solvents adopt a conformational mixture of type I and the type II beta-turns, although the X-ray-determined conformation was an ideal type I beta-turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above beta-turn models has an influence on their conformational properties. Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type II beta-turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnády, G., Hollósi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669-679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 beta-turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major beta-turns by CD analysis. These results

  13. Quantitative image analysis in sonograms of the thyroid gland

    NASA Astrophysics Data System (ADS)

    Catherine, Skouroliakou; Maria, Lyra; Aristides, Antoniou; Lambros, Vlahos

    2006-12-01

    High-resolution, real-time ultrasound is a routine examination for assessing the disorders of the thyroid gland. However, the current diagnosis practice is based mainly on qualitative evaluation of the resulting sonograms, therefore depending on the physician's experience. Computerized texture analysis is widely employed in sonographic images of various organs (liver, breast), and it has been proven to increase the sensitivity of diagnosis by providing a better tissue characterization. The present study attempts to characterize thyroid tissue by automatic texture analysis. The texture features that are calculated are based on co-occurrence matrices as they have been proposed by Haralick. The sample consists of 40 patients. For each patient two sonographic images (one for each lobe) are recorded in DICOM format. The lobe is manually delineated in each sonogram, and the co-occurrence matrices for 52 separation vectors are calculated. The texture features extracted from each one of these matrices are: contrast, correlation, energy and homogeneity. Primary component analysis is used to select the optimal set of features. The statistical analysis resulted in the extraction of 21 optimal descriptors. The optimal descriptors are all co-occurrence parameters as the first-order statistics did not prove to be representative of the images characteristics. The bigger number of components depends mainly on correlation for very close or very far distances. The results indicate that quantitative analysis of thyroid sonograms can provide an objective characterization of thyroid tissue.

  14. In vivo osteogenesis assay: a rapid method for quantitative analysis.

    PubMed

    Dennis, J E; Konstantakos, E K; Arm, D; Caplan, A I

    1998-08-01

    A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale

  15. Phenotypic analysis of Arabidopsis mutants: quantitative analysis of root growth.

    PubMed

    Doerner, Peter

    2008-03-01

    INTRODUCTIONThe growth of plant roots is very easy to measure and is particularly straightforward in Arabidopsis thaliana, because the increase in organ size is essentially restricted to one dimension. The precise measurement of root apical growth can be used to accurately determine growth activity (the rate of growth at a given time) during development in mutants, transgenic backgrounds, or in response to experimental treatments. Root growth is measured in a number of ways, the simplest of which is to grow the seedlings in a Petri dish and record the position of the advancing root tip at appropriate time points. The increase in root length is measured with a ruler and the data are entered into Microsoft Excel for analysis. When dealing with large numbers of seedlings, however, this procedure can be tedious, as well as inaccurate. An alternative approach, described in this protocol, uses "snapshots" of the growing plants, which are taken using gel-documentation equipment (i.e., a video camera with a frame-grabber unit, now commonly used to capture images from ethidium-bromide-stained electrophoresis gels). The images are analyzed using publicly available software (NIH-Image), which allows the user simply to cut and paste data into Microsoft Excel.

  16. Functional Linear Models for Association Analysis of Quantitative Traits

    PubMed Central

    Fan, Ruzong; Wang, Yifan; Mills, James L.; Wilson, Alexander F.; Bailey-Wilson, Joan E.; Xiong, Momiao

    2014-01-01

    Functional linear models are developed in this paper for testing associations between quantitative traits and genetic variants, which can be rare variants or common variants or the combination of the two. By treating multiple genetic variants of an individual in a human population as a realization of a stochastic process, the genome of an individual in a chromosome region is a continuum of sequence data rather than discrete observations. The genome of an individual is viewed as a stochastic function that contains both linkage and linkage disequilibrium (LD) information of the genetic markers. By using techniques of functional data analysis, both fixed and mixed effect functional linear models are built to test the association between quantitative traits and genetic variants adjusting for covariates. After extensive simulation analysis, it is shown that the F-distributed tests of the proposed fixed effect functional linear models have higher power than that of sequence kernel association test (SKAT) and its optimal unified test (SKAT-O) for three scenarios in most cases: (1) the causal variants are all rare, (2) the causal variants are both rare and common, and (3) the causal variants are common. The superior performance of the fixed effect functional linear models is most likely due to its optimal utilization of both genetic linkage and LD information of multiple genetic variants in a genome and similarity among different individuals, while SKAT and SKAT-O only model the similarities and pairwise LD but do not model linkage and higher order LD information sufficiently. In addition, the proposed fixed effect models generate accurate type I error rates in simulation studies. We also show that the functional kernel score tests of the proposed mixed effect functional linear models are preferable in candidate gene analysis and small sample problems. The methods are applied to analyze three biochemical traits in data from the Trinity Students Study. PMID:24130119

  17. Quantitative Analysis of Single-Molecule RNA-Protein Interaction

    PubMed Central

    Fuhrmann, Alexander; Schoening, Jan C.; Anselmetti, Dario; Staiger, Dorothee; Ros, Robert

    2009-01-01

    Abstract RNA-binding proteins impact gene expression at the posttranscriptional level by interacting with cognate cis elements within the transcripts. Here, we apply dynamic single-molecule force spectroscopy to study the interaction of the Arabidopsis glycine-rich RNA-binding protein AtGRP8 with its RNA target. A dwell-time-dependent analysis of the single-molecule data in combination with competition assays and site-directed mutagenesis of both the RNA target and the RNA-binding domain of the protein allowed us to distinguish and quantify two different binding modes. For dwell times <0.21 s an unspecific complex with a lifetime of 0.56 s is observed, whereas dwell times >0.33 s result in a specific interaction with a lifetime of 208 s. The corresponding reaction lengths are 0.28 nm for the unspecific and 0.55 nm for the specific AtGRP8-RNA interactions, indicating formation of a tighter complex with increasing dwell time. These two binding modes cannot be dissected in ensemble experiments. Quantitative titration in RNA bandshift experiments yields an ensemble-averaged equilibrium constant of dissociation of KD = 2 × 10−7 M. Assuming comparable on-rates for the specific and nonspecific binding modes allows us to estimate their free energies as ΔG0 = −42 kJ/mol and ΔG0 = −28 kJ/mol for the specific and nonspecific binding modes, respectively. Thus, we show that single-molecule force spectroscopy with a refined statistical analysis is a potent tool for the analysis of protein-RNA interactions without the drawback of ensemble averaging. This makes it possible to discriminate between different binding modes or sites and to analyze them quantitatively. We propose that this method could be applied to complex interactions of biomolecules in general, and be of particular interest for the investigation of multivalent binding reactions. PMID:19527663

  18. Functional linear models for association analysis of quantitative traits.

    PubMed

    Fan, Ruzong; Wang, Yifan; Mills, James L; Wilson, Alexander F; Bailey-Wilson, Joan E; Xiong, Momiao

    2013-11-01

    Functional linear models are developed in this paper for testing associations between quantitative traits and genetic variants, which can be rare variants or common variants or the combination of the two. By treating multiple genetic variants of an individual in a human population as a realization of a stochastic process, the genome of an individual in a chromosome region is a continuum of sequence data rather than discrete observations. The genome of an individual is viewed as a stochastic function that contains both linkage and linkage disequilibrium (LD) information of the genetic markers. By using techniques of functional data analysis, both fixed and mixed effect functional linear models are built to test the association between quantitative traits and genetic variants adjusting for covariates. After extensive simulation analysis, it is shown that the F-distributed tests of the proposed fixed effect functional linear models have higher power than that of sequence kernel association test (SKAT) and its optimal unified test (SKAT-O) for three scenarios in most cases: (1) the causal variants are all rare, (2) the causal variants are both rare and common, and (3) the causal variants are common. The superior performance of the fixed effect functional linear models is most likely due to its optimal utilization of both genetic linkage and LD information of multiple genetic variants in a genome and similarity among different individuals, while SKAT and SKAT-O only model the similarities and pairwise LD but do not model linkage and higher order LD information sufficiently. In addition, the proposed fixed effect models generate accurate type I error rates in simulation studies. We also show that the functional kernel score tests of the proposed mixed effect functional linear models are preferable in candidate gene analysis and small sample problems. The methods are applied to analyze three biochemical traits in data from the Trinity Students Study.

  19. The Quantitative Analysis of Chennai Automotive Industry Cluster

    NASA Astrophysics Data System (ADS)

    Bhaskaran, Ethirajan

    2016-07-01

    Chennai, also called as Detroit of India due to presence of Automotive Industry producing over 40 % of the India's vehicle and components. During 2001-2002, the Automotive Component Industries (ACI) in Ambattur, Thirumalizai and Thirumudivakkam Industrial Estate, Chennai has faced problems on infrastructure, technology, procurement, production and marketing. The objective is to study the Quantitative Performance of Chennai Automotive Industry Cluster before (2001-2002) and after the CDA (2008-2009). The methodology adopted is collection of primary data from 100 ACI using quantitative questionnaire and analyzing using Correlation Analysis (CA), Regression Analysis (RA), Friedman Test (FMT), and Kruskall Wallis Test (KWT).The CA computed for the different set of variables reveals that there is high degree of relationship between the variables studied. The RA models constructed establish the strong relationship between the dependent variable and a host of independent variables. The models proposed here reveal the approximate relationship in a closer form. KWT proves, there is no significant difference between three locations clusters with respect to: Net Profit, Production Cost, Marketing Costs, Procurement Costs and Gross Output. This supports that each location has contributed for development of automobile component cluster uniformly. The FMT proves, there is no significant difference between industrial units in respect of cost like Production, Infrastructure, Technology, Marketing and Net Profit. To conclude, the Automotive Industries have fully utilized the Physical Infrastructure and Centralised Facilities by adopting CDA and now exporting their products to North America, South America, Europe, Australia, Africa and Asia. The value chain analysis models have been implemented in all the cluster units. This Cluster Development Approach (CDA) model can be implemented in industries of under developed and developing countries for cost reduction and productivity

  20. Advance in orientation microscopy: quantitative analysis of nanocrystalline structures.

    PubMed

    Seyring, Martin; Song, Xiaoyan; Rettenmayr, Markus

    2011-04-26

    The special properties of nanocrystalline materials are generally accepted to be a consequence of the high density of planar defects (grain and twin boundaries) and their characteristics. However, until now, nanograin structures have not been characterized with similar detail and statistical relevance as coarse-grained materials, due to the lack of an appropriate method. In the present paper, a novel method based on quantitative nanobeam diffraction in transmission electron microscopy (TEM) is presented to determine the misorientation of adjacent nanograins and subgrains. Spatial resolution of <5 nm can be achieved. This method is applicable to characterize orientation relationships in wire, film, and bulk materials with nanocrystalline structures. As a model material, nanocrystalline Cu is used. Several important features of the nanograin structure are discovered utilizing quantitative analysis: the fraction of twin boundaries is substantially higher than that observed in bright-field images in the TEM; small angle grain boundaries are prominent; there is an obvious dependence of the grain boundary characteristics on grain size distribution and mean grain size.

  1. Quantitative analysis of tumor burden in mouse lung via MRI.

    PubMed

    Tidwell, Vanessa K; Garbow, Joel R; Krupnick, Alexander S; Engelbach, John A; Nehorai, Arye

    2012-02-01

    Lung cancer is the leading cause of cancer death in the United States. Despite recent advances in screening protocols, the majority of patients still present with advanced or disseminated disease. Preclinical rodent models provide a unique opportunity to test novel therapeutic drugs for targeting lung cancer. Respiratory-gated MRI is a key tool for quantitatively measuring lung-tumor burden and monitoring the time-course progression of individual tumors in mouse models of primary and metastatic lung cancer. However, quantitative analysis of lung-tumor burden in mice by MRI presents significant challenges. Herein, a method for measuring tumor burden based upon average lung-image intensity is described and validated. The method requires accurate lung segmentation; its efficiency and throughput would be greatly aided by the ability to automatically segment the lungs. A technique for automated lung segmentation in the presence of varying tumor burden levels is presented. The method includes development of a new, two-dimensional parametric model of the mouse lungs and a multi-faceted cost function to optimally fit the model parameters to each image. Results demonstrate a strong correlation (0.93), comparable with that of fully manual expert segmentation, between the automated method's tumor-burden metric and the tumor burden measured by lung weight.

  2. Quantitative Analysis of Peripheral Tissue Perfusion Using Spatiotemporal Molecular Dynamics

    PubMed Central

    Lee, Jungsul; Koh, Gou Young; Kwon, Kihwan; Choi, Chulhee

    2009-01-01

    Background Accurate measurement of peripheral tissue perfusion is challenging but necessary to diagnose peripheral vascular insufficiency. Because near infrared (NIR) radiation can penetrate relatively deep into tissue, significant attention has been given to intravital NIR fluorescence imaging. Methodology/Principal Findings We developed a new optical imaging-based strategy for quantitative measurement of peripheral tissue perfusion by time-series analysis of local pharmacokinetics of the NIR fluorophore, indocyanine green (ICG). Time-series NIR fluorescence images were obtained after injecting ICG intravenously in a murine hindlimb ischemia model. Mathematical modeling and computational simulations were used for translating time-series ICG images into quantitative pixel perfusion rates and a perfusion map. We could successfully predict the prognosis of ischemic hindlimbs based on the perfusion profiles obtained immediately after surgery, which were dependent on the preexisting collaterals. This method also reflected increases in perfusion and improvements in prognosis of ischemic hindlimbs induced by treatment with vascular endothelial growth factor and COMP-angiopoietin-1. Conclusions/Significance We propose that this novel NIR-imaging-based strategy is a powerful tool for biomedical studies related to the evaluation of therapeutic interventions directed at stimulating angiogenesis. PMID:19169354

  3. Quantitative analysis of incipient mineral loss in hard tissues

    NASA Astrophysics Data System (ADS)

    Matvienko, Anna; Mandelis, Andreas; Hellen, Adam; Jeon, Raymond; Abrams, Stephen; Amaechi, Bennett

    2009-02-01

    A coupled diffuse-photon-density-wave and thermal-wave theoretical model was developed to describe the biothermophotonic phenomena in multi-layered hard tissue structures. Photothermal Radiometry was applied as a safe, non-destructive, and highly sensitive tool for the detection of early tooth enamel demineralization to test the theory. Extracted human tooth was treated sequentially with an artificial demineralization gel to simulate controlled mineral loss in the enamel. The experimental setup included a semiconductor laser (659 nm, 120 mW) as the source of the photothermal signal. Modulated laser light generated infrared blackbody radiation from teeth upon absorption and nonradiative energy conversion. The infrared flux emitted by the treated region of the tooth surface and sub-surface was monitored with an infrared detector, both before and after treatment. Frequency scans with a laser beam size of 3 mm were performed in order to guarantee one-dimensionality of the photothermal field. TMR images showed clear differences between sound and demineralized enamel, however this technique is destructive. Dental radiographs did not indicate any changes. The photothermal signal showed clear change even after 1 min of gel treatment. As a result of the fittings, thermal and optical properties of sound and demineralized enamel were obtained, which allowed for quantitative differentiation of healthy and non-healthy regions. In conclusion, the developed model was shown to be a promising tool for non-invasive quantitative analysis of early demineralization of hard tissues.

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

    PubMed Central

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

    2016-01-01

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

  5. Quantitative multi-image analysis for biomedical Raman spectroscopic imaging.

    PubMed

    Hedegaard, Martin A B; Bergholt, Mads S; Stevens, Molly M

    2016-05-01

    Imaging by Raman spectroscopy enables unparalleled label-free insights into cell and tissue composition at the molecular level. With established approaches limited to single image analysis, there are currently no general guidelines or consensus on how to quantify biochemical components across multiple Raman images. Here, we describe a broadly applicable methodology for the combination of multiple Raman images into a single image for analysis. This is achieved by removing image specific background interference, unfolding the series of Raman images into a single dataset, and normalisation of each Raman spectrum to render comparable Raman images. Multivariate image analysis is finally applied to derive the contributing 'pure' biochemical spectra for relative quantification. We present our methodology using four independently measured Raman images of control cells and four images of cells treated with strontium ions from substituted bioactive glass. We show that the relative biochemical distribution per area of the cells can be quantified. In addition, using k-means clustering, we are able to discriminate between the two cell types over multiple Raman images. This study shows a streamlined quantitative multi-image analysis tool for improving cell/tissue characterisation and opens new avenues in biomedical Raman spectroscopic imaging.

  6. Quantitative three-dimensional holographic interferometry for flow field analysis

    NASA Astrophysics Data System (ADS)

    Holden, C. M. E.; Parker, S. C. J.; Bryanston-Cross, P. J.

    Holographic interferometry offers the potential for quantitative, wholefield analysis of three-dimensional compressible flows. The technique is non-intrusive, does not require the introduction of seeding particles, and records the entire flow information within the pulse duration of a Q-switched ruby laser (~30ns). At present, however, holographic interferometry is mainly used qualitatively due to the practical restrictions of data recording, acquisition and processing. To address the potential of holographic flow analysis a prototype multi-channel interferometer has been designed and preliminary wind tunnel results have been obtained. The proposed configuration uses specular illumination which, unlike comparable diffuse systems, does not suffer from fringe localisation and speckle noise. Beam collimation and steering through the flow field is achieved in a single operation by the use of holographic optical elements (HOEs). The resulting design is compact, light efficient, has aberration compensation, and the recorded data are conducive to both tomographic analysis and direct comparison to computational fluid dynamics (CFD) predictions. Holograms have been recorded of simple two-dimensional and axisymmetric compressible flows, to compare the accuracy of holographic density measurements with data from conventional pressure sensors and CFD codes. Data extraction from the holograms, and the elimination of rigid body motion, was achieved using digital Fourier transform fringe analysis. The introduction of phase errors by image processing has been investigated by analysing simulated fringe patterns generated from a combination of experimental amplitude information and computer generated phase data.

  7. Multipoint quantitative-trait linkage analysis in general pedigrees.

    PubMed Central

    Almasy, L; Blangero, J

    1998-01-01

    Multipoint linkage analysis of quantitative-trait loci (QTLs) has previously been restricted to sibships and small pedigrees. In this article, we show how variance-component linkage methods can be used in pedigrees of arbitrary size and complexity, and we develop a general framework for multipoint identity-by-descent (IBD) probability calculations. We extend the sib-pair multipoint mapping approach of Fulker et al. to general relative pairs. This multipoint IBD method uses the proportion of alleles shared identical by descent at genotyped loci to estimate IBD sharing at arbitrary points along a chromosome for each relative pair. We have derived correlations in IBD sharing as a function of chromosomal distance for relative pairs in general pedigrees and provide a simple framework whereby these correlations can be easily obtained for any relative pair related by a single line of descent or by multiple independent lines of descent. Once calculated, the multipoint relative-pair IBDs can be utilized in variance-component linkage analysis, which considers the likelihood of the entire pedigree jointly. Examples are given that use simulated data, demonstrating both the accuracy of QTL localization and the increase in power provided by multipoint analysis with 5-, 10-, and 20-cM marker maps. The general pedigree variance component and IBD estimation methods have been implemented in the SOLAR (Sequential Oligogenic Linkage Analysis Routines) computer package. PMID:9545414

  8. Quantitative analysis of the reconstruction performance of interpolants

    NASA Technical Reports Server (NTRS)

    Lansing, Donald L.; Park, Stephen K.

    1987-01-01

    The analysis presented provides a quantitative measure of the reconstruction or interpolation performance of linear, shift-invariant interpolants. The performance criterion is the mean square error of the difference between the sampled and reconstructed functions. The analysis is applicable to reconstruction algorithms used in image processing and to many types of splines used in numerical analysis and computer graphics. When formulated in the frequency domain, the mean square error clearly separates the contribution of the interpolation method from the contribution of the sampled data. The equations provide a rational basis for selecting an optimal interpolant; that is, one which minimizes the mean square error. The analysis has been applied to a selection of frequently used data splines and reconstruction algorithms: parametric cubic and quintic Hermite splines, exponential and nu splines (including the special case of the cubic spline), parametric cubic convolution, Keys' fourth-order cubic, and a cubic with a discontinuous first derivative. The emphasis in this paper is on the image-dependent case in which no a priori knowledge of the frequency spectrum of the sampled function is assumed.

  9. Quantitative multielement analysis using high energy particle bombardment

    NASA Technical Reports Server (NTRS)

    Clark, P. J.; Neal, G. F.; Allen, R. O.

    1974-01-01

    Charged particles ranging in energy from 0.8 to 4.0 MeV are used to induce resonant nuclear reactions, Coulomb excitation (gamma X-rays), and X-ray emission in both thick and thin targets. Quantitative analysis is possible for elements from Li to Pb in complex environmental samples, although the matrix can severely reduce the sensitivity. It is necessary to use a comparator technique for the gamma-rays, while for X-rays an internal standard can be used. A USGS standard rock is analyzed for a total of 28 elements. Water samples can be analyzed either by nebulizing the sample doped with Cs or Y onto a thin formvar film or by extracting the sample (with or without an internal standard) onto ion exchange resin which is pressed into a pellet.

  10. Large-Scale Quantitative Analysis of Painting Arts

    PubMed Central

    Kim, Daniel; Son, Seung-Woo; Jeong, Hawoong

    2014-01-01

    Scientists have made efforts to understand the beauty of painting art in their own languages. As digital image acquisition of painting arts has made rapid progress, researchers have come to a point where it is possible to perform statistical analysis of a large-scale database of artistic paints to make a bridge between art and science. Using digital image processing techniques, we investigate three quantitative measures of images – the usage of individual colors, the variety of colors, and the roughness of the brightness. We found a difference in color usage between classical paintings and photographs, and a significantly low color variety of the medieval period. Interestingly, moreover, the increment of roughness exponent as painting techniques such as chiaroscuro and sfumato have advanced is consistent with historical circumstances. PMID:25501877

  11. Quantitative genetic analysis of injury liability in infants and toddlers

    SciTech Connect

    Phillips, K.; Matheny, A.P. Jr.

    1995-02-27

    A threshold model of latent liability was applied to infant and toddler twin data on total count of injuries sustained during the interval from birth to 36 months of age. A quantitative genetic analysis of estimated twin correlations in injury liability indicated strong genetic dominance effects, but no additive genetic variance was detected. Because interpretations involving overdominance have little research support, the results may be due to low order epistasis or other interaction effects. Boys had more injuries than girls, but this effect was found only for groups whose parents were prompted and questioned in detail about their children`s injuries. Activity and impulsivity are two behavioral predictors of childhood injury, and the results are discussed in relation to animal research on infant and adult activity levels, and impulsivity in adult humans. Genetic epidemiological approaches to childhood injury should aid in targeting higher risk children for preventive intervention. 30 refs., 4 figs., 3 tabs.

  12. Quantitative multielement analysis using high energy particle bombardment

    NASA Technical Reports Server (NTRS)

    Clark, P. J.; Neal, G. F.; Allen, R. O.

    1975-01-01

    Charged particles ranging in energy from 0.8 to 4.0 MeV are used to induce resonant nuclear reactions, Coulomb excitation (gamma-rays), and X-ray emission in both thick and thin targets. Quantitative analysis is possible for elements from Li to Pb in complex environmental samples, although the matrix can severely reduce the sensitivity. It is necessary to use a comparator technique for the gamma-rays while for X-rays an internal standard can be used. A USGS standard rock is analyzed for a total of 28 elements. Water samples can be analyzed either by nebulizing the sample doped with Cs or Y a thin Formvar film or by extracting the sample onto ion exchange resin which is pressed into a pellet.

  13. Large-Scale Quantitative Analysis of Painting Arts

    NASA Astrophysics Data System (ADS)

    Kim, Daniel; Son, Seung-Woo; Jeong, Hawoong

    2014-12-01

    Scientists have made efforts to understand the beauty of painting art in their own languages. As digital image acquisition of painting arts has made rapid progress, researchers have come to a point where it is possible to perform statistical analysis of a large-scale database of artistic paints to make a bridge between art and science. Using digital image processing techniques, we investigate three quantitative measures of images - the usage of individual colors, the variety of colors, and the roughness of the brightness. We found a difference in color usage between classical paintings and photographs, and a significantly low color variety of the medieval period. Interestingly, moreover, the increment of roughness exponent as painting techniques such as chiaroscuro and sfumato have advanced is consistent with historical circumstances.

  14. Quantitative image analysis of WE43-T6 cracking behavior

    NASA Astrophysics Data System (ADS)

    Ahmad, A.; Yahya, Z.

    2013-06-01

    Environment-assisted cracking of WE43 cast magnesium (4.2 wt.% Yt, 2.3 wt.% Nd, 0.7% Zr, 0.8% HRE) in the T6 peak-aged condition was induced in ambient air in notched specimens. The mechanism of fracture was studied using electron backscatter diffraction, serial sectioning and in situ observations of crack propagation. The intermetallic (rare earthed-enriched divorced intermetallic retained at grain boundaries and predominantly at triple points) material was found to play a significant role in initiating cracks which leads to failure of this material. Quantitative measurements were required for this project. The populations of the intermetallic and clusters of intermetallic particles were analyzed using image analysis of metallographic images. This is part of the work to generate a theoretical model of the effect of notch geometry on the static fatigue strength of this material.

  15. Quantitative analysis of gallstones using laser-induced breakdown spectroscopy

    SciTech Connect

    Singh, Vivek K.; Singh, Vinita; Rai, Awadhesh K.; Thakur, Surya N.; Rai, Pradeep K.; Singh, Jagdish P

    2008-11-01

    The utility of laser-induced breakdown spectroscopy (LIBS) for categorizing different types of gallbladder stone has been demonstrated by analyzing their major and minor constituents. LIBS spectra of three types of gallstone have been recorded in the 200-900 nm spectral region. Calcium is found to be the major element in all types of gallbladder stone. The spectrophotometric method has been used to classify the stones. A calibration-free LIBS method has been used for the quantitative analysis of metal elements, and the results have been compared with those obtained from inductively coupled plasma atomic emission spectroscopy (ICP-AES) measurements. The single-shot LIBS spectra from different points on the cross section (in steps of 0.5 mm from one end to the other) of gallstones have also been recorded to study the variation of constituents from the center to the surface. The presence of different metal elements and their possible role in gallstone formation is discussed.

  16. Automatic analysis of quantitative NMR data of pharmaceutical compound libraries.

    PubMed

    Liu, Xuejun; Kolpak, Michael X; Wu, Jiejun; Leo, Gregory C

    2012-08-07

    In drug discovery, chemical library compounds are usually dissolved in DMSO at a certain concentration and then distributed to biologists for target screening. Quantitative (1)H NMR (qNMR) is the preferred method for the determination of the actual concentrations of compounds because the relative single proton peak areas of two chemical species represent the relative molar concentrations of the two compounds, that is, the compound of interest and a calibrant. Thus, an analyte concentration can be determined using a calibration compound at a known concentration. One particularly time-consuming step in the qNMR analysis of compound libraries is the manual integration of peaks. In this report is presented an automated method for performing this task without prior knowledge of compound structures and by using an external calibration spectrum. The script for automated integration is fast and adaptable to large-scale data sets, eliminating the need for manual integration in ~80% of the cases.

  17. Quantitative analysis of forest island pattern in selected Ohio landscapes

    SciTech Connect

    Bowen, G.W.; Burgess, R.L.

    1981-07-01

    The purpose of this study was to quantitatively describe the various aspects of regional distribution patterns of forest islands and relate those patterns to other landscape features. Several maps showing the forest cover of various counties in Ohio were selected as representative examples of forest patterns to be quantified. Ten thousand hectare study areas (landscapes) were delineated on each map. A total of 15 landscapes representing a wide variety of forest island patterns was chosen. Data were converted into a series of continuous variables which contained information pertinent to the sizes, shape, numbers, and spacing of woodlots within a landscape. The continuous variables were used in a factor analysis to describe the variation among landscapes in terms of forest island pattern. The results showed that forest island patterns are related to topography and other environmental features correlated with topography.

  18. Quantitative microstructure analysis of polymer-modified mortars.

    PubMed

    Jenni, A; Herwegh, M; Zurbriggen, R; Aberle, T; Holzer, L

    2003-11-01

    Digital light, fluorescence and electron microscopy in combination with wavelength-dispersive spectroscopy were used to visualize individual polymers, air voids, cement phases and filler minerals in a polymer-modified cementitious tile adhesive. In order to investigate the evolution and processes involved in formation of the mortar microstructure, quantifications of the phase distribution in the mortar were performed including phase-specific imaging and digital image analysis. The required sample preparation techniques and imaging related topics are discussed. As a form of case study, the different techniques were applied to obtain a quantitative characterization of a specific mortar mixture. The results indicate that the mortar fractionates during different stages ranging from the early fresh mortar until the final hardened mortar stage. This induces process-dependent enrichments of the phases at specific locations in the mortar. The approach presented provides important information for a comprehensive understanding of the functionality of polymer-modified mortars.

  19. Quantitative analysis in outcome assessment of instrumented lumbosacral arthrodesis

    PubMed Central

    Mazel, Christian; Mitulescu, Anca

    2007-01-01

    The outcome assessment in instrumented lumbosacral fusion mostly focuses on clinical criteria, complications and scores, with a high variability of imaging means, methods of fusion grading and parameters describing degenerative changes, making comparisons between studies difficult. The aim of this retrospective evaluation was to evaluate the interest of quantified radiographic analysis of lumbar spine in global outcome assessment and to highlight the key biomechanical factors involved. Clinical data and Beaujon–Lassale scores were collected for 49 patients who underwent lumbosacral arthrodesis after prior lumbar discectomy (mean follow-up: 5 years). Sagittal standing and lumbar flexion-extension X-ray films allowed quantifying vertebral, lumbar, pelvic and kinematic parameters of the lumbar spine, which were compared to reference values. Statistics were performed to assess evolution for all variables. At long-term follow-up, 90% of patients presented satisfactory clinical outcomes, associated to normal sagittal alignment; vertebral parameters objectified adjacent level degeneration in four cases (8%). Clinical outcome was correlated (r = 0.8) with fusion that was confirmed in 80% of cases, doubtful in 16% and pseudarthrosis seemed to occur in 4% (2) of cases. In addition to clinical data (outcomes comparable to the literature), quantitative analysis accurately described lumbar spine geometry and kinematics, highlighting parameters related to adjacent level’s degeneration and a significant correlation between clinical outcome and fusion. Furthermore, criteria proposed to quantitatively evaluate fusion from lumbar dynamic radiographs seem to be appropriate and in agreement with surgeon’s qualitative grading in 87% of cases. PMID:17216227

  20. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  1. Automatic quantitative analysis of cardiac MR perfusion images

    NASA Astrophysics Data System (ADS)

    Breeuwer, Marcel M.; Spreeuwers, Luuk J.; Quist, Marcel J.

    2001-07-01

    Magnetic Resonance Imaging (MRI) is a powerful technique for imaging cardiovascular diseases. The introduction of cardiovascular MRI into clinical practice is however hampered by the lack of efficient and accurate image analysis methods. This paper focuses on the evaluation of blood perfusion in the myocardium (the heart muscle) from MR images, using contrast-enhanced ECG-triggered MRI. We have developed an automatic quantitative analysis method, which works as follows. First, image registration is used to compensate for translation and rotation of the myocardium over time. Next, the boundaries of the myocardium are detected and for each position within the myocardium a time-intensity profile is constructed. The time interval during which the contrast agent passes for the first time through the left ventricle and the myocardium is detected and various parameters are measured from the time-intensity profiles in this interval. The measured parameters are visualized as color overlays on the original images. Analysis results are stored, so that they can later on be compared for different stress levels of the heart. The method is described in detail in this paper and preliminary validation results are presented.

  2. QTL analysis for some quantitative traits in bread wheat*

    PubMed Central

    Pushpendra, Kumar Gupta; Harindra, Singh Balyan; Pawan, Laxminarayan Kulwal; Neeraj, Kumar; Ajay, Kumar; Reyazul, Rouf Mir; Amita, Mohan; Jitendra, Kumar

    2007-01-01

    Quantitative trait loci (QTL) analysis was conducted in bread wheat for 14 important traits utilizing data from four different mapping populations involving different approaches of QTL analysis. Analysis for grain protein content (GPC) suggested that the major part of genetic variation for this trait is due to environmental interactions. In contrast, pre-harvest sprouting tolerance (PHST) was controlled mainly by main effect QTL (M-QTL) with very little genetic variation due to environmental interactions; a major QTL for PHST was detected on chromosome arm 3AL. For grain weight, one QTL each was detected on chromosome arms 1AS, 2BS and 7AS. QTL for 4 growth related traits taken together detected by different methods ranged from 37 to 40; nine QTL that were detected by single-locus as well as two-locus analyses were all M-QTL. Similarly, single-locus and two-locus QTL analyses for seven yield and yield contributing traits in two populations respectively allowed detection of 25 and 50 QTL by composite interval mapping (CIM), 16 and 25 QTL by multiple-trait composite interval mapping (MCIM) and 38 and 37 QTL by two-locus analyses. These studies should prove useful in QTL cloning and wheat improvement through marker aided selection. PMID:17973342

  3. A Quantitative Analysis of the Solar Composition Problem

    NASA Astrophysics Data System (ADS)

    Villante, F. L.; Serenelli, A. M.

    We perform a quantitative analysis of the solar composition problem by using a statistical approach that allows us to combine the information provided by helioseismic and solar neutrino data in an effective way. We show that the opacity profile of the Sun is well constrained by the solar observational properties. In the context of a two parameter analysis in which elements are grouped as volatiles (i.e. C, N, O and Ne) and refractories (i.e. Mg, Si, S, Fe), the optimal surface composition is found by increasing the abundance of volatiles by (45 ± 4) % and that of refractories by (19 ± 3) % with respect to the values provided by Asplund et al., 2009. As an additional result of our analysis, we show that the best fit to the observational data is obtained with values of input parameters of the standard solar models (radiative opacities, gravitational settling rate, the astrophysical factors S 34 and S 17) that differ at the ∼ 1σ level from those presently adopted.

  4. An approach for quantitative image quality analysis for CT

    NASA Astrophysics Data System (ADS)

    Rahimi, Amir; Cochran, Joe; Mooney, Doug; Regensburger, Joe

    2016-03-01

    An objective and standardized approach to assess image quality of Compute Tomography (CT) systems is required in a wide variety of imaging processes to identify CT systems appropriate for a given application. We present an overview of the framework we have developed to help standardize and to objectively assess CT image quality for different models of CT scanners used for security applications. Within this framework, we have developed methods to quantitatively measure metrics that should correlate with feature identification, detection accuracy and precision, and image registration capabilities of CT machines and to identify strengths and weaknesses in different CT imaging technologies in transportation security. To that end we have designed, developed and constructed phantoms that allow for systematic and repeatable measurements of roughly 88 image quality metrics, representing modulation transfer function, noise equivalent quanta, noise power spectra, slice sensitivity profiles, streak artifacts, CT number uniformity, CT number consistency, object length accuracy, CT number path length consistency, and object registration. Furthermore, we have developed a sophisticated MATLAB based image analysis tool kit to analyze CT generated images of phantoms and report these metrics in a format that is standardized across the considered models of CT scanners, allowing for comparative image quality analysis within a CT model or between different CT models. In addition, we have developed a modified sparse principal component analysis (SPCA) method to generate a modified set of PCA components as compared to the standard principal component analysis (PCA) with sparse loadings in conjunction with Hotelling T2 statistical analysis method to compare, qualify, and detect faults in the tested systems.

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

    PubMed

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

    2008-06-15

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

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. Quantitative analysis of drug-induced tremor in mice.

    PubMed

    Shinozaki, H

    1984-12-01

    A method of analyzing tremor in mice was developed using a power spectral analysis of the random current induced by the movement of a magnet attached to a mouse, on a wire coil. The power spectral density function defined the frequency composition of the tremor, and the mean square value of the data in any frequency range of concern was determined. It was possible to determine qualitative differences in the tremor caused by various tremorgenic agents, and to differentiate the drug-induced tremor from spontaneous motor activity. The power spectral densities of the tremorine- and oxotremorine-induced tremors were tentatively expressed as the sum of 3 main components (Cauchy distribution) with different peak frequencies, consisting of the spontaneous motor activity component and two tremor components. On the other hand, the power spectral densities of the harmaline-induced tremor were expressed as the sum of two components with two peak frequencies, and the plots of the power spectral densities versus frequency, consisting of the spontaneous motor activity component and a tremor component. The frequency of the peak spectral density was almost independent of the dose of tremorgenic agents, but changed slightly with the lapse of time after their injection. The severity of the tremor was determined quantitatively in terms of the sum of the mean square value. The sum of the mean square value for a period of 45 min after the injection of tremorine, changed in a dose-dependent manner. The severity of the tremor was different for each of the mouse strains. The method studied in the present paper is expected to be utilized for the quantitative examination of the fine motor movement of the experimental animal, particularly, for the screening test of new anti-tremor drugs.

  9. Towards quantitative analysis of retinal features in optical coherence tomography.

    PubMed

    Baroni, Maurizio; Fortunato, Pina; La Torre, Agostino

    2007-05-01

    The purpose of this paper was to propose a new computer method for quantitative evaluation of representative features of the retina using optical coherence tomography (OCT). A multi-step approach was devised and positively tested for segmentation of the three main retinal layers: the vitreo-retinal interface and the inner and outer retina. Following a preprocessing step, three regions of interest were delimited. Significant peaks corresponding to high and low intensity strips were located along the OCT A-scan lines and accurate boundaries between different layers were obtained by maximizing an edge likelihood function. For a quantitative description, thickness measurement, densitometry, texture and curvature analyses were performed. As a first application, the effect of intravitreal injection of triamcinolone acetonide (IVTA) for the treatment of vitreo-retinal interface syndrome was evaluated. Almost all the parameters, measured on a set of 16 pathologic OCT images, were statistically different before and after IVTA injection (p<0.05). Shape analysis of the internal limiting membrane confirmed the reduction of the pathological traction state. Other significant parameters, such as reflectivity and texture contrast, exhibited relevant changes both at the vitreo-retinal interface and in the inner retinal layers. Texture parameters in the inner and outer retinal layers significantly correlated with the visual acuity restoration. According to these findings an IVTA injection might be considered a possible alternative to surgery for selected patients. In conclusion, the proposed approach appeared to be a promising tool for the investigation of tissue changes produced by pathology and/or therapy.

  10. Quantitative analysis and parametric display of regional myocardial mechanics

    NASA Astrophysics Data System (ADS)

    Eusemann, Christian D.; Bellemann, Matthias E.; Robb, Richard A.

    2000-04-01

    Quantitative assessment of regional heart motion has significant potential for more accurate diagnosis of heart disease and/or cardiac irregularities. Local heart motion may be studied from medical imaging sequences. Using functional parametric mapping, regional myocardial motion during a cardiac cycle can be color mapped onto a deformable heart model to obtain better understanding of the structure- to-function relationships in the myocardium, including regional patterns of akinesis or diskinesis associated with ischemia or infarction. In this study, 3D reconstructions were obtained from the Dynamic Spatial Reconstructor at 15 time points throughout one cardiac cycle of pre-infarct and post-infarct hearts. Deformable models were created from the 3D images for each time point of the cardiac cycles. Form these polygonal models, regional excursions and velocities of each vertex representing a unit of myocardium were calculated for successive time-intervals. The calculated results were visualized through model animations and/or specially formatted static images. The time point of regional maximum velocity and excursion of myocardium through the cardiac cycle was displayed using color mapping. The absolute value of regional maximum velocity and maximum excursion were displayed in a similar manner. Using animations, the local myocardial velocity changes were visualized as color changes on the cardiac surface during the cardiac cycle. Moreover, the magnitude and direction of motion for individual segments of myocardium could be displayed. Comparison of these dynamic parametric displays suggest that the ability to encode quantitative functional information on dynamic cardiac anatomy enhances the diagnostic value of 4D images of the heart. Myocardial mechanics quantified this way adds a new dimension to the analysis of cardiac functional disease, including regional patterns of akinesis and diskinesis associated with ischemia and infarction. Similarly, disturbances in

  11. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  12. Quantitative Analysis Of Acoustic Emission From Rock Fracture Experiments

    NASA Astrophysics Data System (ADS)

    Goodfellow, Sebastian David

    This thesis aims to advance the methods of quantitative acoustic emission (AE) analysis by calibrating sensors, characterizing sources, and applying the results to solve engi- neering problems. In the first part of this thesis, we built a calibration apparatus and successfully calibrated two commercial AE sensors. The ErgoTech sensor was found to have broadband velocity sensitivity and the Panametrics V103 was sensitive to surface normal displacement. These calibration results were applied to two AE data sets from rock fracture experiments in order to characterize the sources of AE events. The first data set was from an in situ rock fracture experiment conducted at the Underground Research Laboratory (URL). The Mine-By experiment was a large scale excavation response test where both AE (10 kHz - 1 MHz) and microseismicity (MS) (1 Hz - 10 kHz) were monitored. Using the calibration information, magnitude, stress drop, dimension and energy were successfully estimated for 21 AE events recorded in the tensile region of the tunnel wall. Magnitudes were in the range -7.5 < Mw < -6.8, which is consistent with other laboratory AE results, and stress drops were within the range commonly observed for induced seismicity in the field (0.1 - 10 MPa). The second data set was AE collected during a true-triaxial deformation experiment, where the objectives were to characterize laboratory AE sources and identify issues related to moving the analysis from ideal in situ conditions to more complex laboratory conditions in terms of the ability to conduct quantitative AE analysis. We found AE magnitudes in the range -7.8 < Mw < -6.7 and as with the in situ data, stress release was within the expected range of 0.1 - 10 MPa. We identified four major challenges to quantitative analysis in the laboratory, which in- hibited our ability to study parameter scaling (M0 ∝ fc -3 scaling). These challenges were 0c (1) limited knowledge of attenuation which we proved was continuously evolving, (2

  13. Quantitative evaluation of midpalatal suture maturation via fractal analysis

    PubMed Central

    Kwak, Kyoung Ho; Kim, Yong-Il; Kim, Yong-Deok

    2016-01-01

    Objective The purpose of this study was to determine whether the results of fractal analysis can be used as criteria for midpalatal suture maturation evaluation. Methods The study included 131 subjects aged over 18 years of age (range 18.1–53.4 years) who underwent cone-beam computed tomography. Skeletonized images of the midpalatal suture were obtained via image processing software and used to calculate fractal dimensions. Correlations between maturation stage and fractal dimensions were calculated using Spearman's correlation coefficient. Optimal fractal dimension cut-off values were determined using a receiver operating characteristic curve. Results The distribution of maturation stages of the midpalatal suture according to the cervical vertebrae maturation index was highly variable, and there was a strong negative correlation between maturation stage and fractal dimension (−0.623, p < 0.001). Fractal dimension was a statistically significant indicator of dichotomous results with regard to maturation stage (area under curve = 0.794, p < 0.001). A test in which fractal dimension was used to predict the resulting variable that splits maturation stages into ABC and D or E yielded an optimal fractal dimension cut-off value of 1.0235. Conclusions There was a strong negative correlation between fractal dimension and midpalatal suture maturation. Fractal analysis is an objective quantitative method, and therefore we suggest that it may be useful for the evaluation of midpalatal suture maturation. PMID:27668195

  14. A quantitative risk analysis approach to port hydrocarbon logistics.

    PubMed

    Ronza, A; Carol, S; Espejo, V; Vílchez, J A; Arnaldos, J

    2006-01-16

    A method is presented that allows quantitative risk analysis to be performed on marine hydrocarbon terminals sited in ports. A significant gap was identified in the technical literature on QRA for the handling of hazardous materials in harbours published prior to this work. The analysis is extended to tanker navigation through port waters and loading and unloading facilities. The steps of the method are discussed, beginning with data collecting. As to accident scenario identification, an approach is proposed that takes into account minor and massive spills due to loading arm failures and tank rupture. Frequency estimation is thoroughly reviewed and a shortcut approach is proposed for frequency calculation. This allows for the two-fold possibility of a tanker colliding/grounding at/near the berth or while navigating to/from the berth. A number of probability data defining the possibility of a cargo spill after an external impact on a tanker are discussed. As to consequence and vulnerability estimates, a scheme is proposed for the use of ratios between the numbers of fatal victims, injured and evacuated people. Finally, an example application is given, based on a pilot study conducted in the Port of Barcelona, where the method was tested.

  15. Inside Single Cells: Quantitative Analysis with Advanced Optics and Nanomaterials

    PubMed Central

    Cui, Yi; Irudayaraj, Joseph

    2014-01-01

    Single cell explorations offer a unique window to inspect molecules and events relevant to mechanisms and heterogeneity constituting the central dogma of biology. A large number of nucleic acids, proteins, metabolites and small molecules are involved in determining and fine-tuning the state and function of a single cell at a given time point. Advanced optical platforms and nanotools provide tremendous opportunities to probe intracellular components with single-molecule accuracy, as well as promising tools to adjust single cell activity. In order to obtain quantitative information (e.g. molecular quantity, kinetics and stoichiometry) within an intact cell, achieving the observation with comparable spatiotemporal resolution is a challenge. For single cell studies both the method of detection and the biocompatibility are critical factors as they determine the feasibility, especially when considering live cell analysis. Although a considerable proportion of single cell methodologies depend on specialized expertise and expensive instruments, it is our expectation that the information content and implication will outweigh the costs given the impact on life science enabled by single cell analysis. PMID:25430077

  16. Therapeutic electrical stimulation for spasticity: quantitative gait analysis.

    PubMed

    Pease, W S

    1998-01-01

    Improvement in motor function following electrical stimulation is related to strengthening of the stimulated spastic muscle and inhibition of the antagonist. A 26-year-old man with familial spastic paraparesis presented with gait dysfunction and bilateral lower limb spastic muscle tone. Clinically, muscle strength and sensation were normal. He was considered appropriate for a trial of therapeutic electrical stimulation following failed trials of physical therapy and baclofen. No other treatment was used concurrent with the electrical stimulation. Before treatment, quantitative gait analysis revealed 63% of normal velocity and a crouched gait pattern, associated with excessive electromyographic activity in the hamstrings and gastrocnemius muscles. Based on these findings, bilateral stimulation of the quadriceps and anterior compartment musculature was performed two to three times per week for three months. Repeat gait analysis was conducted three weeks after the cessation of stimulation treatment. A 27% increase in velocity was noted associated with an increase in both cadence and right step length. Right hip and bilateral knee stance motion returned to normal (rather than "crouched"). No change in the timing of dynamic electromyographic activity was seen. These findings suggest a role for the use of electrical stimulation for rehabilitation of spasticity. The specific mechanism of this improvement remains uncertain.

  17. Comprehensive Quantitative Analysis of Ovarian and Breast Cancer Tumor Peptidomes

    SciTech Connect

    Xu, Zhe; Wu, Chaochao; Xie, Fang; Slysz, Gordon W.; Tolic, Nikola; Monroe, Matthew E.; Petyuk, Vladislav A.; Payne, Samuel H.; Fujimoto, Grant M.; Moore, Ronald J.; Fillmore, Thomas L.; Schepmoes, Athena A.; Levine, Douglas; Townsend, Reid; Davies, Sherri; Li, Shunqiang; Ellis, Matthew; Boja, Emily; Rivers, Robert; Rodriguez, Henry; Rodland, Karin D.; Liu, Tao; Smith, Richard D.

    2015-01-02

    Aberrant degradation of proteins is associated with many pathological states, including cancers. Mass spectrometric analysis of tumor peptidomes, the intracellular and intercellular products of protein degradation, has the potential to provide biological insights on proteolytic processing in cancer. However, attempts to use the information on these smaller protein degradation products from tumors for biomarker discovery and cancer biology studies have been fairly limited to date, largely due to the lack of effective approaches for robust peptidomics identification and quantification, and the prevalence of confounding factors and biases associated with sample handling and processing. Herein, we have developed an effective and robust analytical platform for comprehensive analyses of tissue peptidomes, which is suitable for high throughput quantitative studies. The reproducibility and coverage of the platform, as well as the suitability of clinical ovarian tumor and patient-derived breast tumor xenograft samples with post-excision delay of up to 60 min before freezing for peptidomics analysis, have been demonstrated. Moreover, our data also show that the peptidomics profiles can effectively separate breast cancer subtypes, reflecting tumor-associated protease activities. Peptidomics complements results obtainable from conventional bottom-up proteomics, and provides insights not readily obtainable from such approaches.

  18. A Computational Tool for Quantitative Analysis of Vascular Networks

    PubMed Central

    Zudaire, Enrique; Gambardella, Laure; Kurcz, Christopher; Vermeren, Sonja

    2011-01-01

    Angiogenesis is the generation of mature vascular networks from pre-existing vessels. Angiogenesis is crucial during the organism' development, for wound healing and for the female reproductive cycle. Several murine experimental systems are well suited for studying developmental and pathological angiogenesis. They include the embryonic hindbrain, the post-natal retina and allantois explants. In these systems vascular networks are visualised by appropriate staining procedures followed by microscopical analysis. Nevertheless, quantitative assessment of angiogenesis is hampered by the lack of readily available, standardized metrics and software analysis tools. Non-automated protocols are being used widely and they are, in general, time - and labour intensive, prone to human error and do not permit computation of complex spatial metrics. We have developed a light-weight, user friendly software, AngioTool, which allows for quick, hands-off and reproducible quantification of vascular networks in microscopic images. AngioTool computes several morphological and spatial parameters including the area covered by a vascular network, the number of vessels, vessel length, vascular density and lacunarity. In addition, AngioTool calculates the so-called “branching index” (branch points / unit area), providing a measurement of the sprouting activity of a specimen of interest. We have validated AngioTool using images of embryonic murine hindbrains, post-natal retinas and allantois explants. AngioTool is open source and can be downloaded free of charge. PMID:22110636

  19. Quantitative analysis of triple-mutant genetic interactions.

    PubMed

    Braberg, Hannes; Alexander, Richard; Shales, Michael; Xu, Jiewei; Franks-Skiba, Kathleen E; Wu, Qiuqin; Haber, James E; Krogan, Nevan J

    2014-08-01

    The quantitative analysis of genetic interactions between pairs of gene mutations has proven to be effective for characterizing cellular functions, but it can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed triple-mutant analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, which is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principal actors are deleted. TMA has also uncovered double-mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete and measures interactions for up to 30 double mutants against a library of 1,536 single mutants.

  20. Applying Qualitative Hazard Analysis to Support Quantitative Safety Analysis for Proposed Reduced Wake Separation Conops

    NASA Technical Reports Server (NTRS)

    Shortle, John F.; Allocco, Michael

    2005-01-01

    This paper describes a scenario-driven hazard analysis process to identify, eliminate, and control safety-related risks. Within this process, we develop selective criteria to determine the applicability of applying engineering modeling to hypothesized hazard scenarios. This provides a basis for evaluating and prioritizing the scenarios as candidates for further quantitative analysis. We have applied this methodology to proposed concepts of operations for reduced wake separation for closely spaced parallel runways. For arrivals, the process identified 43 core hazard scenarios. Of these, we classified 12 as appropriate for further quantitative modeling, 24 that should be mitigated through controls, recommendations, and / or procedures (that is, scenarios not appropriate for quantitative modeling), and 7 that have the lowest priority for further analysis.

  1. Qualitative and quantitative analysis of atmospheric organosulfates in Centreville, Alabama

    NASA Astrophysics Data System (ADS)

    Hettiyadura, Anusha P. S.; Jayarathne, Thilina; Baumann, Karsten; Goldstein, Allen H.; de Gouw, Joost A.; Koss, Abigail; Keutsch, Frank N.; Skog, Kate; Stone, Elizabeth A.

    2017-01-01

    Organosulfates are components of secondary organic aerosols (SOA) that form from oxidation of volatile organic compounds (VOCs) in the presence of sulfate. In this study, the composition and abundance of organosulfates were determined in fine particulate matter (PM2.5) collected from Centreville, AL, during the Southern Oxidant and Aerosol Study (SOAS) in summer 2013. Six organosulfates were quantified using hydrophilic interaction liquid chromatography (HILIC) with triple quadrupole mass spectrometry (TQD) against authentic standards. Among these, the three most abundant species were glycolic acid sulfate (0.5-52.5 ng m-3), lactic acid sulfate (0.5-36.7 ng m-3), and hydroxyacetone sulfate (0.5-14.3 ng m-3). These three species were strongly inter-correlated, suggesting similar precursors and/or formation pathways. Further correlations with sulfate, isoprene, and isoprene oxidation products indicate important roles for these precursors in organosulfate formation in Centreville. Positive filter sampling artifacts associated with these organosulfates due to gas adsorption or reaction of gas phase precursors of organosulfates with sulfuric acid were assessed for a subset of samples and were less than 7.8 % of their PM2.5 concentrations. Together, the quantified organosulfates accounted for < 0.3 % of organic carbon mass in PM2.5. To gain insights into other organosulfates in PM2.5 collected from Centreville, semi-quantitative analysis was employed by way of monitoring characteristic product ions of organosulfates (HSO4- at m/z 97 and SO4- ṡ at m/z 96) and evaluating relative signal strength by HILIC-TQD. Molecular formulas of organosulfates were determined by high-resolution time-of-flight (TOF) mass spectrometry. The major organosulfate signal across all samples corresponded to 2-methyltetrol sulfates, which accounted for 42-62 % of the total bisulfate ion signal. Conversely, glycolic acid sulfate, the most abundant organosulfate quantified in this study, was 0

  2. Evaluating the Quantitative Capabilities of Metagenomic Analysis Software.

    PubMed

    Kerepesi, Csaba; Grolmusz, Vince

    2016-05-01

    DNA sequencing technologies are applied widely and frequently today to describe metagenomes, i.e., microbial communities in environmental or clinical samples, without the need for culturing them. These technologies usually return short (100-300 base-pairs long) DNA reads, and these reads are processed by metagenomic analysis software that assign phylogenetic composition-information to the dataset. Here we evaluate three metagenomic analysis software (AmphoraNet--a webserver implementation of AMPHORA2--, MG-RAST, and MEGAN5) for their capabilities of assigning quantitative phylogenetic information for the data, describing the frequency of appearance of the microorganisms of the same taxa in the sample. The difficulties of the task arise from the fact that longer genomes produce more reads from the same organism than shorter genomes, and some software assign higher frequencies to species with longer genomes than to those with shorter ones. This phenomenon is called the "genome length bias." Dozens of complex artificial metagenome benchmarks can be found in the literature. Because of the complexity of those benchmarks, it is usually difficult to judge the resistance of a metagenomic software to this "genome length bias." Therefore, we have made a simple benchmark for the evaluation of the "taxon-counting" in a metagenomic sample: we have taken the same number of copies of three full bacterial genomes of different lengths, break them up randomly to short reads of average length of 150 bp, and mixed the reads, creating our simple benchmark. Because of its simplicity, the benchmark is not supposed to serve as a mock metagenome, but if a software fails on that simple task, it will surely fail on most real metagenomes. We applied three software for the benchmark. The ideal quantitative solution would assign the same proportion to the three bacterial taxa. We have found that AMPHORA2/AmphoraNet gave the most accurate results and the other two software were under

  3. The Measles Vaccination Narrative in Twitter: A Quantitative Analysis

    PubMed Central

    Radzikowski, Jacek; Jacobsen, Kathryn H; Croitoru, Arie; Crooks, Andrew; Delamater, Paul L

    2016-01-01

    Background The emergence of social media is providing an alternative avenue for information exchange and opinion formation on health-related issues. Collective discourse in such media leads to the formation of a complex narrative, conveying public views and perceptions. Objective This paper presents a study of Twitter narrative regarding vaccination in the aftermath of the 2015 measles outbreak, both in terms of its cyber and physical characteristics. We aimed to contribute to the analysis of the data, as well as presenting a quantitative interdisciplinary approach to analyze such open-source data in the context of health narratives. Methods We collected 669,136 tweets referring to vaccination from February 1 to March 9, 2015. These tweets were analyzed to identify key terms, connections among such terms, retweet patterns, the structure of the narrative, and connections to the geographical space. Results The data analysis captures the anatomy of the themes and relations that make up the discussion about vaccination in Twitter. The results highlight the higher impact of stories contributed by news organizations compared to direct tweets by health organizations in communicating health-related information. They also capture the structure of the antivaccination narrative and its terms of reference. Analysis also revealed the relationship between community engagement in Twitter and state policies regarding child vaccination. Residents of Vermont and Oregon, the two states with the highest rates of non-medical exemption from school-entry vaccines nationwide, are leading the social media discussion in terms of participation. Conclusions The interdisciplinary study of health-related debates in social media across the cyber-physical debate nexus leads to a greater understanding of public concerns, views, and responses to health-related issues. Further coalescing such capabilities shows promise towards advancing health communication, thus supporting the design of more

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

    PubMed

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

    2010-02-01

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

  5. Quantitative texture analysis of talc in mantle hydrated mylonites

    NASA Astrophysics Data System (ADS)

    Benitez-Perez, J. M.; Gomez Barreiro, J.; Wenk, H. R.; Vogel, S. C.; Soda, Y.; Voltolini, M.; Martinez-Catalan, J. R.

    2014-12-01

    A quantitative texture analysis of talc-serpentinite mylonites developed in highly deformed ultramafic rocks from different orogenic contexts have been done with neutorn diffraction at HIPPO (Los Álamos National Laboratory). Mineral assemblage, metamorphic evolution and deformative fabric of these samples could be correlated with those verified along the shallow levels (<100km; <5GPa) of a subduction zone. The hydration of mantle (ultramafic) rocks at those levels it is likely to occur dynamically, with important implications on seismogenesis. Given the high anisotropy of the major phases in the samples (i.e. talc and antigorite) it is expected to influence seismic anisotropy of the whole system, in the presence of texture. However to date there was no data on the crystallographic preferred orientation of talc and examples of antigorite textures are very limited. We explore the contribution of talc texture to the seismic anisotropy of mantle hydrated mylonites. Acknowledgements: This work has been funded by research project CGL2011-22728 of Spanish Ministry of Economy and Competitiveness. JGB and JMBP are grateful to the Ramón y Cajal and FPI funding programs. Access to HIPPO (LANSCE) to conduct diffraction experiments is kindly acknowledged.

  6. Quantitative Analysis of Cellular Metabolic Dissipative, Self-Organized Structures

    PubMed Central

    de la Fuente, Ildefonso Martínez

    2010-01-01

    One of the most important goals of the postgenomic era is understanding the metabolic dynamic processes and the functional structures generated by them. Extensive studies during the last three decades have shown that the dissipative self-organization of the functional enzymatic associations, the catalytic reactions produced during the metabolite channeling, the microcompartmentalization of these metabolic processes and the emergence of dissipative networks are the fundamental elements of the dynamical organization of cell metabolism. Here we present an overview of how mathematical models can be used to address the properties of dissipative metabolic structures at different organizational levels, both for individual enzymatic associations and for enzymatic networks. Recent analyses performed with dissipative metabolic networks have shown that unicellular organisms display a singular global enzymatic structure common to all living cellular organisms, which seems to be an intrinsic property of the functional metabolism as a whole. Mathematical models firmly based on experiments and their corresponding computational approaches are needed to fully grasp the molecular mechanisms of metabolic dynamical processes. They are necessary to enable the quantitative and qualitative analysis of the cellular catalytic reactions and also to help comprehend the conditions under which the structural dynamical phenomena and biological rhythms arise. Understanding the molecular mechanisms responsible for the metabolic dissipative structures is crucial for unraveling the dynamics of cellular life. PMID:20957111

  7. Separation and quantitative analysis of alkyl sulfate ethoxymers by HPLC.

    PubMed

    Morvan, Julien; Hubert-Roux, Marie; Agasse, Valérie; Cardinael, Pascal; Barbot, Florence; Decock, Gautier; Bouillon, Jean-Philippe

    2008-01-01

    Separation of alkyl sulfate ethoxymers is investigated on various high-performance liquid chromatography (HPLC) stationary phases: Acclaim C18 Surfactant, Surfactant C8, and Hypercarb. For a fixed alkyl chain length, ethoxymers are eluted in the order of increasing number of ethoxylated units on Acclaim C18 Surfactant, whereas a reversed elution order is observed on Surfactant C8 and Hypercarb. Moreover, on an Acclaim C18 Surfactant column, non-ethoxylated compounds are eluted in their ethoxymers distribution and the use of sodium acetate additive in mobile phase leads to a co-elution of ethoxymers. HPLC stationary phases dedicated to surfactants analysis are evaluated by means of the Tanaka test. Surfactant C8 presents a great silanol activity whereas Acclaim C18 Surfactant shows a high steric selectivity. For alkyl sulfates, linearity of the calibration curve and limits of detection and quantitation are evaluated. The amount of sodium laureth sulfate raw material found in commercial body product is in agreement with the specification of the manufacturer.

  8. Quantitative analysis of 3-OH oxylipins in fermentation yeast.

    PubMed

    Potter, Greg; Xia, Wei; Budge, Suzanne M; Speers, R Alex

    2017-02-01

    Despite the ubiquitous distribution of oxylipins in plants, animals, and microbes, and the application of numerous analytical techniques to study these molecules, 3-OH oxylipins have never been quantitatively assayed in yeasts. The formation of heptafluorobutyrate methyl ester derivatives and subsequent analysis with gas chromatography - negative chemical ionization - mass spectrometry allowed for the first determination of yeast 3-OH oxylipins. The concentration of 3-OH 10:0 (0.68-4.82 ng/mg dry cell mass) in the SMA strain of Saccharomyces pastorianus grown in laboratory-scale beverage fermentations was elevated relative to oxylipin concentrations in plant tissues and macroalgae. In fermenting yeasts, the onset of 3-OH oxylipin formation has been related to fermentation progression and flocculation initiation. When the SMA strain was grown in laboratory-scale fermentations, the maximal sugar consumption rate preceded the lowest concentration of 3-OH 10:0 by ∼4.5 h and a distinct increase in 3-OH 10:0 concentration by ∼16.5 h.

  9. Quantitative analysis of brain magnetic resonance imaging for hepatic encephalopathy

    NASA Astrophysics Data System (ADS)

    Syh, Hon-Wei; Chu, Wei-Kom; Ong, Chin-Sing

    1992-06-01

    High intensity lesions around ventricles have recently been observed in T1-weighted brain magnetic resonance images for patients suffering hepatic encephalopathy. The exact etiology that causes magnetic resonance imaging (MRI) gray scale changes has not been totally understood. The objective of our study was to investigate, through quantitative means, (1) the amount of changes to brain white matter due to the disease process, and (2) the extent and distribution of these high intensity lesions, since it is believed that the abnormality may not be entirely limited to the white matter only. Eleven patients with proven haptic encephalopathy and three normal persons without any evidence of liver abnormality constituted our current data base. Trans-axial, sagittal, and coronal brain MRI were obtained on a 1.5 Tesla scanner. All processing was carried out on a microcomputer-based image analysis system in an off-line manner. Histograms were decomposed into regular brain tissues and lesions. Gray scale ranges coded as lesion were then brought back to original images to identify distribution of abnormality. Our results indicated the disease process involved pallidus, mesencephalon, and subthalamic regions.

  10. Quantitative SERS sensors for environmental analysis of naphthalene.

    PubMed

    Péron, O; Rinnert, E; Toury, T; Lamy de la Chapelle, M; Compère, C

    2011-03-07

    In the investigation of chemical pollutants, such as PAHs (Polycyclic Aromatic Hydrocarbons) at low concentration in aqueous medium, Surface-Enhanced Raman Scattering (SERS) stands for an alternative to the inherent low cross-section of normal Raman scattering. Indeed, SERS is a very sensitive spectroscopic technique due to the excitation of the surface plasmon modes of the nanostructured metallic film. The surface of quartz substrates was coated with a hydrophobic film obtained by silanization and subsequently reacted with polystyrene (PS) beads coated with gold nanoparticles. The hydrophobic surface of the SERS substrates pre-concentrates non-polar molecules such as naphthalene. Under laser excitation, the SERS-active substrates allow the detection and the identification of the target molecules localized close to the gold nanoparticles. The morphology of the SERS substrates based on polystyrene beads surrounded by gold nanoparticles was characterized by scanning electron microscopy (SEM). Furthermore, the Raman fingerprint of the polystyrene stands for an internal spectral reference. To this extent, an innovative method to detect and to quantify organic molecules, as naphthalene in the range of 1 to 20 ppm, in aqueous media was carried out. Such SERS-active substrates tend towards an application as quantitative SERS sensors for the environmental analysis of naphthalene.

  11. Quantitative Financial Analysis of Alternative Energy Efficiency Shareholder Incentive Mechanisms

    SciTech Connect

    Cappers, Peter; Goldman, Charles; Chait, Michele; Edgar, George; Schlegel, Jeff; Shirley, Wayne

    2008-08-03

    Rising energy prices and climate change are central issues in the debate about our nation's energy policy. Many are demanding increased energy efficiency as a way to help reduce greenhouse gas emissions and lower the total cost of electricity and energy services for consumers and businesses. Yet, as the National Action Plan on Energy Efficiency (NAPEE) pointed out, many utilities continue to shy away from seriously expanding their energy efficiency program offerings because they claim there is insufficient profit-motivation, or even a financial disincentive, when compared to supply-side investments. With the recent introduction of Duke Energy's Save-a-Watt incentive mechanism and ongoing discussions about decoupling, regulators and policymakers are now faced with an expanded and diverse landscape of financial incentive mechanisms, Determining the 'right' way forward to promote deep and sustainable demand side resource programs is challenging. Due to the renaissance that energy efficiency is currently experiencing, many want to better understand the tradeoffs in stakeholder benefits between these alternative incentive structures before aggressively embarking on a path for which course corrections can be time-consuming and costly. Using a prototypical Southwest utility and a publicly available financial model, we show how various stakeholders (e.g. shareholders, ratepayers, etc.) are affected by these different types of shareholder incentive mechanisms under varying assumptions about program portfolios. This quantitative analysis compares the financial consequences associated with a wide range of alternative incentive structures. The results will help regulators and policymakers better understand the financial implications of DSR program incentive regulation.

  12. Quantitative analysis of plasma interleiukin-6 by immunoassay on microchip

    NASA Astrophysics Data System (ADS)

    Abe, K.; Hashimoto, Y.; Yatsushiro, S.; Yamamura, S.; Tanaka, M.; Ooie, T.; Baba, Y.; Kataoka, M.

    2012-03-01

    Sandwich enzyme-linked immunoassay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. We employed the piezoelectric inkjet printing for deposition and fixation of 1st antibody on the microchannnel surface (300 μm width and 100 μm depth). Model analyte was interleukin-6 (IL-6) which was one of the inflammatory cytokine. After blocking the microchannel, antigen, biotin-labeled 2nd antibody, and avidin-labeled peroxidase were infused into the microchannel and incubated for 20 min, 10 min, and 5 min, respectively. This assay could detect 2 pg/ml and quantitatively measure the range of 0-32 pg/ml. Liner regression analysis of plasma IL-6 concentration obtained by microchip and conventional methods exhibited a significant relationship (R2 = 0.9964). This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. This assay enables us to determine plasma IL-6 with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.

  13. Quantitative produced water analysis using mobile 1H NMR

    NASA Astrophysics Data System (ADS)

    Wagner, Lisabeth; Kalli, Chris; Fridjonsson, Einar O.; May, Eric F.; Stanwix, Paul L.; Graham, Brendan F.; Carroll, Matthew R. J.; Johns, Michael L.

    2016-10-01

    Measurement of oil contamination of produced water is required in the oil and gas industry to the (ppm) level prior to discharge in order to meet typical environmental legislative requirements. Here we present the use of compact, mobile 1H nuclear magnetic resonance (NMR) spectroscopy, in combination with solid phase extraction (SPE), to meet this metrology need. The NMR hardware employed featured a sufficiently homogeneous magnetic field, such that chemical shift differences could be used to unambiguously differentiate, and hence quantitatively detect, the required oil and solvent NMR signals. A solvent system consisting of 1% v/v chloroform in tetrachloroethylene was deployed, this provided a comparable 1H NMR signal intensity for the oil and the solvent (chloroform) and hence an internal reference 1H signal from the chloroform resulting in the measurement being effectively self-calibrating. The measurement process was applied to water contaminated with hexane or crude oil over the range 1-30 ppm. The results were validated against known solubility limits as well as infrared analysis and gas chromatography.

  14. Quantitative analysis of dynamic association in live biological fluorescent samples.

    PubMed

    Ruusuvuori, Pekka; Paavolainen, Lassi; Rutanen, Kalle; Mäki, Anita; Huttunen, Heikki; Marjomäki, Varpu

    2014-01-01

    Determining vesicle localization and association in live microscopy may be challenging due to non-simultaneous imaging of rapidly moving objects with two excitation channels. Besides errors due to movement of objects, imaging may also introduce shifting between the image channels, and traditional colocalization methods cannot handle such situations. Our approach to quantifying the association between tagged proteins is to use an object-based method where the exact match of object locations is not assumed. Point-pattern matching provides a measure of correspondence between two point-sets under various changes between the sets. Thus, it can be used for robust quantitative analysis of vesicle association between image channels. Results for a large set of synthetic images shows that the novel association method based on point-pattern matching demonstrates robust capability to detect association of closely located vesicles in live cell-microscopy where traditional colocalization methods fail to produce results. In addition, the method outperforms compared Iterated Closest Points registration method. Results for fixed and live experimental data shows the association method to perform comparably to traditional methods in colocalization studies for fixed cells and to perform favorably in association studies for live cells.

  15. High throughput, quantitative analysis of human osteoclast differentiation and activity.

    PubMed

    Diepenhorst, Natalie A; Nowell, Cameron J; Rueda, Patricia; Henriksen, Kim; Pierce, Tracie; Cook, Anna E; Pastoureau, Philippe; Sabatini, Massimo; Charman, William N; Christopoulos, Arthur; Summers, Roger J; Sexton, Patrick M; Langmead, Christopher J

    2017-02-15

    Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an 'osteoclast population' is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.

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

    PubMed

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

    2016-10-29

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

  17. Quantitative Analysis of Intracellular Fluorescent Foci in Live Bacteria

    PubMed Central

    Moolman, M. Charl; Kerssemakers, Jacob W.J.; Dekker, Nynke H.

    2015-01-01

    Fluorescence microscopy has revolutionized in vivo cellular biology. Through the specific labeling of a protein of interest with a fluorescent protein, one is able to study movement and colocalization, and even count individual proteins in a live cell. Different algorithms exist to quantify the total intensity and position of a fluorescent focus. Although these algorithms have been rigorously studied for in vitro conditions, which are greatly different than the in-homogenous and variable cellular environments, their exact limits and applicability in the context of a live cell have not been thoroughly and systematically evaluated. In this study, we quantitatively characterize the influence of different background subtraction algorithms on several focus analysis algorithms. We use, to our knowledge, a novel approach to assess the sensitivity of the focus analysis algorithms to background removal, in which simulated and experimental data are combined to maintain full control over the sensitivity of a focus within a realistic background of cellular fluorescence. We demonstrate that the choice of algorithm and the corresponding error are dependent on both the brightness of the focus, and the cellular context. Expectedly, focus intensity estimation and localization accuracy suffer in all algorithms at low focus to background ratios, with the bacteroidal background subtraction in combination with the median excess algorithm, and the region of interest background subtraction in combination with a two-dimensional Gaussian fit algorithm, performing the best. We furthermore show that the choice of background subtraction algorithm is dependent on the expression level of the protein under investigation, and that the localization error is dependent on the distance of a focus from the bacterial edge and pole. Our results establish a set of guidelines for what signals can be analyzed to give a targeted spatial and intensity accuracy within a bacterial cell. PMID:26331246

  18. Communication about vaccinations in Italian websites: a quantitative analysis.

    PubMed

    Tafuri, Silvio; Gallone, Maria S; Gallone, Maria F; Zorico, Ivan; Aiello, Valeria; Germinario, Cinzia

    2014-01-01

    Babies' parents and people who look for information about vaccination often visit anti-vaccine movement's websites, blogs by naturopathic physicians or natural and alternative medicine practitioners. The aim of this work is to provide a quantitative analysis on the type of information available to Italian people regarding vaccination and a quality analysis of websites retrieved through our searches. A quality score was created to evaluate the technical level of websites. A research was performed through Yahoo, Google, and MSN using the keywords "vaccine" and "vaccination," with the function "OR" in order to identify the most frequently used websites. The 2 keywords were input in Italian, and the first 15 pages retrieved by each search engine were analyzed. 149 websites were selected through this methodology. Fifty-three per cent of the websites belonged to associations, groups, or scientific companies, 32.2% (n = 48) consisted of a personal blog and 14.8% (n = 22) belonged to some of the National Health System offices. Among all analyzed websites, 15.4% (n = 23) came from anti-vaccine movement groups. 37.6% reported webmaster name, 67.8% webmaster e-mail, 28.6% indicated the date of the last update and 46.6% the author's name. The quality score for government sites was higher on average than anti-vaccine websites; although, government sites don't use Web 2.0 functions, as the forums.: National Health System institutions who have to promote vaccination cannot avoid investing in web communication because it cannot be managed by private efforts but must be the result of Public Health, private and scientific association, and social movement synergy.

  19. Software for quantitative analysis of radiotherapy: overview, requirement analysis and design solutions.

    PubMed

    Zhang, Lanlan; Hub, Martina; Mang, Sarah; Thieke, Christian; Nix, Oliver; Karger, Christian P; Floca, Ralf O

    2013-06-01

    Radiotherapy is a fast-developing discipline which plays a major role in cancer care. Quantitative analysis of radiotherapy data can improve the success of the treatment and support the prediction of outcome. In this paper, we first identify functional, conceptional and general requirements on a software system for quantitative analysis of radiotherapy. Further we present an overview of existing radiotherapy analysis software tools and check them against the stated requirements. As none of them could meet all of the demands presented herein, we analyzed possible conceptional problems and present software design solutions and recommendations to meet the stated requirements (e.g. algorithmic decoupling via dose iterator pattern; analysis database design). As a proof of concept we developed a software library "RTToolbox" following the presented design principles. The RTToolbox is available as open source library and has already been tested in a larger-scale software system for different use cases. These examples demonstrate the benefit of the presented design principles.

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

    PubMed Central

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

    2013-01-01

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

  1. Column precipitation chromatography: an approach to quantitative analysis of eigencolloids.

    PubMed

    Breynaert, E; Maes, A

    2005-08-01

    A new column precipitation chromatography (CPC) technique, capable of quantitatively measuring technetium eigencolloids in aqueous solutions, is presented. The CPC technique is based on the destabilization and precipitation of eigencolloids by polycations in a confined matrix. Tc(IV) colloids can be quantitatively determined from their precipitation onto the CPC column (separation step) and their subsequent elution upon oxidation to pertechnetate by peroxide (elution step). A clean-bed particle removal model was used to explain the experimental results.

  2. Quantitative analysis of harmonic convergence in mosquito auditory interactions.

    PubMed

    Aldersley, Andrew; Champneys, Alan; Homer, Martin; Robert, Daniel

    2016-04-01

    This article analyses the hearing and behaviour of mosquitoes in the context of inter-individual acoustic interactions. The acoustic interactions of tethered live pairs of Aedes aegypti mosquitoes, from same and opposite sex mosquitoes of the species, are recorded on independent and unique audio channels, together with the response of tethered individual mosquitoes to playbacks of pre-recorded flight tones of lone or paired individuals. A time-dependent representation of each mosquito's non-stationary wing beat frequency signature is constructed, based on Hilbert spectral analysis. A range of algorithmic tools is developed to automatically analyse these data, and used to perform a robust quantitative identification of the 'harmonic convergence' phenomenon. The results suggest that harmonic convergence is an active phenomenon, which does not occur by chance. It occurs for live pairs, as well as for lone individuals responding to playback recordings, whether from the same or opposite sex. Male-female behaviour is dominated by frequency convergence at a wider range of harmonic combinations than previously reported, and requires participation from both partners in the duet. New evidence is found to show that male-male interactions are more varied than strict frequency avoidance. Rather, they can be divided into two groups: convergent pairs, typified by tightly bound wing beat frequencies, and divergent pairs, that remain widely spaced in the frequency domain. Overall, the results reveal that mosquito acoustic interaction is a delicate and intricate time-dependent active process that involves both individuals, takes place at many different frequencies, and which merits further enquiry.

  3. Quantitative analysis of harmonic convergence in mosquito auditory interactions

    PubMed Central

    Aldersley, Andrew; Champneys, Alan; Robert, Daniel

    2016-01-01

    This article analyses the hearing and behaviour of mosquitoes in the context of inter-individual acoustic interactions. The acoustic interactions of tethered live pairs of Aedes aegypti mosquitoes, from same and opposite sex mosquitoes of the species, are recorded on independent and unique audio channels, together with the response of tethered individual mosquitoes to playbacks of pre-recorded flight tones of lone or paired individuals. A time-dependent representation of each mosquito's non-stationary wing beat frequency signature is constructed, based on Hilbert spectral analysis. A range of algorithmic tools is developed to automatically analyse these data, and used to perform a robust quantitative identification of the ‘harmonic convergence’ phenomenon. The results suggest that harmonic convergence is an active phenomenon, which does not occur by chance. It occurs for live pairs, as well as for lone individuals responding to playback recordings, whether from the same or opposite sex. Male–female behaviour is dominated by frequency convergence at a wider range of harmonic combinations than previously reported, and requires participation from both partners in the duet. New evidence is found to show that male–male interactions are more varied than strict frequency avoidance. Rather, they can be divided into two groups: convergent pairs, typified by tightly bound wing beat frequencies, and divergent pairs, that remain widely spaced in the frequency domain. Overall, the results reveal that mosquito acoustic interaction is a delicate and intricate time-dependent active process that involves both individuals, takes place at many different frequencies, and which merits further enquiry. PMID:27053654

  4. Quantitative analysis of flavanones and chalcones from willow bark.

    PubMed

    Freischmidt, A; Untergehrer, M; Ziegler, J; Knuth, S; Okpanyi, S; Müller, J; Kelber, O; Weiser, D; Jürgenliemk, G

    2015-09-01

    Willow bark extracts are used for the treatment of fever, pain and inflammation. Recent clinical and pharmacological research revealed that not only the salicylic alcohol derivatives, but also the polyphenols significantly contribute to these effects. Quantitative analysis of the European Pharmacopoeia still focuses on the determination of the salicylic alcohol derivatives. The objective of the present study was the development of an effective quantification method for the determination of as many flavanone and chalcone glycosides as possible in Salix purpurea and other Salix species as well as commercial preparations thereof. As Salix species contain a diverse spectrum of the glycosidated flavanones naringenin, eriodictyol, and the chalcone chalconaringenin, a subsequent acidic and enzymatic hydrolysis was developed to yield naringenin and eriodictyol as aglycones, which were quantified by HPLC. The 5-O-glucosides were cleaved with 11.5% TFA before subsequent hydrolysis of the 7-O-glucosides with an almond β-glucosidase at pH 6-7. The method was validated with regard to LOD, LOQ, intraday and interday precision, accuracy, stability, recovery, time of hydrolysis, robustness and applicability to extracts. All 5-O- and 7-O-glucosides of naringenin, eriodictyol and chalconaringenin were completely hydrolysed and converted to naringenin and eriodictyol. The LOD of the HPLC method was 0.77 μM of naringenin and 0.45 μM of eriodictyol. The LOQ was 2.34 μM of naringenin and 1.35 μM for eriodictyol. The method is robust with regard to sample weight, but susceptible concerning enzyme deterioration. The developed method is applicable to the determination of flavanone and chalcone glycosides in willow bark and corresponding preparations.

  5. Hydrocarbons on Phoebe, Iapetus, and Hyperion: Quantitative Analysis

    NASA Technical Reports Server (NTRS)

    Cruikshank, Dale P.; MoreauDalleOre, Cristina; Pendleton, Yvonne J.; Clark, Roger Nelson

    2012-01-01

    We present a quantitative analysis of the hydrocarbon spectral bands measured on three of Saturn's satellites, Phoebe, Iaperus, and Hyperion. These bands, measured with the Cassini Visible-Infrared Mapping Spectrometer on close fly-by's of these satellites, are the C-H stretching modes of aromatic hydrocarbons at approximately 3.28 micrometers (approximately 3050 per centimeter), and the are four blended bands of aliphatic -CH2- and -CH3 in the range approximately 3.36-3.52 micrometers (approximately 2980- 2840 per centimeter) bably indicating the presence of polycyclic aromatic hydrocarbons (PAH), is unusually strong in comparison to the aliphatic bands, resulting in a unique signarure among Solar System bodies measured so far, and as such offers a means of comparison among the three satellites. The ratio of the C-H bands in aromatic molecules to those in aliphatic molecules in the surface materials of Phoebe, NAro:NAliph approximately 24; for Hyperion the value is approximately 12, while laperus shows an intermediate value. In view of the trend of the evolution (dehydrogenation by heat and radiation) of aliphatic complexes toward more compact molecules and eventually to aromatics, the relative abundances of aliphatic -CH2- and -CH3- is an indication of the lengths of the molecular chain structures, hence the degree of modification of the original material. We derive CH2:CH3 approximately 2.2 in the spectrum of low-albedo material on laperus; this value is the same within measurement errors to the ratio in the diffuse interstellar medium. The similarity in the spectral signatures of the three satellites, plus the apparent weak trend of aromatic/aliphatic abundance from Phoebe to Hyperion, is consistent with, and effectively confirms that the source of the hydrocarbon-bearing material is Phoebe, and that the appearance of that material on the other two satellites arises from the deposition of the inward-spiraling dust that populates the Phoebe ring.

  6. Quantitative Analysis of the Effective Functional Structure in Yeast Glycolysis

    PubMed Central

    De la Fuente, Ildefonso M.; Cortes, Jesus M.

    2012-01-01

    The understanding of the effective functionality that governs the enzymatic self-organized processes in cellular conditions is a crucial topic in the post-genomic era. In recent studies, Transfer Entropy has been proposed as a rigorous, robust and self-consistent method for the causal quantification of the functional information flow among nonlinear processes. Here, in order to quantify the functional connectivity for the glycolytic enzymes in dissipative conditions we have analyzed different catalytic patterns using the technique of Transfer Entropy. The data were obtained by means of a yeast glycolytic model formed by three delay differential equations where the enzymatic rate equations of the irreversible stages have been explicitly considered. These enzymatic activity functions were previously modeled and tested experimentally by other different groups. The results show the emergence of a new kind of dynamical functional structure, characterized by changing connectivity flows and a metabolic invariant that constrains the activity of the irreversible enzymes. In addition to the classical topological structure characterized by the specific location of enzymes, substrates, products and feedback-regulatory metabolites, an effective functional structure emerges in the modeled glycolytic system, which is dynamical and characterized by notable variations of the functional interactions. The dynamical structure also exhibits a metabolic invariant which constrains the functional attributes of the enzymes. Finally, in accordance with the classical biochemical studies, our numerical analysis reveals in a quantitative manner that the enzyme phosphofructokinase is the key-core of the metabolic system, behaving for all conditions as the main source of the effective causal flows in yeast glycolysis. PMID:22393350

  7. Quantitative PCR analysis of salivary pathogen burden in periodontitis

    PubMed Central

    Salminen, Aino; Kopra, K. A. Elisa; Hyvärinen, Kati; Paju, Susanna; Mäntylä, Päivi; Buhlin, Kåre; Nieminen, Markku S.; Sinisalo, Juha; Pussinen, Pirkko J.

    2015-01-01

    Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4–5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39–4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51–4.52). The highest OR 3.59 (95% CI 1.94–6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and

  8. Quantitative PCR analysis of salivary pathogen burden in periodontitis.

    PubMed

    Salminen, Aino; Kopra, K A Elisa; Hyvärinen, Kati; Paju, Susanna; Mäntylä, Päivi; Buhlin, Kåre; Nieminen, Markku S; Sinisalo, Juha; Pussinen, Pirkko J

    2015-01-01

    Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4-5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39-4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51-4.52). The highest OR 3.59 (95% CI 1.94-6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and T

  9. Visual analysis and dynamical control of phosphoproteomic networks

    NASA Astrophysics Data System (ADS)

    Meyer-Bäse, Anke; Görke, Robert; Lobbes, Marc; Emmett, Mark R.; Nilsson, Carol L.

    2013-05-01

    This paper presents novel graph algorithms and modern control solutions applied to the graph networks resulting from specific experiments to discover disease-related pathways and drug targets in glioma cancer stem cells (GSCs). The theoretical framework applies to many other high-throughput data from experiments relevant to a variety of diseases. In addition to developing novel graph and control networks to predict therapeutic targets, these algorithms will provide biochemists with techniques to identify more metabolic regions and biological pathways for complex diseases, and design and test novel therapeutic solutions.

  10. A Quantitative Analysis of the Behavioral Checklist of the Movement ABC Motor Test

    ERIC Educational Resources Information Center

    Ruiz, Luis Miguel; Gomez, Marta; Graupera, Jose Luis; Gutierrez, Melchor; Linaza, Jose Luis

    2007-01-01

    The fifth section of the Henderson and Sugden's Movement ABC Checklist is part of the general Checklist that accompanies The Movement ABC Battery. The authors maintain that the analysis of this section must be mainly qualitative instead of quantitative. The main objective of this study was to employ a quantitative analysis of this behavioural…

  11. Quantitative proton nuclear magnetic resonance for the structural and quantitative analysis of atropine sulfate.

    PubMed

    Shen, Shi; Yao, Jing; Shi, Yaqin

    2014-02-01

    This study assessed a general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of atropine sulfate (Active Pharmaceutical Ingredient, API) as reference standard. The spectra were acquired in D2O using maleic acid as the internal standard. Conformational behaviors of tropane ring were observed and studied by means of NMR and ROESY experiments at different temperature, which showed that the azine methyl group was at equilibrium for axial and equatorial conformations at room temperature. Signal delay and monitor signals of qNMR experimentation were optimized for quantification. The study reported here validated the method's linearity, range, limit of quantification, stability and precision. The results were consistent with the results obtained from mass balance approach.

  12. Quantitative Analysis by Isotopic Dilution Using Mass Spectroscopy: The Determination of Caffeine by GC-MS.

    ERIC Educational Resources Information Center

    Hill, Devon W.; And Others

    1988-01-01

    Describes a laboratory technique for quantitative analysis of caffeine by an isotopic dilution method for coupled gas chromatography-mass spectroscopy. Discusses caffeine analysis and experimental methodology. Lists sample caffeine concentrations found in common products. (MVL)

  13. Microchromatography of hemoglobins. VIII. A general qualitative and quantitative method in plastic drinking straws and the quantitative analysis of Hb-F.

    PubMed

    Schroeder, W A; Pace, L A

    1978-03-01

    The microchromatographic procedure for the quantitative analysis of the hemoglobin components in a hemolysate uses columns of DEAE-cellulose in a plastic drinking straw with a glycine-KCN-NaCl developer. Not only may the method be used for the quantitative analysis of Hb-F but also for the analysis of the varied components in mixtures of hemoglobins.

  14. Geographical classification of Epimedium based on HPLC fingerprint analysis combined with multi-ingredients quantitative analysis.

    PubMed

    Xu, Ning; Zhou, Guofu; Li, Xiaojuan; Lu, Heng; Meng, Fanyun; Zhai, Huaqiang

    2017-05-01

    A reliable and comprehensive method for identifying the origin and assessing the quality of Epimedium has been developed. The method is based on analysis of HPLC fingerprints, combined with similarity analysis, hierarchical cluster analysis (HCA), principal component analysis (PCA) and multi-ingredient quantitative analysis. Nineteen batches of Epimedium, collected from different areas in the western regions of China, were used to establish the fingerprints and 18 peaks were selected for the analysis. Similarity analysis, HCA and PCA all classified the 19 areas into three groups. Simultaneous quantification of the five major bioactive ingredients in the Epimedium samples was also carried out to confirm the consistency of the quality tests. These methods were successfully used to identify the geographical origin of the Epimedium samples and to evaluate their quality.

  15. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

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

    PubMed

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

    2014-01-01

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

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

    SciTech Connect

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

    2013-09-22

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

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

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-05

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

  19. Teaching Quantitative Reasoning for Nonscience Majors through Carbon Footprint Analysis

    ERIC Educational Resources Information Center

    Boose, David L.

    2014-01-01

    Quantitative reasoning is a key intellectual skill, applicable across disciplines and best taught in the context of authentic, relevant problems. Here, I describe and assess a laboratory exercise that has students calculate their "carbon footprint" and evaluate the impacts of various behavior choices on that footprint. Students gather…

  20. Quantitative proteomic analysis by accurate mass retention time pairs.

    PubMed

    Silva, Jeffrey C; Denny, Richard; Dorschel, Craig A; Gorenstein, Marc; Kass, Ignatius J; Li, Guo-Zhong; McKenna, Therese; Nold, Michael J; Richardson, Keith; Young, Phillip; Geromanos, Scott

    2005-04-01

    Current methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods to name only a few. The current literature illustrates both pros and cons for each of the previously mentioned methodologies. Keeping with the teachings of William of Ockham, "with all things being equal the simplest solution tends to be correct", a simple LC/MS based methodology is presented that allows relative changes in abundance of proteins in highly complex mixtures to be determined. Utilizing a reproducible chromatographic separations system along with the high mass resolution and mass accuracy of an orthogonal time-of-flight mass spectrometer, the quantitative comparison of tens of thousands of ions emanating from identically prepared control and experimental samples can be made. Using this configuration, we can determine the change in relative abundance of a small number of ions between the two conditions solely by accurate mass and retention time. Employing standard operating procedures for both sample preparation and ESI-mass spectrometry, one typically obtains under 5 ppm mass precision and quantitative variations between 10 and 15%. The principal focus of this paper will demonstrate the quantitative aspects of the methodology and continue with a discussion of the associated, complementary qualitative capabilities.

  1. MOLD SPECIFIC QUANTITATIVE PCR: THE EMERGING STANDARD IN MOLD ANALYSIS

    EPA Science Inventory

    Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to human...

  2. Quantitative and Qualitative Analysis of Biomarkers in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, a combination HPLC-DART-TOF-MS system was utilized to identify and quantitatively analyze carbohydrates in wild type and mutant strains of Fusarium verticillioides. Carbohydrate fractions were isolated from F. verticillioides cellular extracts by HPLC using a cation-exchange size-excl...

  3. A new quantitative method for gunshot residue analysis by ion beam analysis.

    PubMed

    Christopher, Matthew E; Warmenhoeven, John-William; Romolo, Francesco S; Donghi, Matteo; Webb, Roger P; Jeynes, Christopher; Ward, Neil I; Kirkby, Karen J; Bailey, Melanie J

    2013-08-21

    Imaging and analyzing gunshot residue (GSR) particles using the scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDS) is a standard technique that can provide important forensic evidence, but the discrimination power of this technique is limited due to low sensitivity to trace elements and difficulties in obtaining quantitative results from small particles. A new, faster method using a scanning proton microbeam and Particle Induced X-ray Emission (μ-PIXE), together with Elastic Backscattering Spectrometry (EBS) is presented for the non-destructive, quantitative analysis of the elemental composition of single GSR particles. In this study, the GSR particles were all Pb, Ba, Sb. The precision of the method is assessed. The grouping behaviour of different makes of ammunition is determined using multivariate analysis. The protocol correctly groups the cartridges studied here, with a confidence >99%, irrespective of the firearm or population of particles selected.

  4. Structural and Quantitative Analysis of Three C-Glycosylflavones by Variable Temperature Proton Quantitative Nuclear Magnetic Resonance

    PubMed Central

    Liu, Yang; Dai, Zhong

    2017-01-01

    Quantitative nuclear magnetic resonance is a powerful tool in drug analysis because of its speed, precision, and efficiency. In present study, the application of variable temperature proton quantitative nuclear magnetic resonance (VT-1H-qNMR) for the calibration of three C-glycosylflavones including orientin, isoorientin, and schaftoside as reference substances was reported. Since there was conformational equilibrium due to the restricted rotation around the C(sp3)-C(sp2) bond in C-glycosylflavones, the conformational behaviors were investigated by VT-NMR and verified by molecular mechanics (MM) calculation. The VT-1H-qNMR method was validated including the linearity, limit of quantification, precision, and stability. The results were consistent with those obtained from mass balance approach. VT-1H-qNMR can be deployed as an effective tool in analyzing C-glycosylflavones. PMID:28243484

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

    PubMed Central

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

    2015-01-01

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

  6. Quantitative sectioning and noise analysis for structured illumination microscopy

    PubMed Central

    Hagen, Nathan; Gao, Liang; Tkaczyk, Tomasz S.

    2011-01-01

    Structured illumination (SI) has long been regarded as a nonquantitative technique for obtaining sectioned microscopic images. Its lack of quantitative results has restricted the use of SI sectioning to qualitative imaging experiments, and has also limited researchers’ ability to compare SI against competing sectioning methods such as confocal microscopy. We show how to modify the standard SI sectioning algorithm to make the technique quantitative, and provide formulas for calculating the noise in the sectioned images. The results indicate that, for an illumination source providing the same spatially-integrated photon flux at the object plane, and for the same effective slice thicknesses, SI sectioning can provide higher SNR images than confocal microscopy for an equivalent setup when the modulation contrast exceeds about 0.09. PMID:22274364

  7. Quantitative analysis of immobilized metalloenzymes by atomic absorption spectroscopy.

    PubMed

    Opwis, Klaus; Knittel, Dierk; Schollmeyer, Eckhard

    2004-12-01

    A new, sensitive assay for the quantitative determination of immobilized metal containing enzymes has been developed using atomic absorption spectroscopy (AAS). In contrast with conventionally used indirect methods the described quantitative AAS assay for metalloenzymes allows more exact analyses, because the carrier material with the enzyme is investigated directly. As an example, the validity and reliability of the method was examined by fixing the iron-containing enzyme catalase on cotton fabrics using different immobilization techniques. Sample preparation was carried out by dissolving the loaded fabrics in sulfuric acid before oxidising the residues with hydrogen peroxide. The iron concentrations were determined by flame atomic absorption spectrometry after calibration of the spectrometer with solutions of the free enzyme at different concentrations.

  8. Semi-quantitative spectrographic analysis and rank correlation in geochemistry

    USGS Publications Warehouse

    Flanagan, F.J.

    1957-01-01

    The rank correlation coefficient, rs, which involves less computation than the product-moment correlation coefficient, r, can be used to indicate the degree of relationship between two elements. The method is applicable in situations where the assumptions underlying normal distribution correlation theory may not be satisfied. Semi-quantitative spectrographic analyses which are reported as grouped or partly ranked data can be used to calculate rank correlations between elements. ?? 1957.

  9. Quantitative analysis of cortical pyramidal neurons after corpus callosotomy.

    PubMed

    Jacobs, Bob; Creswell, Johanna; Britt, Jonathan P; Ford, Kevin L; Bogen, Joseph E; Zaidel, Eran

    2003-07-01

    This study quantitatively explored the dendritic/spine extent of supragranular pyramidal neurons across several cortical areas in two adult male subjects who had undergone a callosotomy several decades before death. In all cortical areas, there were numerous atypical, supragranular pyramidal neurons with elongated "tap root" basilar dendrites. These atypical cells could be associated with an underlying epileptic condition and/or could represent a compensatory mechanism in response to deafferentation after callosotomy.

  10. Fluorescent microscopy approaches of quantitative soil microbial analysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Konstantin; Polyanskaya, Lubov

    2015-04-01

    Classical fluorescent microscopy method was used during the last decades in various microbiological studies of terrestrial ecosystems. The method provides representative results and simple application which is allow to use it both as routine part of amplitudinous research and in small-scaled laboratories. Furthermore, depending on research targets a lot of modifications of fluorescent microscopy method were established. Combination and comparison of several approaches is an opportunity of quantitative estimation of microbial community in soil. The first analytical part of the study was dedicated to soil bacterial density estimation by fluorescent microscopy in dynamic of several 30-days experiments. The purpose of research was estimation of changes in soil bacterial community on the different soil horizons under aerobic and anaerobic conditions with adding nutrients in two experimental sets: cellulose and chitin. Was modified the nalidixic acid method for inhibition of DNA division of gram-negative bacteria, and the method provides the quantification of this bacterial group by fluorescent microscopy. Established approach allowed to estimate 3-4 times more cells of gram-negative bacteria in soil. The functions of actinomyces in soil polymer destruction are traditionally considered as dominant in comparison to gram-negative bacterial group. However, quantification of gram-negative bacteria in chernozem and peatland provides underestimation of classical notion for this bacterial group. Chitin introduction had no positive effect to gram-negative bacterial population density changes in chernozem but concurrently this nutrient provided the fast growing dynamics at the first 3 days of experiment both under aerobic and anaerobic conditions. This is confirming chitinolytic activity of gram-negative bacteria in soil organic matter decomposition. At the next part of research modified method for soil gram-negative bacteria quantification was compared to fluorescent in situ

  11. The Effects of Selection on Linkage Analysis for Quantitative Traits

    PubMed Central

    Mackinnon, M. J.; Georges, MAJ.

    1992-01-01

    The effects of within-sample selection on the outcome of analyses detecting linkage between genetic markers and quantitative traits were studied. It was found that selection by truncation for the trait of interest significantly reduces the differences between marker genotype means thus reducing the power to detect linked quantitative trait loci (QTL). The size of this reduction is a function of proportion selected, the magnitude of the QTL effect, recombination rate between the marker locus and the QTL, and the allele frequency of the QTL. Proportion selected was the most influential of these factors on bias, e.g., for an allele substitution effect of one standard deviation unit, selecting the top 80%, 50% or 20% of the population required 2,6 or 24 times the number of progeny, respectively, to offset the loss of power caused by this selection. The effect on power was approximately linear with respect to the size of gene effect, almost invariant to recombination rate, and a complex function of QTL allele frequency. It was concluded that experimental samples from animal populations which have been subjected to even minor amounts of selection will be inefficient in yielding information on linkage between markers and loci influencing the quantitative trait under selection. PMID:1459434

  12. An Inexpensive Electrodeposition Device and Its Use in a Quantitative Analysis Laboratory Exercise

    ERIC Educational Resources Information Center

    Parker, Richard H.

    2011-01-01

    An experimental procedure, using an apparatus that is easy to construct, was developed to incorporate a quantitative electrogravimetric determination of the solution nickel content into an undergraduate or advanced high school quantitative analysis laboratory. This procedure produces results comparable to the procedure used for the gravimetric…

  13. Quantitative Analysis of Single Amino Acid Variant Peptides Associated with Pancreatic Cancer in Serum by an Isobaric Labeling Quantitative Method

    PubMed Central

    2015-01-01

    Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value < 0.05). Significant differences in the variant peptide SDNCEDTPEAGYFAVAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research. PMID:25393578

  14. Quantitative image analysis in the assessment of diffuse large B-cell lymphoma.

    PubMed

    Chabot-Richards, Devon S; Martin, David R; Myers, Orrin B; Czuchlewski, David R; Hunt, Kristin E

    2011-12-01

    Proliferation rates in diffuse large B-cell lymphoma have been associated with conflicting outcomes in the literature, more often with high proliferation associated with poor prognosis. In most studies, the proliferation rate was estimated by a pathologist using an immunohistochemical stain for the monoclonal antibody Ki-67. We hypothesized that a quantitative image analysis algorithm would give a more accurate estimate of the proliferation rate, leading to better associations with survival. In all, 84 cases of diffuse large B-cell lymphoma were selected according to the World Health Organization criteria. Ki-67 percentage positivity estimated by the pathologist was recorded from the original report. The same slides were then scanned using an Aperio ImageScope, and Ki-67 percentage positivity was calculated using a computer-based quantitative immunohistochemistry nuclear algorithm. In addition, chart review was performed and survival time was recorded. The Ki-67 percentage estimated by the pathologist from the original report versus quantitative image analysis was significantly correlated (P<0.001), but pathologist Ki-67 percentages were significantly higher than quantitative image analysis (P=0.021). There was less agreement at lower Ki-67 percentages. Comparison of Ki-67 percentage positivity versus survival did not show significant association either with pathologist estimate or quantitative image analysis. However, although not significant, there was a trend of worse survival at higher proliferation rates detected by the pathologist but not by quantitative image analysis. Interestingly, our data suggest that the Ki-67 percentage positivity as assessed by the pathologist may be more closely associated with survival outcome than that identified by quantitative image analysis. This may indicate that pathologists are better at selecting appropriate areas of the slide. More cases are needed to assess whether this finding would be statistically significant. Due to

  15. MCM - 2 and Ki - 67 as proliferation markers in renal cell carcinoma: A quantitative and semi - quantitative analysis

    PubMed Central

    Mehdi, Muhammad Zain; Nagi, Abdul Hanan; Naseem, Nadia

    2016-01-01

    ABSTRACT Introduction/Background: Fuhrman nuclear grade is the most important histological parameter to predict prognosis in a patient of renal cell carcinoma (RCC). However, it suffers from inter-observer and intra-observer variation giving rise to need of a parameter that not only correlates with nuclear grade but is also objective and reproducible. Proliferation is the measure of aggressiveness of a tumour and it is strongly correlated with Fuhrman nuclear grade, clinical survival and recurrence in RCC. Ki-67 is conventionally used to assess proliferation. Mini-chromosome maintenance 2 (MCM-2) is a lesser known marker of proliferation and identifies a greater proliferation faction. This study was designed to assess the prognostic significance of MCM-2 by comparing it with Fuhrman nuclear grade and Ki-67. Material and Methods: n=50 cases of various ages, stages, histological subtypes and grades of RCC were selected for this study. Immunohistochemical staining using Ki-67(MIB-1, Mouse monoclonal antibody, Dako) and MCM-2 (Mouse monoclonal antibody, Thermo) was performed on the paraffin embedded blocks in the department of Morbid anatomy and Histopathology, University of Health Sciences, Lahore. Labeling indices (LI) were determined by two pathologists independently using quantitative and semi-quantitative analysis. Statistical analysis was carried out using SPSS 20.0. Kruskall-Wallis test was used to determine a correlation of proliferation markers with grade, and Pearson's correlate was used to determine correlation between the two proliferation markers. Results: Labeling index of MCM-2 (median=24.29%) was found to be much higher than Ki-67(median=13.05%). Both markers were significantly related with grade (p=0.00; Kruskall-Wallis test). LI of MCM-2 was found to correlate significantly with LI of Ki-67(r=0.0934;p=0.01 with Pearson's correlate). Results of semi-quantitative analysis correlated well with quantitative analysis. Conclusion: Both Ki-67 and MCM-2 are

  16. Study of proper conditions for quantitative atom-probe analysis

    NASA Astrophysics Data System (ADS)

    Rolander, Ulf; Andrén, Hans-Olof

    1994-03-01

    Atom-probe microanalysis is a truly quantitative method only if certain requirements are fulfilled. Field evaporation must only happen when the detector system is active; ions must travel from specimen to detector without being obstructed; and ions must be detected with the same probability regardless of mass and energy. Designs and methods to achieve these requirements are presented in the paper, such as a controlled high-voltage pulser, a detector with good and variable multi-hit resolution, ion optical alignment procedures, and a method to statistically correct for pile-up in the detector.

  17. Quantitative analysis of Euclidean distance to complement qualitative analysis of facial expression during deception

    PubMed Central

    Mondal, Ananya; Mukhopadhyay, Pritha; Basu, Nabanita; Bandyopadhyay, Samir Kumar; Chatterjee, Tanima

    2016-01-01

    Background: Accurate evaluation of an individuals' veracity is a fundamental aspect of social functioning that allows individuals to act in adaptive ways. The domain of deception detection ability is still young, and many components in this field are yet to be touched which demands more research in this field. Aims: The present study aims at deciphering the structural composition of face during felt, posed, and deceived emotions in facial expression unique to Indian culture, using Facial Action Coding System (FACS). Quantitative analysis of Euclidean distance has been done to complement qualitative FACS analysis. Methods: In this study, thirty female, young adults with age range of 23–27 years were chosen randomly for portraying their (felt, posed, and deceived) facial expression. All facial expressions were captured through instruction, and videos were converted into static images. The static images were coded on the basis of FACS to decipher the felt, posed, and deceived expressions. Quantitative analysis of the data has been done using MATLAB to meet the objectives of the study and to complement the qualitative analysis. Results: Felt and posed emotions differ in terms of intensity of the expression and subjective experience. Posed emotional and deceived expressions differ in intent. Facial asymmetry is an important indicator for detecting deception. PMID:28163412

  18. Chemical Fingerprint Analysis and Quantitative Analysis of Rosa rugosa by UPLC-DAD.

    PubMed

    Mansur, Sanawar; Abdulla, Rahima; Ayupbec, Amatjan; Aisa, Haji Akbar

    2016-12-21

    A method based on ultra performance liquid chromatography with a diode array detector (UPLC-DAD) was developed for quantitative analysis of five active compounds and chemical fingerprint analysis of Rosa rugosa. Ten batches of R. rugosa collected from different plantations in the Xinjiang region of China were used to establish the fingerprint. The feasibility and advantages of the used UPLC fingerprint were verified for its similarity evaluation by systematically comparing chromatograms with professional analytical software recommended by State Food and Drug Administration (SFDA) of China. In quantitative analysis, the five compounds showed good regression (R² = 0.9995) within the test ranges, and the recovery of the method was in the range of 94.2%-103.8%. The similarities of liquid chromatography fingerprints of 10 batches of R. rugosa were more than 0.981. The developed UPLC fingerprint method is simple, reliable, and validated for the quality control and identification of R. rugosa. Additionally, simultaneous quantification of five major bioactive ingredients in the R. rugosa samples was conducted to interpret the consistency of the quality test. The results indicated that the UPLC fingerprint, as a characteristic distinguishing method combining similarity evaluation and quantification analysis, can be successfully used to assess the quality and to identify the authenticity of R. rugosa.

  19. A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin*

    PubMed Central

    Völker-Albert, Moritz Carl; Pusch, Miriam Caroline; Fedisch, Andreas; Schilcher, Pierre; Schmidt, Andreas; Imhof, Axel

    2016-01-01

    The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner. PMID:26811354

  20. Quantitative analysis of biopolymers by matrix-assisted laser desorption

    SciTech Connect

    Tang, K.; Allman, S.L.; Jones, R.B.; Chen, C.H. )

    1993-08-01

    During the past few years, major efforts have been made to use mass spectrometry to measure biopolymers because of the great potential benefit to biological and medical research. Although the theoretical details of laser desorption and ionization mechanisms of MALDI are not yet fully understood, several models have been presented to explain the production of large biopolymer ions. In brief, it is very difficult to obtain reliable measurements of the absolute quantity of analytes by MALDI. If MALDI is going to become a routine analytical tool, it is obvious that quantitative measurement capability must be pursued. Oligonucleotides and protein samples used in this work were purchased from commercial sources. Nicotinic acid was used as matrix for both types of biopolymers. From this experiment, it is seen that it is difficult to obtain absolute quantitative measurements of biopolymers using MALDI. However, internal calibration with molecules having similar chemical properties can be used to resolve these difficulties. Chemical reactions between biopolymers must be avoided to prevent the destruction of the analyte materials. 10 refs., 8 figs.

  1. Quantitative Spectral Analysis of Evolved Low-Mass Stars

    NASA Astrophysics Data System (ADS)

    Werner, Klaus; Rauch, Thomas; Kruk, Jeffrey W.

    2009-09-01

    The hydrogen-deficiency in extremely hot post-AGB stars of spectral class PG1159 is probably caused by a (very) late helium-shell flash or a AGB final thermal pulse that consumes the hydrogen envelope, exposing the usually-hidden intershell region. Thus, the photospheric element abundances of these stars allow us to draw conclusions about details of nuclear burning and mixing processes in the precursor AGB stars. We compare predicted element abundances to those determined by quantitative spectral analyses performed with advanced non-LTE model atmospheres. A good qualitative and quantitative agreement is found for many species (He, C, N, O, Ne, F, Si, Ar) but discrepancies for others (P, S, Fe) point at shortcomings in stellar evolution models for AGB stars. Almost all of the chemical trace elements in these hot stars can only be identified in the UV spectral range. The Far Ultraviolet Spectroscopic Explorer and the Hubble Space Telescope played a crucial role for this research.

  2. Quantitative analysis of radiation-induced changes in sperm morphology

    SciTech Connect

    Young, I.T.; Gledhill, B.L.; Lake, S.; Wyrobek, A.J.

    1982-09-01

    When developing spermatogenic cells are exposed to radiation, chemical carcinogens or mutagens, the transformation in the morphology of the mature sperm can be used to determine the severity of the exposure. In this study five groups of mice with three mice per group received testicular doses of X irradiation at dosage levels ranging from 0 rad to 120 rad. A random sample of 100 mature sperm per mouse was analyzed five weeks later for the quantitative morphologic transformation as a function of dosage level. The cells were stained with gallocyanin chrome alum (GCA) so that only the DNA in the sperm head was visible. The ACUity quantitative microscopy system at Lawrence Livermore National Laboratory was used to scan the sperm at a sampling density of 16 points per linear micrometer and with 256 brightness levels per point. The contour of each cell was extracted using conventional thresholding techniques on the high-contrast images. For each contour a variety of shape features was then computed to characterize the morphology of that cell. Using the control group and the distribution of their shape features to establish the variability of a normal sperm population, the 95% limits on normal morphology were established. Using only four shape features, a doubling dose of approximately 39 rad was determined. That is, at 39 rad exposure the percentage of abnormal cells was twice that occurring in the control population. This compared to a doubling dose of approximately 70 rad obtained from a concurrent visual procedure.

  3. Quantitative Raman spectroscopy for the analysis of carrot bioactives.

    PubMed

    Killeen, Daniel P; Sansom, Catherine E; Lill, Ross E; Eason, Jocelyn R; Gordon, Keith C; Perry, Nigel B

    2013-03-20

    Rapid quantitative near-infrared Fourier transform Raman analyses of the key phytonutrients in carrots, polyacetylenes and carotenoids, are reported here for the first time. Solvent extracts of 31 carrot lines were analyzed for these phytonutrients by conventional methods, polyacetylenes by GC-FID and carotenoids by visible spectrophotometry. Carotenoid concentrations were 0-5586 μg g(-1) dry weight (DW). Polyacetylene concentrations were 74-4846 μg g(-1) DW, highest in wild carrots. The polyacetylenes were falcarinol, 6-1237 μg g(-1) DW; falcarindiol, 42-3475 μg g(-1) DW; and falcarindiol 3-acetate, 27-649 μg g(-1) DW. Strong Raman bands for carotenoids gave good correlation to results by visible spectrophotometry. A chemometric model capable of quantitating carotenoids from Raman data was developed. A classification model for rapidly distinguishing carrots with high and low polyacetylene (limit of detection = 1400 μg g(-1)) concentrations based on Raman spectral intensity in the region of 2250 cm(-1) was produced.

  4. Quantitative characterization of surface topography using spectral analysis

    NASA Astrophysics Data System (ADS)

    Jacobs, Tevis D. B.; Junge, Till; Pastewka, Lars

    2017-03-01

    Roughness determines many functional properties of surfaces, such as adhesion, friction, and (thermal and electrical) contact conductance. Recent analytical models and simulations enable quantitative prediction of these properties from knowledge of the power spectral density (PSD) of the surface topography. The utility of the PSD is that it contains statistical information that is unbiased by the particular scan size and pixel resolution chosen by the researcher. In this article, we first review the mathematical definition of the PSD, including the one- and two-dimensional cases, and common variations of each. We then discuss strategies for reconstructing an accurate PSD of a surface using topography measurements at different size scales. Finally, we discuss detecting and mitigating artifacts at the smallest scales, and computing upper/lower bounds on functional properties obtained from models. We accompany our discussion with virtual measurements on computer-generated surfaces. This discussion summarizes how to analyze topography measurements to reconstruct a reliable PSD. Analytical models demonstrate the potential for tuning functional properties by rationally tailoring surface topography—however, this potential can only be achieved through the accurate, quantitative reconstruction of the PSDs of real-world surfaces.

  5. QUANTITATIVE CT ANALYSIS, AIRFLOW OBSTRUCTION AND LUNG CANCER IN THE PITTSBURGH LUNG SCREENING STUDY

    PubMed Central

    Wilson, David O; Leader, Joseph K; Fuhrman, Carl R; Reilly, John J; Sciurba, Frank C.; Weissfeld, Joel L

    2011-01-01

    Background To study the relationship between emphysema, airflow obstruction and lung cancer in a high risk population we performed quantitative analysis of screening computed tomography (CT) scans. Methods Subjects completed questionnaires, spirometry and low-dose helical chest CT. Analyses compared cases and controls according to automated quantitative analysis of lung parenchyma and airways measures. Results Our case-control study of 117 matched pairs of lung cancer cases and controls did not reveal any airway or lung parenchymal findings on quantitative analysis of screening CT scans that were associated with increased lung cancer risk. Airway measures including wall area %, lumen perimeter, lumen area and average wall HU, and parenchymal measures including lung fraction < −910 Hounsfield Units (HU), were not statistically different between cases and controls. Conclusions The relationship between visual assessment of emphysema and increased lung cancer risk could not be verified by quantitative analysis of low-dose screening CT scans in a high risk tobacco exposed population. PMID:21610523

  6. Grid-Enabled Quantitative Analysis of Breast Cancer

    DTIC Science & Technology

    2010-10-01

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

  7. Analysis of Artifacts Suggests DGGE Should Not Be Used For Quantitative Diversity Analysis

    PubMed Central

    Neilson, Julia W.; Jordan, Fiona L.; Maier, Raina M.

    2014-01-01

    PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with α-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real time PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis. PMID:23313091

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

    PubMed

    Nakagami, Hirofumi

    2014-01-01

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

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

    PubMed Central

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

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

  10. Quantitative Anatomic Analysis of the Native Ligamentum Teres

    PubMed Central

    Mikula, Jacob D.; Slette, Erik L.; Chahla, Jorge; Brady, Alex W.; Locks, Renato; Trindade, Christiano A. C.; Rasmussen, Matthew T.; LaPrade, Robert F.; Philippon, Marc J.

    2017-01-01

    Background: While recent studies have addressed the biomechanical function of the ligamentum teres and provided descriptions of ligamentum teres reconstruction techniques, its detailed quantitative anatomy remains relatively undocumented. Moreover, there is a lack of consensus in the literature regarding the number and morphology of the acetabular attachments of the ligamentum teres. Purpose: To provide a clinically relevant quantitative anatomic description of the native human ligamentum teres. Study Design: Descriptive laboratory study. Methods: Ten human cadaveric hemipelvises, complete with femurs (mean age, 59.6 years; range, 47-65 years), were dissected free of all extra-articular soft tissues to isolate the ligamentum teres and its attachments. A coordinate measuring device was used to quantify the attachment areas and their relationships to pertinent open and arthroscopic landmarks on both the acetabulum and the femur. The clock face reference system was utilized to describe acetabular anatomy, and all anatomic relationships were described using the mean and 95% confidence intervals. Results: There were 6 distinct attachments to the acetabulum and 1 to the femur. The areas of the acetabular and femoral attachment footprints of the ligamentum teres were 434 mm2 (95% CI, 320-549 mm2) and 84 mm2 (95% CI, 65-104 mm2), respectively. The 6 acetabular clock face locations were as follows: anterior attachment, 4:53 o’clock (95% CI, 4:45-5:02); posterior attachment, 6:33 o’clock (95% CI, 6:23-6:43); ischial attachment, 8:07 o’clock (95% CI, 7:47-8:26); iliac attachment, 1:49 o’clock (95% CI, 1:04-2:34); and a smaller pubic attachment that was located at 3:50 o’clock (95% CI, 3:41-4:00). The ischial attachment possessed the largest cross-sectional attachment area (127.3 mm2; 95% CI, 103.0-151.7 mm2) of all the acetabular attachments of the ligamentum teres. Conclusion: The most important finding of this study was that the human ligamentum teres had 6

  11. Bridging the gaps for global sustainable development: a quantitative analysis.

    PubMed

    Udo, Victor E; Jansson, Peter Mark

    2009-09-01

    Global human progress occurs in a complex web of interactions between society, technology and the environment as driven by governance and infrastructure management capacity among nations. In our globalizing world, this complex web of interactions over the last 200 years has resulted in the chronic widening of economic and political gaps between the haves and the have-nots with consequential global cultural and ecosystem challenges. At the bottom of these challenges is the issue of resource limitations on our finite planet with increasing population. The problem is further compounded by pleasure-driven and poverty-driven ecological depletion and pollution by the haves and the have-nots respectively. These challenges are explored in this paper as global sustainable development (SD) quantitatively; in order to assess the gaps that need to be bridged. Although there has been significant rhetoric on SD with very many qualitative definitions offered, very few quantitative definitions of SD exist. The few that do exist tend to measure SD in terms of social, energy, economic and environmental dimensions. In our research, we used several human survival, development, and progress variables to create an aggregate SD parameter that describes the capacity of nations in three dimensions: social sustainability, environmental sustainability and technological sustainability. Using our proposed quantitative definition of SD and data from relatively reputable secondary sources, 132 nations were ranked and compared. Our comparisons indicate a global hierarchy of needs among nations similar to Maslow's at the individual level. As in Maslow's hierarchy of needs, nations that are struggling to survive are less concerned with environmental sustainability than advanced and stable nations. Nations such as the United States, Canada, Finland, Norway and others have higher SD capacity, and thus, are higher on their hierarchy of needs than nations such as Nigeria, Vietnam, Mexico and other

  12. Quantitative interpretation of mineral hyperspectral images based on principal component analysis and independent component analysis methods.

    PubMed

    Jiang, Xiping; Jiang, Yu; Wu, Fang; Wu, Fenghuang

    2014-01-01

    Interpretation of mineral hyperspectral images provides large amounts of high-dimensional data, which is often complicated by mixed pixels. The quantitative interpretation of hyperspectral images is known to be extremely difficult when three types of information are unknown, namely, the number of pure pixels, the spectrum of pure pixels, and the mixing matrix. The problem is made even more complex by the disturbance of noise. The key to interpreting abstract mineral component information, i.e., pixel unmixing and abundance inversion, is how to effectively reduce noise, dimension, and redundancy. A three-step procedure is developed in this study for quantitative interpretation of hyperspectral images. First, the principal component analysis (PCA) method can be used to process the pixel spectrum matrix and keep characteristic vectors with larger eigenvalues. This can effectively reduce the noise and redundancy, which facilitates the abstraction of major component information. Second, the independent component analysis (ICA) method can be used to identify and unmix the pixels based on the linear mixed model. Third, the pure-pixel spectrums can be normalized for abundance inversion, which gives the abundance of each pure pixel. In numerical experiments, both simulation data and actual data were used to demonstrate the performance of our three-step procedure. Under simulation data, the results of our procedure were compared with theoretical values. Under the actual data measured from core hyperspectral images, the results obtained through our algorithm are compared with those of similar software (Mineral Spectral Analysis 1.0, Nanjing Institute of Geology and Mineral Resources). The comparisons show that our method is effective and can provide reference for quantitative interpretation of hyperspectral images.

  13. Torpedo electromotor system development: a quantitative analysis of synaptogenesis.

    PubMed

    Fox, G Q; Kötting, D

    1984-04-10

    Synaptogenesis in the electric organ of Torpedo marmorato has been studied quantitatively at the ultrastructural level of observation. In addition to establishing the normal developmental time course for this event we were interested in determining whether a gradient of synaptogenesis might be present because the electric organ produces several morphologically recognizable spatiotemporal gradients during its early ontogeny. These gradients genesis of electrocyte columns, both gradients of which are operative for periods of weeks. No gradient of synaptogenesis was found, indicating this to be a synchronous process. The idea is advanced that synaptogenesis in the electric organ is modulated by extrinsic influences, many of which may originate from the target electrocytes which, by this time, have become synchronized in their development.

  14. Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

    PubMed

    Boneschansker, Leo; Yan, Jun; Wong, Elisabeth; Briscoe, David M; Irimia, Daniel

    2014-09-03

    Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

  15. MICROFLUIDIC PLATFORM FOR THE QUANTITATIVE ANALYSIS OF LEUKOCYTE MIGRATION SIGNATURES

    PubMed Central

    Wong, Elisabeth; Briscoe, David M.; Irimia, Daniel

    2014-01-01

    Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyze four qualitative migration patterns: chemo-attraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence, and fraction of cells responding. We find that established chemokines C5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T-lymphocytes migrate away than towards SDF-1 and their directional migration patterns are not persistent. Overall, our platform characterizes migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators. PMID:25183261

  16. Comparative analysis of quantitative methodologies for Vibrionaceae biofilms

    PubMed Central

    Chavez-Dozal, Alba A.; Nourabadi, Neda; Erken, Martina; McDougald, Diane; Nishiguchi, Michele K.

    2016-01-01

    Multiple symbiotic and free living Vibrio sp. grow as a form of microbial community known as biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct Colony Forming Unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic activity and vitality of cells within the biofilm matrix. This study systematically tested five techniques, among which the XTT assay and wrinkled colony measurement provided the most reproducible, accurate, and efficient methods for the quantitative estimation of Vibrionaceae biofilms. PMID:27009592

  17. Quantitative Analysis of Matrine in Liquid Crystalline Nanoparticles by HPLC

    PubMed Central

    Peng, Xinsheng; Hu, Min; Ling, Yahao; Tian, Yuan; Zhou, Yanxing; Zhou, Yanfang

    2014-01-01

    A reversed-phase high-performance liquid chromatographic method has been developed to quantitatively determine matrine in liquid crystal nanoparticles. The chromatographic method is carried out using an isocratic system. The mobile phase was composed of methanol-PBS(pH6.8)-triethylamine (50 : 50 : 0.1%) with a flow rate of 1 mL/min with SPD-20A UV/vis detector and the detection wavelength was at 220 nm. The linearity of matrine is in the range of 1.6 to 200.0 μg/mL. The regression equation is y = 10706x − 2959 (R2 = 1.0). The average recovery is 101.7%; RSD = 2.22%  (n = 9). This method provides a simple and accurate strategy to determine matrine in liquid crystalline nanoparticle. PMID:24834359

  18. Copper in silicon: Quantitative analysis of internal and proximity gettering

    SciTech Connect

    McHugo, S.A.; Flink, C.; Weber, E.R.

    1997-08-01

    The behavior of copper in the presence of a proximity gettering mechanism and a standard internal gettering mechanism in silicon was studied. He implantation-induced cavities in the near surface region were used as a proximity gettering mechanism and oxygen precipitates in the bulk of the material provided internal gettering sites. Moderate levels of copper contamination were introduced by ion implantation such that the copper was not supersaturated during the anneals, thus providing realistic copper contamination/gettering conditions. Copper concentrations at cavities and internal gettering sites were quantitatively measured after the annealings. In this manner, the gettering effectiveness of cavities was measured when in direct competition with internal gettering sites. The cavities were found to be the dominant gettering mechanism with only a small amount of copper gettered at the internal gettering sites. These results reveal the benefits of a segregation-type gettering mechanism for typical contamination conditions.

  19. Quantitative analysis of peptide-MHC class II interaction.

    PubMed

    Fleckenstein, B; Jung, G; Wiesmüller, K H

    1999-12-01

    The tremendous progress in the field of basic immunology and immunochemistry made in the last decade has significantly advanced our understanding of antigen processing and presentation by MHC class I and II proteins. In this review different techniques to study peptide interaction with MHC class II molecules are summarized and their impact on the elucidation of quantitative parameters, like affinities or kinetic data, is discussed. A recently introduced method based on synthetic combinatorial peptide libraries allows to quantify the binding contribution of each amino acid residue in a class II ligand and is presented in more detail. As this knowledge is fundamental for current investigations in modern medicine, e.g. for novel immune system based therapy concepts, further aspects like the design of new high affinity MHC class II ligands and the prediction of peptide antigens are discussed.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-20

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

  4. Grid-Enabled Quantitative Analysis of Breast Cancer

    DTIC Science & Technology

    2009-10-01

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

  5. Response Neighborhoods in Online Learning Networks: A Quantitative Analysis

    ERIC Educational Resources Information Center

    Aviv, Reuven; Erlich, Zippy; Ravid, Gilad

    2005-01-01

    Theoretical foundation of Response mechanisms in networks of online learners are revealed by Statistical Analysis of p* Markov Models for the Networks. Our comparative analysis of two networks shows that the minimal-effort hunt-for-social-capital mechanism controls a major behavior of both networks: negative tendency to respond. Differences in…

  6. Afghanistan Reconstruction - A Quantitative Analysis of the International Effort

    DTIC Science & Technology

    2008-03-01

    Trinkunas Chairman, Department of National Security Affairs iv THIS PAGE INTENTIONALLY LEFT BLANK v ABSTRACT Since the start of Operation Enduring...10 4. Chapter V - Provincial Level Analysis - Part II ........................11 5. Chapter VI - Conclusions...Level Approach.......80 C. PRESENTATION OF RESULTS................................................................82 V . PROVINCIAL LEVEL ANALYSIS

  7. Quantitative analysis of numerical solvers for oscillatory biomolecular system models

    PubMed Central

    Quo, Chang F; Wang, May D

    2008-01-01

    Background This article provides guidelines for selecting optimal numerical solvers for biomolecular system models. Because various parameters of the same system could have drastically different ranges from 10-15 to 1010, the ODEs can be stiff and ill-conditioned, resulting in non-unique, non-existing, or non-reproducible modeling solutions. Previous studies have not examined in depth how to best select numerical solvers for biomolecular system models, which makes it difficult to experimentally validate the modeling results. To address this problem, we have chosen one of the well-known stiff initial value problems with limit cycle behavior as a test-bed system model. Solving this model, we have illustrated that different answers may result from different numerical solvers. We use MATLAB numerical solvers because they are optimized and widely used by the modeling community. We have also conducted a systematic study of numerical solver performances by using qualitative and quantitative measures such as convergence, accuracy, and computational cost (i.e. in terms of function evaluation, partial derivative, LU decomposition, and "take-off" points). The results show that the modeling solutions can be drastically different using different numerical solvers. Thus, it is important to intelligently select numerical solvers when solving biomolecular system models. Results The classic Belousov-Zhabotinskii (BZ) reaction is described by the Oregonator model and is used as a case study. We report two guidelines in selecting optimal numerical solver(s) for stiff, complex oscillatory systems: (i) for problems with unknown parameters, ode45 is the optimal choice regardless of the relative error tolerance; (ii) for known stiff problems, both ode113 and ode15s are good choices under strict relative tolerance conditions. Conclusions For any given biomolecular model, by building a library of numerical solvers with quantitative performance assessment metric, we show that it is possible

  8. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting.

    PubMed

    Rumlow, Annekathrin; Keunen, Els; Klein, Jan; Pallmann, Philip; Riemenschneider, Anja; Cuypers, Ann; Papenbrock, Jutta

    Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes.

  9. Quantitative proteomic analysis of the Salmonella-lettuce interaction

    PubMed Central

    Zhang, Yuping; Nandakumar, Renu; Bartelt-Hunt, Shannon L; Snow, Daniel D; Hodges, Laurie; Li, Xu

    2014-01-01

    Human pathogens can internalize food crops through root and surface uptake and persist inside crop plants. The goal of the study was to elucidate the global modulation of bacteria and plant protein expression after Salmonella internalizes lettuce. A quantitative proteomic approach was used to analyse the protein expression of Salmonella enterica serovar Infantis and lettuce cultivar Green Salad Bowl 24 h after infiltrating S. Infantis into lettuce leaves. Among the 50 differentially expressed proteins identified by comparing internalized S. Infantis against S. Infantis grown in Luria Broth, proteins involved in glycolysis were down-regulated, while one protein involved in ascorbate uptake was up-regulated. Stress response proteins, especially antioxidant proteins, were up-regulated. The modulation in protein expression suggested that internalized S. Infantis might utilize ascorbate as a carbon source and require multiple stress response proteins to cope with stresses encountered in plants. On the other hand, among the 20 differentially expressed lettuce proteins, proteins involved in defense response to bacteria were up-regulated. Moreover, the secreted effector PipB2 of S. Infantis and R proteins of lettuce were induced after bacterial internalization into lettuce leaves, indicating human pathogen S. Infantis triggered the defense mechanisms of lettuce, which normally responds to plant pathogens. PMID:24512637

  10. [Device for quantitative analysis of perception and pain sensation].

    PubMed

    Arita, Hideko; Kato, Jitsu; Ogawa, Setsuro; Hanaoka, Kazuo

    2014-07-01

    The article describes an analysing device that measures the perception and intensity of pain quantitatively. While it is not necessarily true that psychological aspect is totally irrelevant to pain measurement, this device is remarkable in that it is capable of measuring the intensity of pain felt by the patient more objectively by using electric stimuli. The feature of this device is that it uses a non-pain heteresthesia for measuring the intensity of pain. The device is compact, light-weight, and portable. Unlike VAS that requires only a scale, the device requires a person to carry out the measurement. Nevertheless, as the National Health Insurance (NHI) coverage has been approved, introduction of the device may be facilitated in terms of budget for the purchase and labor. The device is useful to better understand not only the intensity of pain but also the pathological conditions, resulting in more appropriate treatment, by (1) comparing degree of pain or VAS values taken by a multicenter study with those of a patient; (2) using both degree of pain and VAS; and (3) multiple measurements of degree of pain and VAS in one case.

  11. Quantitative analysis of virus and plasmid trafficking in cells

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Dauty, Emmanuel; Holcman, David

    2009-01-01

    Intracellular transport of DNA carriers is a fundamental step of gene delivery. By combining both theoretical and numerical approaches we study here single and several viruses and DNA particles trafficking in the cell cytoplasm to a small nuclear pore. We present a physical model to account for certain aspects of cellular organization, starting with the observation that a viral trajectory consists of epochs of pure diffusion and epochs of active transport along microtubules. We define a general degradation rate to describe the limitations of the delivery of plasmid or viral particles to a nuclear pore imposed by various types of direct and indirect hydrolysis activity inside the cytoplasm. By replacing the switching dynamics by a single steady state stochastic description, we obtain estimates for the probability and the mean time for the first one of many particles to go from the cell membrane to a small nuclear pore. Computational simulations confirm that our model can be used to analyze and interpret viral trajectories and estimate quantitatively the success of nuclear delivery.

  12. Quantitative Analysis of CME Deflections in the Corona

    NASA Astrophysics Data System (ADS)

    Gui, Bin; Shen, Chenglong; Wang, Yuming; Ye, Pinzhong; Liu, Jiajia; Wang, Shui; Zhao, Xuepu

    2011-07-01

    In this paper, ten CME events viewed by the STEREO twin spacecraft are analyzed to study the deflections of CMEs during their propagation in the corona. Based on the three-dimensional information of the CMEs derived by the graduated cylindrical shell (GCS) model (Thernisien, Howard, and Vourlidas in Astrophys. J. 652, 1305, 2006), it is found that the propagation directions of eight CMEs had changed. By applying the theoretical method proposed by Shen et al. ( Solar Phys. 269, 389, 2011) to all the CMEs, we found that the deflections are consistent, in strength and direction, with the gradient of the magnetic energy density. There is a positive correlation between the deflection rate and the strength of the magnetic energy density gradient and a weak anti-correlation between the deflection rate and the CME speed. Our results suggest that the deflections of CMEs are mainly controlled by the background magnetic field and can be quantitatively described by the magnetic energy density gradient (MEDG) model.

  13. Space-to-Ground Communication for Columbus: A Quantitative Analysis.

    PubMed

    Uhlig, Thomas; Mannel, Thurid; Fortunato, Antonio; Illmer, Norbert

    2015-01-01

    The astronauts on board the International Space Station (ISS) are only the most visible part of a much larger team engaged around the clock in the performance of science and technical activities in space. The bulk of such team is scattered around the globe in five major Mission Control Centers (MCCs), as well as in a number of smaller payload operations centres. Communication between the crew in space and the flight controllers at those locations is an essential element and one of the key drivers to efficient space operations. Such communication can be carried out in different forms, depending on available technical assets and the selected operational approach for the activity at hand. This paper focuses on operational voice communication and provides a quantitative overview of the balance achieved in the Columbus program between collaborative space/ground operations and autonomous on-board activity execution. An interpretation of the current situation is provided, together with a description of potential future approaches for deep space exploration missions.

  14. Quantitative analysis of task selection for brain-computer interfaces

    NASA Astrophysics Data System (ADS)

    Llera, Alberto; Gómez, Vicenç; Kappen, Hilbert J.

    2014-10-01

    Objective. To assess quantitatively the impact of task selection in the performance of brain-computer interfaces (BCI). Approach. We consider the task-pairs derived from multi-class BCI imagery movement tasks in three different datasets. We analyze for the first time the benefits of task selection on a large-scale basis (109 users) and evaluate the possibility of transferring task-pair information across days for a given subject. Main results. Selecting the subject-dependent optimal task-pair among three different imagery movement tasks results in approximately 20% potential increase in the number of users that can be expected to control a binary BCI. The improvement is observed with respect to the best task-pair fixed across subjects. The best task-pair selected for each subject individually during a first day of recordings is generally a good task-pair in subsequent days. In general, task learning from the user side has a positive influence in the generalization of the optimal task-pair, but special attention should be given to inexperienced subjects. Significance. These results add significant evidence to existing literature that advocates task selection as a necessary step towards usable BCIs. This contribution motivates further research focused on deriving adaptive methods for task selection on larger sets of mental tasks in practical online scenarios.

  15. Quantitative proteomic analysis of the Salmonella-lettuce interaction.

    PubMed

    Zhang, Yuping; Nandakumar, Renu; Bartelt-Hunt, Shannon L; Snow, Daniel D; Hodges, Laurie; Li, Xu

    2014-11-01

    Human pathogens can internalize food crops through root and surface uptake and persist inside crop plants. The goal of the study was to elucidate the global modulation of bacteria and plant protein expression after Salmonella internalizes lettuce. A quantitative proteomic approach was used to analyse the protein expression of Salmonella enterica serovar Infantis and lettuce cultivar Green Salad Bowl 24 h after infiltrating S. Infantis into lettuce leaves. Among the 50 differentially expressed proteins identified by comparing internalized S. Infantis against S. Infantis grown in Luria Broth, proteins involved in glycolysis were down-regulated, while one protein involved in ascorbate uptake was up-regulated. Stress response proteins, especially antioxidant proteins, were up-regulated. The modulation in protein expression suggested that internalized S. Infantis might utilize ascorbate as a carbon source and require multiple stress response proteins to cope with stresses encountered in plants. On the other hand, among the 20 differentially expressed lettuce proteins, proteins involved in defense response to bacteria were up-regulated. Moreover, the secreted effector PipB2 of S. Infantis and R proteins of lettuce were induced after bacterial internalization into lettuce leaves, indicating human pathogen S. Infantis triggered the defense mechanisms of lettuce, which normally responds to plant pathogens.

  16. Quantitative proteomic analysis of amphotericin B resistance in Leishmania infantum.

    PubMed

    Brotherton, Marie-Christine; Bourassa, Sylvie; Légaré, Danielle; Poirier, Guy G; Droit, Arnaud; Ouellette, Marc

    2014-08-01

    Amphotericin B (AmB) in its liposomal form is now considered as either first- or second-line treatment against Leishmania infections in different part of the world. Few cases of AmB resistance have been reported and resistance mechanisms toward AmB are still poorly understood. This paper reports a large-scale comparative proteomic study in the context of AmB resistance. Quantitative proteomics using stable isotope labeling of amino acids in cell culture (SILAC) was used to better characterize cytoplasmic and membrane-enriched (ME) proteomes of the in vitro generated Leishmania infantum AmB resistant mutant AmB1000.1. In total, 97 individual proteins were found as differentially expressed between the mutant and its parental sensitive strain (WT). More than half of these proteins were either metabolic enzymes or involved in transcription or translation processes. Key energetic pathways such as glycolysis and TCA cycle were up-regulated in the mutant. Interestingly, many proteins involved in reactive oxygen species (ROS) scavenging and heat-shock proteins were also up-regulated in the resistant mutant. This work provides a basis for further investigations to understand the roles of proteins differentially expressed in relation with AmB resistance.

  17. Space-to-Ground Communication for Columbus: A Quantitative Analysis

    PubMed Central

    Uhlig, Thomas; Mannel, Thurid; Fortunato, Antonio; Illmer, Norbert

    2015-01-01

    The astronauts on board the International Space Station (ISS) are only the most visible part of a much larger team engaged around the clock in the performance of science and technical activities in space. The bulk of such team is scattered around the globe in five major Mission Control Centers (MCCs), as well as in a number of smaller payload operations centres. Communication between the crew in space and the flight controllers at those locations is an essential element and one of the key drivers to efficient space operations. Such communication can be carried out in different forms, depending on available technical assets and the selected operational approach for the activity at hand. This paper focuses on operational voice communication and provides a quantitative overview of the balance achieved in the Columbus program between collaborative space/ground operations and autonomous on-board activity execution. An interpretation of the current situation is provided, together with a description of potential future approaches for deep space exploration missions. PMID:26290898

  18. Quantitative analysis on electric dipole energy in Rashba band splitting

    PubMed Central

    Hong, Jisook; Rhim, Jun-Won; Kim, Changyoung; Ryong Park, Seung; Hoon Shim, Ji

    2015-01-01

    We report on quantitative comparison between the electric dipole energy and the Rashba band splitting in model systems of Bi and Sb triangular monolayers under a perpendicular electric field. We used both first-principles and tight binding calculations on p-orbitals with spin-orbit coupling. First-principles calculation shows Rashba band splitting in both systems. It also shows asymmetric charge distributions in the Rashba split bands which are induced by the orbital angular momentum. We calculated the electric dipole energies from coupling of the asymmetric charge distribution and external electric field, and compared it to the Rashba splitting. Remarkably, the total split energy is found to come mostly from the difference in the electric dipole energy for both Bi and Sb systems. A perturbative approach for long wave length limit starting from tight binding calculation also supports that the Rashba band splitting originates mostly from the electric dipole energy difference in the strong atomic spin-orbit coupling regime. PMID:26323493

  19. Quantitative Analysis of Synaptic Release at the Photoreceptor Synapse

    PubMed Central

    Duncan, Gabriel; Rabl, Katalin; Gemp, Ian; Heidelberger, Ruth; Thoreson, Wallace B.

    2010-01-01

    Abstract Exocytosis from the rod photoreceptor is stimulated by submicromolar Ca2+ and exhibits an unusually shallow dependence on presynaptic Ca2+. To provide a quantitative description of the photoreceptor Ca2+ sensor for exocytosis, we tested a family of conventional and allosteric computational models describing the final Ca2+-binding steps leading to exocytosis. Simulations were fit to two measures of release, evoked by flash-photolysis of caged Ca2+: exocytotic capacitance changes from individual rods and postsynaptic currents of second-order neurons. The best simulations supported the occupancy of only two Ca2+ binding sites on the rod Ca2+ sensor rather than the typical four or five. For most models, the on-rates for Ca2+ binding and maximal fusion rate were comparable to those of other neurons. However, the off-rates for Ca2+ unbinding were unexpectedly slow. In addition to contributing to the high-affinity of the photoreceptor Ca2+ sensor, slow Ca2+ unbinding may support the fusion of vesicles located at a distance from Ca2+ channels. In addition, partial sensor occupancy due to slow unbinding may contribute to the linearization of the first synapse in vision. PMID:20483317

  20. Quantitative analysis of ultrasound images for computer-aided diagnosis

    PubMed Central

    Wu, Jie Ying; Tuomi, Adam; Beland, Michael D.; Konrad, Joseph; Glidden, David; Grand, David; Merck, Derek

    2016-01-01

    Abstract. We propose an adaptable framework for analyzing ultrasound (US) images quantitatively to provide computer-aided diagnosis using machine learning. Our preliminary clinical targets are hepatic steatosis, adenomyosis, and craniosynostosis. For steatosis and adenomyosis, we collected US studies from 288 and 88 patients, respectively, as well as their biopsy or magnetic resonanceconfirmed diagnosis. Radiologists identified a region of interest (ROI) on each image. We filtered the US images for various texture responses and use the pixel intensity distribution within each ROI as feature parameterizations. Our craniosynostosis dataset consisted of 22 CT-confirmed cases and 22 age-matched controls. One physician manually measured the vectors from the center of the skull to the outer cortex at every 10 deg for each image and we used the principal directions as shape features for parameterization. These parameters and the known diagnosis were used to train classifiers. Testing with cross-validation, we obtained 72.74% accuracy and 0.71 area under receiver operating characteristics curve for steatosis (p<0.0001), 77.27% and 0.77 for adenomyosis (p<0.0001), and 88.63% and 0.89 for craniosynostosis (p=0.0006). Our framework is able to detect a variety of diseases with high accuracy. We hope to include it as a routinely available support system in the clinic. PMID:26835502

  1. Quantitative analysis of chromosome condensation in fission yeast.

    PubMed

    Petrova, Boryana; Dehler, Sascha; Kruitwagen, Tom; Hériché, Jean-Karim; Miura, Kota; Haering, Christian H

    2013-03-01

    Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ε, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote.

  2. Quantitative analysis of a frequency-domain nonlinearity indicator.

    PubMed

    Reichman, Brent O; Gee, Kent L; Neilsen, Tracianne B; Miller, Kyle G

    2016-05-01

    In this paper, quantitative understanding of a frequency-domain nonlinearity indicator is developed. The indicator is derived from an ensemble-averaged, frequency-domain version of the generalized Burgers equation, which can be rearranged in order to directly compare the effects of nonlinearity, absorption, and geometric spreading on the pressure spectrum level with frequency and distance. The nonlinear effect is calculated using pressure-squared-pressure quadspectrum. Further theoretical development has given an expression for the role of the normalized quadspectrum, referred to as Q/S by Morfey and Howell [AIAA J. 19, 986-992 (1981)], in the spatial rate of change of the pressure spectrum level. To explore this finding, an investigation of the change in level for initial sinusoids propagating as plane waves through inviscid and thermoviscous media has been conducted. The decibel change with distance, calculated through Q/S, captures the growth and decay of the harmonics and indicates that the most significant changes in level occur prior to sawtooth formation. At large distances, the inviscid case results in a spatial rate of change that is uniform across all harmonics. For thermoviscous media, large positive nonlinear gains are observed but offset by absorption, which leads to a greater overall negative spatial rate of change for higher harmonics.

  3. Temporal Kinetics and Quantitative Analysis of Cryptococcus neoformans Nonlytic Exocytosis

    PubMed Central

    Stukes, Sabriya A.; Cohen, Hillel W.

    2014-01-01

    Cryptococcus neoformans is a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems. C. neoformans has the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies of C. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macrophages containing intracellular C. neoformans and identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P < 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection. PMID:24595144

  4. Quantitative analysis of pheromone-binding protein specificity

    PubMed Central

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

  5. Quantitative assessment of human motion using video motion analysis

    NASA Technical Reports Server (NTRS)

    Probe, John D.

    1990-01-01

    In the study of the dynamics and kinematics of the human body, a wide variety of technologies was developed. Photogrammetric techniques are well documented and are known to provide reliable positional data from recorded images. Often these techniques are used in conjunction with cinematography and videography for analysis of planar motion, and to a lesser degree three-dimensional motion. Cinematography has been the most widely used medium for movement analysis. Excessive operating costs and the lag time required for film development coupled with recent advances in video technology have allowed video based motion analysis systems to emerge as a cost effective method of collecting and analyzing human movement. The Anthropometric and Biomechanics Lab at Johnson Space Center utilizes the video based Ariel Performance Analysis System to develop data on shirt-sleeved and space-suited human performance in order to plan efficient on orbit intravehicular and extravehicular activities. The system is described.

  6. From quantitative protein complex analysis to disease mechanism.

    PubMed

    Texier, Y; Kinkl, N; Boldt, K; Ueffing, M

    2012-12-15

    Interest in the field of cilia biology and cilia-associated diseases - ciliopathies - has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers.

  7. [Quantitative spectrum analysis of characteristic gases of spontaneous combustion coal].

    PubMed

    Liang, Yun-Tao; Tang, Xiao-Jun; Luo, Hai-Zhu; Sun, Yong

    2011-09-01

    Aimed at the characteristics of spontaneous combustion gas such as a variety of gases, lou limit of detection, and critical requirement of safety, Fourier transform infrared (FTIR) spectral analysis is presented to analyze characteristic gases of spontaneous combustion In this paper, analysis method is introduced at first by combing characteristics of absorption spectra of analyte and analysis requirement. Parameter setting method, sample preparation, feature variable abstract and analysis model building are taken into consideration. The methods of sample preparation, feature abstraction and analysis model are introduced in detail. And then, eleven kinds of gases were tested with Tensor 27 spectrometer. CH4, C2H6, C3H8, iC4H10, nC4H10, C2 H4, C3 H6, C3 H2, SF6, CO and CO2 were included. The optical path length was 10 cm while the spectra resolution was set as 1 cm(-1). The testing results show that the detection limit of all analytes is less than 2 x 10(-6). All the detection limits fit the measurement requirement of spontaneous combustion gas, which means that FTIR may be an ideal instrument and the analysis method used in this paper is competent for spontaneous combustion gas measurement on line.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-12-01

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

  10. Software applications toward quantitative metabolic flux analysis and modeling.

    PubMed

    Dandekar, Thomas; Fieselmann, Astrid; Majeed, Saman; Ahmed, Zeeshan

    2014-01-01

    Metabolites and their pathways are central for adaptation and survival. Metabolic modeling elucidates in silico all the possible flux pathways (flux balance analysis, FBA) and predicts the actual fluxes under a given situation, further refinement of these models is possible by including experimental isotopologue data. In this review, we initially introduce the key theoretical concepts and different analysis steps in the modeling process before comparing flux calculation and metabolite analysis programs such as C13, BioOpt, COBRA toolbox, Metatool, efmtool, FiatFlux, ReMatch, VANTED, iMAT and YANA. Their respective strengths and limitations are discussed and compared to alternative software. While data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific changes are all possible, we highlight the considerations that need to be taken into account before deciding on a specific software. Current challenges in the field include the computation of large-scale networks (in elementary mode analysis), regulatory interactions and detailed kinetics, and these are discussed in the light of powerful new approaches.

  11. Quantitative assessment of human motion using video motion analysis

    NASA Technical Reports Server (NTRS)

    Probe, John D.

    1993-01-01

    In the study of the dynamics and kinematics of the human body a wide variety of technologies has been developed. Photogrammetric techniques are well documented and are known to provide reliable positional data from recorded images. Often these techniques are used in conjunction with cinematography and videography for analysis of planar motion, and to a lesser degree three-dimensional motion. Cinematography has been the most widely used medium for movement analysis. Excessive operating costs and the lag time required for film development, coupled with recent advances in video technology, have allowed video based motion analysis systems to emerge as a cost effective method of collecting and analyzing human movement. The Anthropometric and Biomechanics Lab at Johnson Space Center utilizes the video based Ariel Performance Analysis System (APAS) to develop data on shirtsleeved and space-suited human performance in order to plan efficient on-orbit intravehicular and extravehicular activities. APAS is a fully integrated system of hardware and software for biomechanics and the analysis of human performance and generalized motion measurement. Major components of the complete system include the video system, the AT compatible computer, and the proprietary software.

  12. Geysers of Enceladus: Quantitative analysis of qualitative models

    NASA Astrophysics Data System (ADS)

    Brilliantov, Nikolai V.; Schmidt, Jürgen; Spahn, Frank

    2008-11-01

    Aspects of two qualitative models of Enceladus' dust plume - the so-called "Cold Faithful" [Porco, C.C., et al., 2006. Cassini observes the active south pole of Enceladus. Science 311, 1393-1401; Ingersoll, A.P., et al., 2006. Models of the Enceladus plumes. In: Bulletin of the American Astronomical Society, vol. 38, p. 508] and "Frigid Faithful" [Kieffer, S.W., et al., 2006. A clathrate reservoir hypothesis for Enceladus' south polar plume. Science 314, 1764; Gioia, G., et al., 2007. Unified model of tectonics and heat transport in a Frigid Enceladus. Proc. Natl. Acad. Sci. 104, 13578-13591] models - are analyzed quantitatively. The former model assumes an explosive boiling of subsurface liquid water, when pressure exerted by the ice crust is suddenly released due to an opening crack. In the latter model the existence of a deep shell of clathrates below Enceladus' south pole is conjectured; clathrates can decompose explosively when exposed to vacuum through a fracture in the outer icy shell. For the Cold Faithful model we estimate the maximal velocity of ice grains, originating from water splashing in explosive boiling. We find that for water near the triple point this velocity is far too small to explain the observed plume properties. For the Frigid Faithful model we consider the problem of momentum transfer from gas to ice particles. It arises since any change in the direction of the gas flow in the cracks of the shell requires re-acceleration of the entrained grains. While this effect may explain the observed speed difference of gas and grains if the gas evaporates from triple point temperature (273.15 K) [Schmidt, J., et al., 2008. Formation of Enceladus dust plume. Nature 451, 685], the low temperatures of the Frigid Faithful model (˜140-170K) imply a too dilute vapor to support the observed high particle fluxes in Enceladus' plume.

  13. Quantitative Analysis of Shoot Development and Branching Patterns in Actinidia

    PubMed Central

    SELEZNYOVA, ALLA N.; THORP, T. GRANT; BARNETT, ANDREW M.; COSTES, EVELYNE

    2002-01-01

    We developed a framework for the quantitative description of Actinidia vine architecture, classifying shoots into three types (short, medium and long) corresponding to the modes of node number distribution and the presence/absence of neoformed nodes. Short and medium shoots were self‐terminated and had only preformed nodes. Based on the cut‐off point between their two modes of node number distribution, short shoots were defined as having nine or less nodes, and medium shoots as having more than nine nodes. Long shoots were non‐terminated and had a number of neoformed nodes; the total number of nodes per shoot was up to 90. Branching patterns for each parent shoot type were represented by a succession of branching zones. Probabilities of different types of axillary production (latent bud, short, medium or long shoot) and the distributions of length for each branching zone were estimated from experimental data using hidden semi‐Markov chain stochastic models. Branching was acrotonic on short and medium parent shoots, with most axillary shoots being located near the shoot tip. For long parent shoots, branching was mesotonic, with most long axillary shoots being located in the transition zone between the preformed and neoformed part of the parent shoot. Although the shoot classification is based on node number distribution there was a marked difference in average (per shoot) internode length between the shoot types, with mean values of 9, 27 and 47 mm for short, medium and long shoots, respectively. Bud and shoot development is discussed in terms of environmental controls. PMID:12096808

  14. Quantitative analysis of lymphangiogenic markers in human colorectal cancer.

    PubMed

    Parr, C; Jiang, W G

    2003-08-01

    Lymphatic spread of colorectal cancer cells to regional lymph nodes is one of the early events in metastatic cancer, and is often associated with distant metastatic spread and a poor prognosis. This study examined lymphangiogenic factors, and in particular a panel of newly discovered lymphangiogenic markers, in colorectal cancer tissues from a cohort of patients. Paired samples (background normal mucosa and cancer) of colon tissue were obtained from patients with colorectal cancer. The expression and levels of the VEGF-C and VEGF-D cytokines, the VEGF receptors VEGFR-2 and VEGFR-3, and newly described lymphatic endothelial markers, LYVE-1, Prox-1, podoplanin and 5'-nucleotidase were assessed. RNA was extracted from the frozen colon tissues. The level of expression for each factor/marker was determined using RT-PCR and quantified using a real-time quantitative PCR (RT-QPCR) technique, with respective cloned cDNA plasmids as internal standards. VEGF-D was expressed to a significantly higher degree in the colon tumour tissues. There was no significant difference between the expression levels for both VEGF-C and its receptor, VEGFR-2, in background and cancer tissues. However, levels of the VEGFR-3 receptor were found to be significantly higher in colon cancer than the normal background tissues. LYVE-1 levels were below detection in most cases. There was a significant increase in the degree of Prox-1 and 5'-nucleotidase expression in colon cancer tissue. Podoplanin expression was also increased in the cancer samples. These markers indicate an increase in lymphangiogenesis in colon cancer, and may therefore have prognostic value for colon cancer patients.

  15. Dynamic and still microcirculatory image analysis for quantitative microcirculation research

    NASA Astrophysics Data System (ADS)

    Ying, Xiaoyou; Xiu, Rui-juan

    1994-05-01

    Based on analyses of various types of digital microcirculatory image (DMCI), we summed up the image features of DMCI, the digitizing demands for digital microcirculatory imaging, and the basic characteristics of the DMCI processing. A dynamic and still imaging separation processing (DSISP) mode was designed for developing a DMCI workstation and the DMCI processing. Original images in this study were clinical microcirculatory images from human finger nail-bed and conjunctiva microvasculature, and intravital microvascular network images from animal tissue or organs. A series of dynamic and still microcirculatory image analysis functions were developed in this study. The experimental results indicate most of the established analog video image analysis methods for microcirculatory measurement could be realized in a more flexible way based on the DMCI. More information can be rapidly extracted from the quality improved DMCI by employing intelligence digital image analysis methods. The DSISP mode is very suitable for building a DMCI workstation.

  16. Automated quantitative analysis of ventilation-perfusion lung scintigrams

    SciTech Connect

    Burton, G.H.; Vernon, P.; Seed, W.A.

    1984-05-01

    An automated computer analysis of ventilation (Kr-81m) and perfusion (Tc-99m) lung images has been devised that produces a graphical image of the distribution of ventilation and perfusion, and of ventilation-perfusion ratios. The analysis has overcome the following problems: the identification of the midline between two lungs and the lung boundaries, the exclusion of extrapulmonary radioactivity, the superimposition of lung images of different sizes, and the format for presentation of the data. Therefore, lung images of different sizes and shapes may be compared with each other. The analysis has been used to develop normal ranges from 55 volunteers. Comparison of younger and older age groups of men and women show small but significant differences in the distribution of ventilation and perfusion, but no differences in ventilation-perfusion ratios.

  17. Quantitative spectroscopy for the analysis of GOME data

    NASA Technical Reports Server (NTRS)

    Chance, K.

    1997-01-01

    Accurate analysis of the global ozone monitoring experiment (GOME) data to obtain atmospheric constituents requires reliable, traceable spectroscopic parameters for atmospheric absorption and scattering. Results are summarized for research that includes: the re-determination of Rayleigh scattering cross sections and phase functions for the 200 nm to 1000 nm range; the analysis of solar spectra to obtain a high-resolution reference spectrum with excellent absolute vacuum wavelength calibration; Ring effect cross sections and phase functions determined directly from accurate molecular parameters of N2 and O2; O2 A band line intensities and pressure broadening coefficients; and the analysis of absolute accuracies for ultraviolet and visible absorption cross sections of O3 and other trace species measurable by GOME.

  18. Single radial immunodiffusion analysis for quantitation of colostral immunoglobulin concentration.

    PubMed

    Fleenor, W A; Stott, G H

    1981-05-01

    Relative accuracy of the single radial immunodiffusion technique to measure immunoglobulin concentrations of colostral preparations (whey, whole, or fat-free) has been assessed. Fresh colostrum samples were analyzed for major constituents. Gammaglobulin as a standard was compared to total immunoglobulin concentration derived from single radial immunodiffusion analysis of colostral preparations with no differences except between standard and whey. Differences were in part from either enhancement or interference of immunoglobulin diffusion by colostral constituents. Removal of casein and fat during whey preparations caused a concentrating effect upon immunoglobulin constituents resulting in exaggerated precipitin rings. Whey has produced unreliable results: therefore, whole colostrum is recommended for single radial immunodiffusion analysis.

  19. EXPLoRA-web: linkage analysis of quantitative trait loci using bulk segregant analysis

    PubMed Central

    Pulido-Tamayo, Sergio; Duitama, Jorge; Marchal, Kathleen

    2016-01-01

    Identification of genomic regions associated with a phenotype of interest is a fundamental step toward solving questions in biology and improving industrial research. Bulk segregant analysis (BSA) combined with high-throughput sequencing is a technique to efficiently identify these genomic regions associated with a trait of interest. However, distinguishing true from spuriously linked genomic regions and accurately delineating the genomic positions of these truly linked regions requires the use of complex statistical models currently implemented in software tools that are generally difficult to operate for non-expert users. To facilitate the exploration and analysis of data generated by bulked segregant analysis, we present EXPLoRA-web, a web service wrapped around our previously published algorithm EXPLoRA, which exploits linkage disequilibrium to increase the power and accuracy of quantitative trait loci identification in BSA analysis. EXPLoRA-web provides a user friendly interface that enables easy data upload and parallel processing of different parameter configurations. Results are provided graphically and as BED file and/or text file and the input is expected in widely used formats, enabling straightforward BSA data analysis. The web server is available at http://bioinformatics.intec.ugent.be/explora-web/. PMID:27105844

  20. Regression Commonality Analysis: A Technique for Quantitative Theory Building

    ERIC Educational Resources Information Center

    Nimon, Kim; Reio, Thomas G., Jr.

    2011-01-01

    When it comes to multiple linear regression analysis (MLR), it is common for social and behavioral science researchers to rely predominately on beta weights when evaluating how predictors contribute to a regression model. Presenting an underutilized statistical technique, this article describes how organizational researchers can use commonality…

  1. Reflectance spectroscopy: quantitative analysis techniques for remote sensing applications.

    USGS Publications Warehouse

    Clark, R.N.; Roush, T.L.

    1984-01-01

    Several methods for the analysis of remotely sensed reflectance data are compared, including empirical methods and scattering theories, both of which are important for solving remote sensing problems. The concept of the photon mean path length and the implications for use in modeling reflectance spectra are presented.-from Authors

  2. Identification and quantitative analysis of tissue particulate burden.

    PubMed

    Abraham, J L

    1984-01-01

    The analysis of particles in tissue samples, especially in lung tissue samples, has been of great use diagnostically and in the search for more precise information on the etiology of occupational/environmental diseases. There is a strong need for an interdisciplinary approach involving patients, epidemiologists, clinicians, pathologists, toxicologists, and microscopists if advancement in the recognition, diagnosis, and prevention of disease is to proceed.

  3. Quantitative analysis of cell migration using optical flow.

    PubMed

    Boric, Katica; Orio, Patricio; Viéville, Thierry; Whitlock, Kathleen

    2013-01-01

    Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.

  4. Efficacy of Rational-Emotive Therapy: A Quantitative Analysis.

    ERIC Educational Resources Information Center

    Engels, Gemma I.; And Others

    1993-01-01

    Meta-analysis of 28 controlled studies on rational-emotive therapy (RET) showed RET superior to placebo and no treatment, but equally effective in comparison with treatments such as combination therapies and systematic desensitization. No support was found for view that RET with main or balanced emphasis on behavioral techniques is more…

  5. Quantitative histology analysis of the ovarian tumour microenvironment

    PubMed Central

    Lan, Chunyan; Heindl, Andreas; Huang, Xin; Xi, Shaoyan; Banerjee, Susana; Liu, Jihong; Yuan, Yinyin

    2015-01-01

    Concerted efforts in genomic studies examining RNA transcription and DNA methylation patterns have revealed profound insights in prognostic ovarian cancer subtypes. On the other hand, abundant histology slides have been generated to date, yet their uses remain very limited and largely qualitative. Our goal is to develop automated histology analysis as an alternative subtyping technology for ovarian cancer that is cost-efficient and does not rely on DNA quality. We developed an automated system for scoring primary tumour sections of 91 late-stage ovarian cancer to identify single cells. We demonstrated high accuracy of our system based on expert pathologists’ scores (cancer = 97.1%, stromal = 89.1%) as well as compared to immunohistochemistry scoring (correlation = 0.87). The percentage of stromal cells in all cells is significantly associated with poor overall survival after controlling for clinical parameters including debulking status and age (multivariate analysis p = 0.0021, HR = 2.54, CI = 1.40–4.60) and progression-free survival (multivariate analysis p = 0.022, HR = 1.75, CI = 1.09–2.82). We demonstrate how automated image analysis enables objective quantification of microenvironmental composition of ovarian tumours. Our analysis reveals a strong effect of the tumour microenvironment on ovarian cancer progression and highlights the potential of therapeutic interventions that target the stromal compartment or cancer-stroma signalling in the stroma-high, late-stage ovarian cancer subset. PMID:26573438

  6. A quantitative analysis of fish consumption and stroke risk.

    PubMed

    Bouzan, Colleen; Cohen, Joshua T; Connor, William E; Kris-Etherton, Penny M; Gray, George M; König, Ariane; Lawrence, Robert S; Savitz, David A; Teutsch, Steven M

    2005-11-01

    Although a rich source of n-3 polyunsaturated fatty acids (PUFAs) that may confer multiple health benefits, some fish contain methyl mercury (MeHg), which may harm the developing fetus. U.S. government recommendations for women of childbearing age are to modify consumption of high-MeHg fish to reduce MeHg exposure, while recommendations encourage fish consumption among the general population because of the nutritional benefits. The Harvard Center for Risk Analysis convened an expert panel (see acknowledgements) to quantify the net impact of resulting hypothetical changes in fish consumption across the population. This paper estimates the impact of fish consumption on stroke risk. Other papers quantify coronary heart disease mortality risk and the impacts of both prenatal MeHg exposure and maternal intake of n-3 PUFAs on cognitive development. This analysis identified articles in a recent qualitative literature review that are appropriate for the development of a dose-response relationship between fish consumption and stroke risk. Studies had to satisfy quality criteria, quantify fish intake, and report the precision of the relative risk estimates. The analysis combined the relative risk results, weighting each proportionately to its precision. Six studies were identified as appropriate for inclusion in this analysis, including five prospective cohort studies and one case-control study (total of 24 exposure groups). Our analysis indicates that any fish consumption confers substantial relative risk reduction compared to no fish consumption (12% for the linear model), with the possibility that additional consumption confers incremental benefits (central estimate of 2.0% per serving per week).

  7. Quantitative analysis of night skyglow amplification under cloudy conditions

    NASA Astrophysics Data System (ADS)

    Kocifaj, Miroslav; Solano Lamphar, Héctor Antonio

    2014-10-01

    The radiance produced by artificial light is a major source of nighttime over-illumination. It can, however, be treated experimentally using ground-based and satellite data. These two types of data complement each other and together have a high information content. For instance, the satellite data enable upward light emissions to be normalized, and this in turn allows skyglow levels at the ground to be modelled under cloudy or overcast conditions. Excessive night lighting imposes an unacceptable burden on nature, humans and professional astronomy. For this reason, there is a pressing need to determine the total amount of downwelling diffuse radiation. Undoubtedly, cloudy periods can cause a significant increase in skyglow as a result of amplification owing to diffuse reflection from clouds. While it is recognized that the amplification factor (AF) varies with cloud cover, the effects of different types of clouds, of atmospheric turbidity and of the geometrical relationships between the positions of an individual observer, the cloud layer, and the light source are in general poorly known. In this paper the AF is quantitatively analysed considering different aerosol optical depths (AODs), urban layout sizes and cloud types with specific albedos and altitudes. The computational results show that the AF peaks near the edges of a city rather than at its centre. In addition, the AF appears to be a decreasing function of AOD, which is particularly important when modelling the skyglow in regions with apparent temporal or seasonal variability of atmospheric turbidity. The findings in this paper will be useful to those designing engineering applications or modelling light pollution, as well as to astronomers and environmental scientists who aim to predict the amplification of skyglow caused by clouds. In addition, the semi-analytical formulae can be used to estimate the AF levels, especially in densely populated metropolitan regions for which detailed computations may be CPU

  8. Quantitative full time course analysis of nonlinear enzyme cycling kinetics.

    PubMed

    Cao, Wenxiang; De La Cruz, Enrique M

    2013-01-01

    Enzyme inhibition due to the reversible binding of reaction products is common and underlies the origins of negative feedback inhibition in many metabolic and signaling pathways. Product inhibition generates non-linearity in steady-state time courses of enzyme activity, which limits the utility of well-established enzymology approaches developed under the assumption of irreversible product release. For more than a century, numerous attempts to find a mathematical solution for analysis of kinetic time courses with product inhibition have been put forth. However, no practical general method capable of extracting common enzymatic parameters from such non-linear time courses has been successfully developed. Here we present a simple and practical method of analysis capable of efficiently extracting steady-state enzyme kinetic parameters and product binding constants from non-linear kinetic time courses with product inhibition and/or substrate depletion. The method is general and applicable to all enzyme systems, independent of reaction schemes and pathways.

  9. Efficacy of rational-emotive therapy: a quantitative analysis.

    PubMed

    Engels, G I; Garnefski, N; Diekstra, R F

    1993-12-01

    Meta-analysis of 28 controlled studies on the efficacy of rational-emotive therapy (RET) showed RET to be superior to placebo and no treatment but equally effective in comparison with other types of treatment such as combination therapies and systematic desensitization. No support was found for the view that RET with a main or balanced emphasis on behavioral techniques is more efficacious than RET with a primarily or exclusively cognitive approach. However, given the heterogeneity of the set of studies and its relatively small sample size, interpretations of results have to be made with caution, particularly because evidence was found for a relationship between study characteristics and magnitude of effect size. For the sake of meta-analysis, outcome studies should be required to report at least the means and standard deviations for all experimental groups.

  10. African Primary Care Research: quantitative analysis and presentation of results.

    PubMed

    Mash, Bob; Ogunbanjo, Gboyega A

    2014-06-06

    This article is part of a series on Primary Care Research Methods. The article describes types of continuous and categorical data, how to capture data in a spreadsheet, how to use descriptive and inferential statistics and, finally, gives advice on how to present the results in text, figures and tables. The article intends to help Master's level students with writing the data analysis section of their research proposal and presenting their results in their final research report.

  11. [Quantitative image analysis in pulmonary pathology - digitalization of preneoplastic lesions in human bronchial epithelium (author's transl)].

    PubMed

    Steinbach, T; Müller, K M; Kämper, H

    1979-01-01

    The report concerns the first phase of a quantitative study of normal and abnormal bronchial epithelium with the objective of establishing the digitalization of histologic patterns. Preparative methods, data collecting and handling, and further mathematical analysis are described. In cluster and discriminatory analysis the digitalized histologic features can be used to separate and classify the individual cases into the respective diagnostic groups.

  12. Digitally Enhanced Thin-Layer Chromatography: An Inexpensive, New Technique for Qualitative and Quantitative Analysis

    ERIC Educational Resources Information Center

    Hess, Amber Victoria Irish

    2007-01-01

    A study conducted shows that if digital photography is combined with regular thin-layer chromatography (TLC), it could perform highly improved qualitative analysis as well as make accurate quantitative analysis possible for a much lower cost than commercial equipment. The findings suggest that digitally enhanced TLC (DE-TLC) is low-cost and easy…

  13. Kinetic Analysis of Amylase Using Quantitative Benedict's and Iodine Starch Reagents

    ERIC Educational Resources Information Center

    Cochran, Beverly; Lunday, Deborah; Miskevich, Frank

    2008-01-01

    Quantitative analysis of carbohydrates is a fundamental analytical tool used in many aspects of biology and chemistry. We have adapted a technique developed by Mathews et al. using an inexpensive scanner and open-source image analysis software to quantify amylase activity using both the breakdown of starch and the appearance of glucose. Breakdown…

  14. Revisiting the quantitative features of surface-assisted laser desorption/ionization mass spectrometric analysis.

    PubMed

    Wu, Ching-Yi; Lee, Kai-Chieh; Kuo, Yen-Ling; Chen, Yu-Chie

    2016-10-28

    Surface-assisted laser desorption/ionization (SALDI) coupled with mass spectrometry (MS) is frequently used to analyse small organics owing to its clean background. Inorganic materials can be used as energy absorbers and the transfer medium to facilitate the desorption/ionization of analytes; thus, they are used as SALDI-assisting materials. Many studies have demonstrated the usefulness of SALDI-MS in quantitative analysis of small organics. However, some characteristics occurring in SALDI-MS require certain attention to ensure the reliability of the quantitative analysis results. The appearance of a coffee-ring effect in SALDI sample preparation is the primary factor that can affect quantitative SALDI-MS analysis results. However, to the best of our knowledge, there are no reports relating to quantitative SALDI-MS analysis that discuss or consider this effect. In this study, the coffee-ring effect is discussed using nanoparticles and nanostructured substrates as SALDI-assisting materials to show how this effect influences SALDI-MS analysis results. Potential solutions for overcoming the existing problems are also suggested.This article is part of the themed issue 'Quantitative mass spectrometry'.

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

    PubMed

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

    2014-12-05

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

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

    PubMed

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

    2014-07-01

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

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

    PubMed Central

    2015-01-01

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

  18. [Study on the multivariate quantitative analysis method for steel alloy elements using LIBS].

    PubMed

    Gu, Yan-hong; Li, Ying; Tian, Ye; Lu, Yuan

    2014-08-01

    Quantitative analysis of steel alloys was carried out using laser induced breakdown spectroscopy (LIBS) taking into account the complex matrix effects in steel alloy samples. The laser induced plasma was generated by a Q-switched Nd:YAG laser operating at 1064 nm with pulse width of 10 ns and repeated frequency of 10 Hz. The LIBS signal was coupled to the echelle spectrometer and recorded by a high sensitive ICCD detector. To get the best experimental conditions, some parameters, such as the detection delay, the CCDs integral gate width and the detecting position from the sample surface, were optimized. The experimental results showed that the optimum detection delay time was 1.5 micros, the optimal CCDs integral gate width was 2 micros and the best detecting position was 1.5 mm below the alloy sample's surface. The samples used in the experiments are ten standard steel alloy samples and two unknown steel alloy samples. The quantitative analysis was investigated with the optimum experimental parameters. Elements Cr and Ni in steel alloy samples were taken as the detection targets. The analysis was carried out with the methods based on conditional univariate quantitative analysis, multiple linear regression and partial least squares (PLS) respectively. It turned out that the correlation coefficients of calibration curves are not very high in the conditional univariate calibration method. The analysis results were obtained with the unsatisfied relative errors for the two predicted samples. So the con- ditional univariate quantitative analysis method can't effectively serve the quantitative analysis purpose for multi-components and complex matrix steel alloy samples. And with multiple linear regression method, the analysis accuracy was improved effectively. The method based on partial least squares (PLS) turned out to be the best method among all the three quantitative analysis methods applied. Based on PLS, the correlation coefficient of calibration curve for Cr is 0

  19. Some remarks on the quantitative analysis of behavior

    PubMed Central

    Marr, M. Jackson

    1989-01-01

    This paper discusses similarities between the mathematization of operant behavior and the early history of the most mathematical of sciences—physics. Galileo explored the properties of motion without dealing with the causes of motion, focusing on changes in motion. Newton's dynamics were concerned with the action of forces as causes of change. Skinner's rationale for using rate to describe behavior derived from an interest in changes in rate. Reinforcement has played the role of force in the dynamics of behavior. Behavioral momentum and maximization have received mathematical formulations in behavior analysis. Yet to be worked out are the relations between molar and molecular formulations of behavioral theory. PMID:22478028

  20. Quantitative Computed Tomography and Image Analysis for Advanced Muscle Assessment

    PubMed Central

    Edmunds, Kyle Joseph; Gíslason, Magnus K.; Arnadottir, Iris D.; Marcante, Andrea; Piccione, Francesco; Gargiulo, Paolo

    2016-01-01

    Medical imaging is of particular interest in the field of translational myology, as extant literature describes the utilization of a wide variety of techniques to non-invasively recapitulate and quantity various internal and external tissue morphologies. In the clinical context, medical imaging remains a vital tool for diagnostics and investigative assessment. This review outlines the results from several investigations on the use of computed tomography (CT) and image analysis techniques to assess muscle conditions and degenerative process due to aging or pathological conditions. Herein, we detail the acquisition of spiral CT images and the use of advanced image analysis tools to characterize muscles in 2D and 3D. Results from these studies recapitulate changes in tissue composition within muscles, as visualized by the association of tissue types to specified Hounsfield Unit (HU) values for fat, loose connective tissue or atrophic muscle, and normal muscle, including fascia and tendon. We show how results from these analyses can be presented as both average HU values and compositions with respect to total muscle volumes, demonstrating the reliability of these tools to monitor, assess and characterize muscle degeneration. PMID:27478562

  1. Quantitative assessment of human body shape using Fourier analysis

    NASA Astrophysics Data System (ADS)

    Friess, Martin; Rohlf, F. J.; Hsiao, Hongwei

    2004-04-01

    Fall protection harnesses are commonly used to reduce the number and severity of injuries. Increasing the efficiency of harness design requires the size and shape variation of the user population to be assessed as detailed and as accurately as possible. In light of the unsatisfactory performance of traditional anthropometry with respect to such assessments, we propose the use of 3D laser surface scans of whole bodies and the statistical analysis of elliptic Fourier coefficients. Ninety-eight male and female adults were scanned. Key features of each torso were extracted as a 3D curve along front, back and the thighs. A 3D extension of Elliptic Fourier analysis4 was used to quantify their shape through multivariate statistics. Shape change as a function of size (allometry) was predicted by regressing the coefficients onto stature, weight and hip circumference. Upper and lower limits of torso shape variation were determined and can be used to redefine the design of the harness that will fit most individual body shapes. Observed allometric changes are used for adjustments to the harness shape in each size. Finally, the estimated outline data were used as templates for a free-form deformation of the complete torso surface using NURBS models (non-uniform rational B-splines).

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

    PubMed Central

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

    2013-01-01

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

  3. [Quantitative analysis of seven phenolic acids in eight Yinqiao Jiedu serial preparations by quantitative analysis of multi-components with single-marker].

    PubMed

    Wang, Jun-jun; Zhang, Li; Guo, Qing; Kou, Jun-ping; Yu, Bo-yang; Gu, Dan-hua

    2015-04-01

    The study aims to develop a unified method to determine seven phenolic acids (neochlorogenic acid, chlorogenic acid, 4-caffeoylquinic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C) contained in honeysuckle flower that is the monarch drug of all the eight Yinqiao Jiedu serial preparations using quantitative analysis of multi-components by single-marker (QAMS). Firstly, chlorogenic acid was used as a reference to get the average relative correction factors (RCFs) of the other phenolic acids in ratios to the reference; columns and instruments from different companies were used to validate the durability of the achieved RCFs in different levels of standard solutions; and honeysuckle flower extract was used as the reference substance to fix the positions of chromatographic peaks. Secondly, the contents of seven phenolic acids in eight different Yinqiao Jiedu serial preparations samples were calculated based on the RCFs durability. Finally, the quantitative results were compared between QAMS and the external standard (ES) method. The results have showed that the durability of the achieved RCFs is good (RSD during 0.80% - 2.56%), and there are no differences between the quantitative results of QAMS and ES (the relative average deviation < 0.93%). So it can be successfully used to the quantitative control of honeysuckle flower principally prescribed in Yinqiao Jiedu serial preparations.

  4. Insights into Primary Immune Deficiency from quantitative microscopy analysis

    PubMed Central

    Mace, Emily M.; Orange, Jordan S.

    2015-01-01

    Recent advances in genomics based technology have resulted in an increase in our understanding of the molecular basis of many primary immune deficiencies. Along with this increased knowledge comes an increased responsibility to understand the underlying mechanism of disease, and thus increasingly sophisticated technologies are being used to investigate the cell biology of human immune deficiencies. One such technology, which has, itself, undergone a recent explosion in innovation, is that of high-resolution microscopy and image analysis. These advances complement innovative studies that have previously shed light on critical cell biological processes that are perturbed by single gene mutations in primary immune deficiency. Here, we highlight advances made specifically in the following cell biological processes: 1) cytoskeletal-related processes; 2) cell signaling; 3) intercellular trafficking; and 4) cellular host defense. PMID:26078103

  5. Automated monitoring and quantitative analysis of feeding behaviour in Drosophila

    PubMed Central

    Itskov, Pavel M.; Moreira, José-Maria; Vinnik, Ekaterina; Lopes, Gonçalo; Safarik, Steve; Dickinson, Michael H.; Ribeiro, Carlos

    2014-01-01

    Food ingestion is one of the defining behaviours of all animals, but its quantification and analysis remain challenging. This is especially the case for feeding behaviour in small, genetically tractable animals such as Drosophila melanogaster. Here, we present a method based on capacitive measurements, which allows the detailed, automated and high-throughput quantification of feeding behaviour. Using this method, we were able to measure the volume ingested in single sips of an individual, and monitor the absorption of food with high temporal resolution. We demonstrate that flies ingest food by rhythmically extending their proboscis with a frequency that is not modulated by the internal state of the animal. Instead, hunger and satiety homeostatically modulate the microstructure of feeding. These results highlight similarities of food intake regulation between insects, rodents, and humans, pointing to a common strategy in how the nervous systems of different animals control food intake. PMID:25087594

  6. Digital photogrammetry for quantitative wear analysis of retrieved TKA components.

    PubMed

    Grochowsky, J C; Alaways, L W; Siskey, R; Most, E; Kurtz, S M

    2006-11-01

    The use of new materials in knee arthroplasty demands a way in which to accurately quantify wear in retrieved components. Methods such as damage scoring, coordinate measurement, and in vivo wear analysis have been used in the past. The limitations in these methods illustrate a need for a different methodology that can accurately quantify wear, which is relatively easy to perform and uses a minimal amount of expensive equipment. Off-the-shelf digital photogrammetry represents a potentially quick and easy alternative to what is readily available. Eighty tibial inserts were visually examined for front and backside wear and digitally photographed in the presence of two calibrated reference fields. All images were segmented (via manual and automated algorithms) using Adobe Photoshop and National Institute of Health ImageJ. Finally, wear was determined using ImageJ and Rhinoceros software. The absolute accuracy of the method and repeatability/reproducibility by different observers were measured in order to determine the uncertainty of wear measurements. To determine if variation in wear measurements was due to implant design, 35 implants of the three most prevalent designs were subjected to retrieval analysis. The overall accuracy of area measurements was 97.8%. The error in automated segmentation was found to be significantly lower than that of manual segmentation. The photogrammetry method was found to be reasonably accurate and repeatable in measuring 2-D areas and applicable to determining wear. There was no significant variation in uncertainty detected among different implant designs. Photogrammetry has a broad range of applicability since it is size- and design-independent. A minimal amount of off-the-shelf equipment is needed for the procedure and no proprietary knowledge of the implant is needed.

  7. Scalp Surgery: Quantitative Analysis of Follicular Unit Growth

    PubMed Central

    Caruana, Giorgia

    2015-01-01

    Background: Over the years, different kinds of hair transplantation have been compared in an attempt to overcome male pattern alopecia and, at the same time, maximize both the survival and growth rate of grafted hair. In this study, we have assessed the survival and growth rate of follicular units (FU) in an in vitro model, as compared with that of conventional hair micrografts, to experimentally evaluate and elaborate on the differences between these 2 approaches in hair transplantation procedures. Methods: Group A (control; n = 100 follicles) was composed of hair micrografts, whereas FUs were assigned to Group B (experimental; n = 100 follicles, n = 35 FUs). Each group was cultured for a period of 10 days; the total stretch of follicles was measured soon after the harvest and 10 days later. The Kruskal-Wallis one-way analysis of variance on ranks test was used to perform statistical analysis. Results: The growth rate of follicles from Group A (mean 10-day shaft growth rate = 0.30 mm) proved to be statistically different compared with that of Group B (mean 10-day shaft growth rate = 0.23 mm). Conversely, our data did not show any significant difference between the survival rate of hair grafts from these 2 groups. Conclusions: Our data highlighted a reduced FU shaft growth compared with that of hair micrografts, corroborating, to a certain extent, the hypothesis that a significant amount of adipose tissue surrounding the follicle included in the graft may result in an inadequate nourishment supply to follicular cells. PMID:26579345

  8. Quantitative proteomic analysis of the brainstem following lethal sarin exposure.

    PubMed

    Meade, Mitchell L; Hoffmann, Andrea; Makley, Meghan K; Snider, Thomas H; Schlager, John J; Gearhart, Jeffery M

    2015-06-22

    The brainstem represents a major tissue area affected by sarin organophosphate poisoning due to its function in respiratory and cardiovascular control. While the acute toxic effects of sarin on brainstem-related responses are relatively unknown, other brain areas e.g., cortex or cerebellum, have been studied more extensively. The study objective was to analyze the guinea pig brainstem toxicology response following sarin (2×LD50) exposure by proteome pathway analysis to gain insight into the complex regulatory mechanisms that lead to impairment of respiratory and cardiovascular control. Guinea pig exposure to sarin resulted in the typical acute behavior/physiology outcomes with death between 15 and 25min. In addition, brain and blood acetylcholinesterase activity was significantly reduced in the pres