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Sample records for quantitative phosphoproteomics dissection

  1. A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation*

    PubMed Central

    Nguyen, Vinh; Cao, Lulu; Lin, Jonathan T.; Hung, Norris; Ritz, Anna; Yu, Kebing; Jianu, Radu; Ulin, Samuel P.; Raphael, Benjamin J.; Laidlaw, David H.; Brossay, Laurent; Salomon, Arthur R.

    2009-01-01

    Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy. PMID:19605366

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

    PubMed

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina; Sarwar, Uzma; Bøgebo, Rikke; Sheikh, Søren P; Gammeltoft, Steen; Olsen, Jesper V; Hansen, Jakob L

    2010-07-01

    Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks Galpha(q) protein activity and activates G protein-independent pathways of the AT(1)R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future. PMID:20363803

  3. Phosphoproteomics

    PubMed Central

    Huang, Paul H.; White, Forest M.

    2009-01-01

    In recent years, phosphoproteomic technologies have increased our understanding of cellular signaling networks. Here, we frame recent phosphoproteomics-based advances in the context of the DNA damage response and ErbB receptor family signaling and offer a perspective on how the molecular insights arising from the integration of such proteomic approaches might be used for clinical applications. PMID:18922462

  4. Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics.

    PubMed

    Cheng, Lei; Pisitkun, Trairak; Knepper, Mark A; Hoffert, Jason D

    2016-01-01

    Isobaric tagging reagents have become an invaluable tool for multiplexed quantitative proteomic analysis. These reagents can label multiple, distinct peptide samples from virtually any source material (e.g., tissue, cell line, purified proteins), allowing users the opportunity to assess changes in peptide abundances across many different time points or experimental conditions. Here, we describe the application of isobaric peptide labeling, specifically 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ), for quantitative phosphoproteomic analysis of cultured cells or tissue suspensions. For this particular protocol, labeled samples are pooled, fractionated by strong cation exchange chromatography, enriched for phosphopeptides, and analyzed by tandem mass spectrometry (LC-MS/MS) for both peptide identification and quantitation. PMID:26584918

  5. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling.

    PubMed

    Wandinger, Sebastian K; Lahortiga, Idoya; 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

  6. 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

  7. 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. PMID:21491946

  8. 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

    2016-04-01

    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. PMID:26960075

  9. Quantitative proteomic and phosphoproteomic analysis of Trypanosoma cruzi amastigogenesis.

    PubMed

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

    2014-12-01

    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

  10. Quantitative phosphoproteomic analysis of prion-infected neuronal cells

    PubMed Central

    2010-01-01

    Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal diseases associated with the conversion of the cellular prion protein (PrPC) to the abnormal prion protein (PrPSc). Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS)-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture) analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease. PMID:20920157

  11. Quantitative Phosphoproteomics of Cytotoxic T Cells to Reveal Protein Kinase D 2 Regulated Networks*

    PubMed Central

    Navarro, Mara N.; Goebel, Juergen; Hukelmann, Jens L.; Cantrell, Doreen A.

    2014-01-01

    The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages. PMID:25266776

  12. Quantitative phosphoproteomics of cytotoxic T cells to reveal protein kinase d 2 regulated networks.

    PubMed

    Navarro, María N; Goebel, Juergen; Hukelmann, Jens L; Cantrell, Doreen A

    2014-12-01

    The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages. PMID:25266776

  13. Quantitative Phosphoproteomics Revealed Glucose-Stimulated Responses of Islet Associated with Insulin Secretion.

    PubMed

    Li, Jiaming; Li, Qingrun; Tang, Jiashu; Xia, Fangying; Wu, Jiarui; Zeng, Rong

    2015-11-01

    As central tissue of glucose homeostasis, islet has been an important focus of diabetes research. Phosphorylation plays pivotal roles in islet function, especially in islet glucose-stimulated insulin secretion. A systematic view on how phosphorylation networks were coordinately regulated in this process remains lacking, partially due to the limited amount of islets from an individual for a phosphoproteomic analysis. Here we optimized the in-tip and best-ratio phosphopeptide enrichment strategy and a SILAC-based workflow for processing rat islet samples. With limited islet lysates from each individual rat (20-47 ?g), we identified 8539 phosphosites on 2487 proteins. Subsequent quantitative analyses uncovered that short-term (30 min) high glucose stimulation induced coordinate responses of islet phosphoproteome on multiple biological levels, including insulin secretion related pathways, cytoskeleton dynamics, protein processing in ER and Golgi, transcription and translation, and so on. Furthermore, three glucose-responsive phosphosites (Prkar1a pT75pS77 and Tagln2 pS163) from the data set were proved to be correlated with insulin secretion. Overall, we initially gave an in-depth map of islet phosphoproteome regulated by glucose on individual rat level. This was a significant addition to our knowledge about how phosphorylation networks responded in insulin secretion. Also, the list of changed phosphosites was a valuable resource for molecular researchers in diabetes field. PMID:26437020

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2016-01-01

    The invasive blood-stage malaria parasite – the merozoite – induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  16. 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

    2016-01-01

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  17. Quantitative analysis of the TNF-?-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKK? substrate.

    PubMed

    Krishnan, Ramesh K; Nolte, Hendrik; Sun, Tianliang; Kaur, Harmandeep; Sreenivasan, Krishnamoorthy; Looso, Mario; Offermanns, Stefan; Krger, Marcus; Swiercz, Jakub M

    2015-01-01

    The inhibitor of the nuclear factor-?B (I?B) kinase (IKK) complex is a key regulator of the canonical NF-?B signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-?B activation; however, there is increasing evidence for NF-?B pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-?-IKK?-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ?1% are regulated up on TNF-? stimulation. We identify various potential novel IKK? substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKK? on serine 298. We provide evidence that IKK?-mediated AEG-1 phosphorylation is essential for I?B? degradation as well as NF-?B-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. PMID:25849741

  18. Quantitative analysis of the TNF-?-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKK? substrate

    PubMed Central

    Krishnan, Ramesh K.; Nolte, Hendrik; Sun, Tianliang; Kaur, Harmandeep; Sreenivasan, Krishnamoorthy; Looso, Mario; Offermanns, Stefan; Krger, Marcus; Swiercz, Jakub M.

    2015-01-01

    The inhibitor of the nuclear factor-?B (I?B) kinase (IKK) complex is a key regulator of the canonical NF-?B signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-?B activation; however, there is increasing evidence for NF-?B pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-?-IKK?-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ?1% are regulated up on TNF-? stimulation. We identify various potential novel IKK? substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKK? on serine 298. We provide evidence that IKK?-mediated AEG-1 phosphorylation is essential for I?B? degradation as well as NF-?B-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. PMID:25849741

  19. Quantitative Phosphoproteomics Analysis of Nitric Oxide–Responsive Phosphoproteins in Cotton Leaf

    PubMed Central

    Song, Meizhen; Pang, Chaoyou; Wei, Hengling; Liu, Ji; Zhan, Xianjin; Lan, Jiayang; Feng, Changhui; Zhang, Shengxi; Yu, Shuxun

    2014-01-01

    Knowledge of phosphorylation events and their regulation is crucial to understanding the functional biology of plant proteins, but very little is currently known about nitric oxide–responsive phosphorylation in plants. Here, we report the first large-scale, quantitative phosphoproteome analysis of cotton (Gossypium hirsutum) treated with sodium nitroprusside (nitric oxide donor) by utilizing the isobaric tag for relative and absolute quantitation (iTRAQ) method. A total of 1315 unique phosphopeptides, spanning 1528 non-redundant phosphorylation sites, were detected from 1020 cotton phosphoproteins. Among them, 183 phosphopeptides corresponding to 167 phosphoproteins were found to be differentially phosphorylated in response to sodium nitroprusside. Several of the phosphorylation sites that we identified, including RQxS, DSxE, TxxxxSP and SPxT, have not, to our knowledge, been reported to be protein kinase sites in other species. The phosphoproteins identified are involved in a wide range of cellular processes, including signal transduction, RNA metabolism, intracellular transport and so on. This study reveals unique features of the cotton phosphoproteome and provides new insight into the biochemical pathways that are regulated by nitric oxide. PMID:24714030

  20. Glycoprotein capture and quantitative phosphoproteomics indicate coordinated regulation of cell migration upon lysophosphatidic acid stimulation.

    PubMed

    Mäusbacher, Nina; Schreiber, Thiemo B; Daub, Henrik

    2010-11-01

    The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior. PMID:20639409

  1. 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, 7days 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. PMID:26613898

  2. Quantitative phosphoproteomic profiling of PINK1-deficient cells identifies phosphorylation changes in nuclear proteins

    PubMed Central

    Qin, Xiaoyan; Zheng, Chaoya; Yates, John R.; Liao, Lujian

    2014-01-01

    The Parkinson's disease (PD) associated gene PINK1 encodes a protein kinase that mediates the phosphorylation of multiple proteins involved in mitochondrial homeostasis. The broader downstream signaling events mediated by PINK1 kinase activity have not been well documented. We combine quantitative phosphoproteomic strategies with siRNA mediated PINK1 knock down in mammalian cells to identify alterations of phosphorylation events downstream of PINK1. Although down-regulation of PINK1 has no major effect on the proteome expression in these cells, phosphorylation of over one hundred proteins was reduced reflecting basal levels of phosphorylation signaling events downstream of PINK1. Motif analysis of the residues flanking the phosphorylation sites indicates proline-directed kinase specificity. Surprisingly, we found that the downstream signaling nodes included many transcription factors, as well as nuclear proteins involved in DNA and RNA metabolism. Thus, PINK1 dependent phosphorylation signaling may regulate nuclear activities. PMID:24626860

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

    PubMed Central

    Doubleday, Peter F.; Ballif, Bryan A.

    2014-01-01

    Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX) chromatography, coupled to immobilized metal affinity chromatography (IMAC), we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain. PMID:25177544

  4. A quantitative map of the liver mitochondrial phosphoproteome reveals post-translational control of ketogenesis

    PubMed Central

    Grimsrud, Paul A.; Carson, Joshua J.; Hebert, Alex S.; Hubler, Shane L.; Niemi, Natalie M.; Bailey, Derek J.; Jochem, Adam; Stapleton, Donald S.; Keller, Mark P.; Westphall, Michael S.; Yandell, Brian S.; Attie, Alan D.; Coon, Joshua J.; Pagliarini, David J.

    2012-01-01

    Summary Mitochondria are dynamic organelles that play a central role in a diverse array of metabolic processes. Elucidating mitochondrial adaptations to changing metabolic demands and the pathogenic alterations that underlie metabolic disorders represent principal challenges in cell biology. Here, we performed multiplexed quantitative mass spectrometry-based proteomics to chart the remodeling of the mouse liver mitochondrial proteome and phosphoproteome during both acute and chronic physiological transformations in more than 50 mice. Our analyses reveal that reversible phosphorylation is widespread in mitochondria, and is a key mechanism for regulating ketogenesis during the onset of obesity and type 2 diabetes. Specifically, we have demonstrated that phosphorylation of a conserved serine on Hmgcs2 (S456) significantly enhances its catalytic activity in response to increased ketogenic demand. Collectively, our work describes the plasticity of this organelle at high resolution and provides a framework for investigating the roles of proteome restructuring and reversible phosphorylation in mitochondrial adaptation. PMID:23140645

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

    PubMed

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

    2016-02-01

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

  6. Quantitative phosphoproteomics analysis reveals broad regulatory role of heparan sulfate on endothelial signaling.

    PubMed

    Qiu, Hong; Jiang, Jun-Lin; Liu, Miao; Huang, Xin; Ding, Shi-Jian; Wang, Lianchun

    2013-08-01

    Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling. PMID:23649490

  7. Research Resource: Identification of Novel Growth Hormone-Regulated Phosphorylation Sites by Quantitative Phosphoproteomics

    PubMed Central

    Ray, Bridgette N.; Kweon, Hye Kyong; Argetsinger, Lawrence S.; Fingar, Diane C.; Andrews, Philip C.

    2012-01-01

    GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na+/H+ exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly broadens our thinking of GH-regulated cell functions. PMID:22570334

  8. Quantitative phosphoproteome analysis unveils LAT as a modulator of CD3? and ZAP-70 tyrosine phosphorylation.

    PubMed

    Salek, Mogjiborahman; McGowan, Simon; Trudgian, David C; Dushek, Omer; de Wet, Ben; Efstathiou, Georgios; Acuto, Oreste

    2013-01-01

    Signaling through the T cell receptor (TCR) initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLC?1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3? and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR) accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341. PMID:24204825

  9. Quantitative Phosphoproteomic Profiling of Human Non-Small Cell Lung Cancer Tumors

    PubMed Central

    Schweppe, Devin K.; Rigas, James R.; Gerber, Scott A.

    2013-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Within the molecular scope of NCSLC, a complex landscape of dysregulated cellular signaling has emerged, defined largely by mutations in select mediators of signal transduction, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma (ALK) kinases. Consequently, these mutant kinases become constitutively activated and targets for chemotherapeutic intervention. Encouragingly, small molecule inhibitors of these pathways have shown promise in clinical trials or are approved for clinical use. However, many protein kinases are dysregulated in NSCLC without genetic mutations. To quantify differences in tumor cell signaling that are transparent to genomic methods, we established a super-SILAC internal standard derived from NSCLC cell lines grown in vitro and labeled with heavy lysine and arginine, and deployed them in a phosphoproteomics workflow. We identified 9019 and 8753 phosphorylation sites in two separate tumors. Relative quantification of phosphopeptide abundance between tumor samples allowed for the determination of specific hubs and pathways differing between each tumor. Sites downstream of Ras showed decreased inhibitory phosphorylation (Raf/Mek) and increased activating phosphorylation (Erk1/2) in one tumor versus another. In this way, we were able to quantitatively access oncogenic kinase signaling in primary human tumors. PMID:23911959

  10. 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.

  11. Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

    PubMed

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

    2014-02-01

    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

  12. Automated, Reproducible, Titania-Based Phosphopeptide Enrichment Strategy for Label-Free Quantitative Phosphoproteomics

    PubMed Central

    Richardson, Brenna McJury; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur

    2013-01-01

    An automated phosphopeptide enrichment strategy is described using titanium dioxide (TiO2)-packed, fused silica capillaries for use with liquid chromatography (LC)-mass spectrometry (MS)/MS-based, label-free proteomics workflows. To correlate an optimum peptide:TiO2 loading ratio between different particle types, the ratio of phenyl phosphate-binding capacities was used. The optimum loading for the column was then verified through replicate enrichments of a range of quantities of digested rat brain tissue cell lysate. Fractions were taken during sample loading, multiple wash steps, and the elution steps and analyzed by LC-MS/MS to gauge the efficiency and reproducibility of the enrichment. Greater than 96% of the total phosphopeptides were detected in the elution fractions, indicating efficient trapping of the phosphopeptides on the first pass of enrichment. The quantitative reproducibility of the automated setup was also improved greatly with phosphopeptide intensities from replicate enrichments exhibiting a median coefficient of variation (CV) of 5.8%, and 80% of the identified phosphopeptides had CVs below 11.1%, while maintaining >85% specificity. By providing this high degree of analytical reproducibility, this method allows for label-free phosphoproteomics over large sample sets with complex experimental designs (multiple biological conditions, multiple biological replicates, multiple time-points, etc.), including large-scale clinical cohorts. PMID:23542237

  13. Quantitative Phosphoproteome Profiling of Iron-Deficient Arabidopsis Roots1[C][W

    PubMed Central

    Lan, Ping; Li, Wenfeng; Wen, Tuan-Nan; Schmidt, Wolfgang

    2012-01-01

    Iron (Fe) is an essential mineral nutrient for plants, but often it is not available in sufficient quantities to sustain optimal growth. To gain insights into adaptive processes to low Fe availability at the posttranslational level, we conducted a quantitative analysis of Fe deficiency-induced changes in the phosphoproteome profile of Arabidopsis (Arabidopsis thaliana) roots. Isobaric tags for relative and absolute quantitation-labeled phosphopeptides were analyzed by liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap with collision-induced dissociation and high-energy collision dissociation capabilities. Using a combination of titanium dioxide and immobilized metal affinity chromatography to enrich phosphopeptides, we extracted 849 uniquely identified phosphopeptides corresponding to 425 proteins and identified several not previously described phosphorylation motifs. A subset of 45 phosphoproteins was defined as being significantly changed in abundance upon Fe deficiency. Kinase motifs in Fe-responsive proteins matched to protein kinase A/calcium calmodulin-dependent kinase II, casein kinase II, and proline-directed kinase, indicating a possible critical function of these kinase classes in Fe homeostasis. To validate our analysis, we conducted site-directed mutagenesis on IAA-CONJUGATE-RESISTANT4 (IAR4), a protein putatively functioning in auxin homeostasis. iar4 mutants showed compromised root hair formation and developed shorter primary roots. Changing serine-296 in IAR4 to alanine resulted in a phenotype intermediate between mutant and wild type, whereas acidic substitution to aspartate to mimic phosphorylation was either lethal or caused an extreme dwarf phenotype, supporting the critical importance of this residue in Fe homeostasis. Our analyses further disclose substantial changes in the abundance of phosphoproteins involved in primary carbohydrate metabolism upon Fe deficiency, complementing the picture derived from previous proteomic and transcriptomic profiling studies. PMID:22438062

  14. Quantitative phosphoproteomics reveals crosstalk between phosphorylation and O-GlcNAc in the DNA damage response pathway.

    PubMed

    Zhong, Jun; Martinez, Marissa; Sengupta, Srona; Lee, Albert; Wu, Xinyan; Chaerkady, Raghothama; Chatterjee, Aditi; O'Meally, Robert N; Cole, Robert N; Pandey, Akhilesh; Zachara, Natasha E

    2015-01-01

    The modification of intracellular proteins by monosaccharides of O-linked ?-N-acetylglucosamine (O-GlcNAc) is an essential and dynamic PTM of metazoans. The addition and removal of O-GlcNAc is catalyzed by the O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. One mechanism by which O-GlcNAc is thought to mediate proteins is by regulating phosphorylation. To provide insight into the pathways regulated by O-GlcNAc, we have utilized SILAC-based quantitative proteomics to carry out comparisons of site-specific phosphorylation in OGT wild-type and Null cells. Quantitation of the phosphoproteome demonstrated that of 5529 phosphoserine, phosphothreonine, and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc. Collectively, these data suggest that deletion of OGT has a profound effect on the phosphorylation of cell cycle and DNA damage response proteins. Key events were confirmed by biochemical analyses and demonstrate an increase in the activating autophosphorylation event on ATM (Ser1987) and on ATM's downstream targets p53, H2AX, and Chk2. Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis. All MS data have been deposited in the ProteomeXchange with identifier PXD001153 (http://proteomecentral.proteomexchange.org/dataset/PXD001153). PMID:25263469

  15. Quantitative Phosphoproteomics Reveals Crosstalk Between Phosphorylation and O-GlcNAc in the DNA Damage Response Pathway

    PubMed Central

    Zhong, Jun; Martinez, Marissa; Sengupta, Srona; Lee, Albert; Wu, Xinyan; Chaerkady, Raghothama; Chatterjee, Aditi; OMeally, Robert N.; Cole, Robert N.; Pandey, Akhilesh; Zachara, Natasha E.

    2015-01-01

    The modification of intracellular proteins by monosaccharides of O-linked ?-N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification of metazoans. The addition and removal of O-GlcNAc is catalyzed by the O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. One mechanism by which O-GlcNAc is thought to mediate proteins is by regulating phosphorylation. To provide insight into the pathways regulated by O-GlcNAc, we have utilized stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics to carry out comparisons of site-specific phosphorylation in OGT wild-type (WT) and Null cells. Quantitation of the phosphoproteome demonstrated that out of 5,529 phosphoserine, phosphothreonine and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc. Collectively, these data suggest that deletion of OGT has a profound effect on the phosphorylation of cell cycle and DNA damage response proteins. Key events were confirmed by biochemical analyses and demonstrate a increase in the activating autophosphorylation event on ATM (Ser1987) and on ATMs downstream targets p53, H2AX and Chk2. Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis. PMID:25263469

  16. 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

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2015-12-01

    West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies. PMID:26485063

  19. 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, Ren 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 ?g 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. PMID:25619855

  20. Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF-M/H) in Alzheimer's disease by iTRAQ

    PubMed Central

    Rudrabhatla,*, Parvathi; Grant,*, Philip; Jaffe, Howard; Strong, Michael J.; Pant, Harish C.

    2010-01-01

    Aberrant hyperphosphorylation of neuronal cytoskeletal proteins is one of the major pathological hallmarks of neurodegenerative disorders such as Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Human NF-M/H display a large number of multiple KSP repeats in the carboxy-terminal tail domain, which are phosphorylation sites of proline-directed serine/threonine (pSer/Thr-Pro, KS/T-P) kinases. The phosphorylation sites of NF-M/H have not been characterized in AD brain. Here, we use quantitative phosphoproteomic methodology, isobaric tag for relative and absolute quantitation (iTRAQ), for the characterization of NF-M/H phosphorylation sites in AD brain. We identified 13 hyperphosphorylated sites of NF-M; 9 Lys-Ser-Pro (KSP) sites; 2 variant motifs, Glu-Ser-Pro (ESP) Ser-736 and Leu-Ser-Pro (LSP) Ser-837; and 2 non-S/T-P motifs, Ser-783 and Ser-788. All the Ser/Thr residues are phosphorylated at significantly greater abundance in AD brain compared with control brain. Ten hyperphosphorylated KSP sites have been identified on the C-terminal tail domain of NF-H, with greater abundance of phosphorylation in AD brain compared with control brain. Our data provide the direct evidence that NF-M/H are hyperphosphorylated in AD compared with control brain and suggest the role of both proline-directed and non-proline-directed protein kinases in AD. This study represents the first comprehensive iTRAQ analyses and quantification of phosphorylation sites of human NF-M and NF-H from AD brain and suggests that aberrant hyperphosphorylation of neuronal intermediate filament proteins is involved in AD.—Rudrabhatla, P., Grant, P., Jaffe, H., Strong, M. J., Pant, H. C. Quantitative phosphoproteomic analysis of neuronal intermediate filament proteins (NF-M/H) in Alzheimer's disease by iTRAQ. PMID:20624930

  1. Dissecting the architecture of a quantitative trait locus in yeast.

    PubMed

    Steinmetz, Lars M; Sinha, Himanshu; Richards, Dan R; Spiegelman, Jamie I; Oefner, Peter J; McCusker, John H; Davis, Ronald W

    2002-03-21

    Most phenotypic diversity in natural populations is characterized by differences in degree rather than in kind. Identification of the actual genes underlying these quantitative traits has proved difficult. As a result, little is known about their genetic architecture. The failures are thought to be due to the different contributions of many underlying genes to the phenotype and the ability of different combinations of genes and environmental factors to produce similar phenotypes. This study combined genome-wide mapping and a new genetic technique named reciprocal-hemizygosity analysis to achieve the complete dissection of a quantitative trait locus (QTL) in Saccharomyces cerevisiae. A QTL architecture was uncovered that was more complex than expected. Functional linkages both in cis and in trans were found between three tightly linked quantitative trait genes that are neither necessary nor sufficient in isolation. This arrangement of alleles explains heterosis (hybrid vigour), the increased fitness of the heterozygote compared with homozygotes. It also demonstrates a deficiency in current approaches to QTL dissection with implications extending to traits in other organisms, including human genetic diseases. PMID:11907579

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

    PubMed Central

    Nguyen, Tran Hong Nha; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese R.; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Paa-Toli?, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-01-01

    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 eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 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. PMID:22843990

  3. 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.

  4. 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 2448 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 (4872 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ?Sterne 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 diagnostic biomarkers of human anthrax detectable within circulating immune cells, and could aid in the identification of pathogenic mechanisms and therapeutic targets. PMID:21189417

  5. 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

  6. 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

    2015-01-01

    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

  7. Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei.

    PubMed

    Urbaniak, Michael D; Martin, David M A; Ferguson, Michael A J

    2013-05-01

    We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3-4 fold over-represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the T. brucei kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in T. brucei is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism. PMID:23485197

  8. Quantitative phosphoproteomics reveals link between Helicobacter pylori infection and RNA splicing modulation in host cells.

    PubMed

    Holland, Carsten; Schmid, Monika; Zimny-Arndt, Ursula; Rohloff, John; Stein, Robert; Jungblut, Peter R; Meyer, Thomas F

    2011-07-01

    The Gram-negative, spiral-shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2-DE, and MALDI TOF/TOF-MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one-third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine-rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection-dependent protein species-specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer. PMID:21717572

  9. 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

    2015-01-01

    Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between 'normal' cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as 'asymptomatic' or 'preclinical' AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain. PMID:26444780

  10. Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1/CXCR4 in breast cancer stem cells

    PubMed Central

    Yi, Tingfang; Zhai, Bo; Yu, Yonghao; Kiyotsugu, Yoshikawa; Raschle, Thomas; Etzkorn, Manuel; Seo, Hee-Chan; Nagiec, Michal; Luna, Rafael E.; Reinherz, Ellis L.; Blenis, John; Gygi, Steven P.; Wagner, Gerhard

    2014-01-01

    Breast cancer is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.7 million new cases and 522,000 deaths around the world in 2012 alone. Cancer stem cells (CSCs) are essential for tumor reoccurrence and metastasis which is the major source of cancer lethality. G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) is critical for tumor metastasis. However, stromal cell-derived factor 1 (SDF-1)/CXCR4–mediated signaling pathways in breast CSCs are largely unknown. Using isotope reductive dimethylation and large-scale MS-based quantitative phosphoproteome analysis, we examined protein phosphorylation induced by SDF-1/CXCR4 signaling in breast CSCs. We quantified more than 11,000 phosphorylation sites in 2,500 phosphoproteins. Of these phosphosites, 87% were statistically unchanged in abundance in response to SDF-1/CXCR4 stimulation. In contrast, 545 phosphosites in 266 phosphoproteins were significantly increased, whereas 113 phosphosites in 74 phosphoproteins were significantly decreased. SDF-1/CXCR4 increases phosphorylation in 60 cell migration- and invasion-related proteins, of them 43 (>70%) phosphoproteins are unrecognized. In addition, SDF-1/CXCR4 upregulates the phosphorylation of 44 previously uncharacterized kinases, 8 phosphatases, and 1 endogenous phosphatase inhibitor. Using computational approaches, we performed system-based analyses examining SDF-1/CXCR4–mediated phosphoproteome, including construction of kinase–substrate network and feedback regulation loops downstream of SDF-1/CXCR4 signaling in breast CSCs. We identified a previously unidentified SDF-1/CXCR4-PKA-MAP2K2-ERK signaling pathway and demonstrated the feedback regulation on MEK, ERK1/2, δ-catenin, and PPP1Cα in SDF-1/CXCR4 signaling in breast CSCs. This study gives a system-wide view of phosphorylation events downstream of SDF-1/CXCR4 signaling in breast CSCs, providing a resource for the study of CSC-targeted cancer therapy. PMID:24782546

  11. Identification of novel signaling components in N,N-Dinitrosopiperazine-mediated metastasis of nasopharyngeal Carcinoma by quantitative phosphoproteomics

    PubMed Central

    2014-01-01

    Background Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic cancer. N,N-dinitrosopiperazine (DNP), a carcinogen with specificity for nasopharyngeal epithelium, facilitates NPC metastasis. However, the underlying mechanism is not known. Methods Quantitative phosphoproteomics, using stable isotope labeling of amino acids in cell cultures, was employed to identify phosphoproteins associated with NPC metastasis mediated by DNP. NPC cell line 6-10B, which is relatively less metastatic, was used to investigate DNP-mediated metastasis. Boyden chamber invasion assay was used to measure DNP-induced motility and invasion, and nude mice were used to verify DNP-mediated metastasis in vivo. Several different phosphoproteins detected by proteomics analysis were verified by immunoblotting. DNP-mediated metastasis facilitated by lysine-rich CEACAM1 co-isolated protein (LYRIC) phosphorylation at serine 568 was confirmed using mutations targeting the phosphorylation site of LYRIC. DNP-mediated metastasis through LYRIC phosphorylation was confirmed in the NPC cell line CNE1. DNP-mediated LYRIC phosphorylation at serine 568 was also verified in metastatic tumors of BABL/c nude mice. Results Boyden chamber invasion assay indicated that DNP mediated cell motility and invasion of NPC cell 6-10B in vitro, and experiments with nude mice indicated that DNP increased 6-10B metastasis in vivo. In the phosphoproteomics analysis, we detected 216 phosphorylation sites on 130 proteins; among these, 48 phosphorylation sites on 30 unique phosphopeptides were modulated by DNP by at least 1.5-fold. DNP mediated the expression of phosphorylated GTPase, ferritin, LYRIC, and RNA polymerase, and it decreased the expression of phosphorylated torsin-1A protein 1. Furthermore, DNP induced LYRIC phosphorylation at serine 568 to facilitate cell motility and invasion, whereas DNP-mediated motility and invasion was decreased when serine 568 in LYRIC was mutated. In another NPC cell line, CNE1, DNP also mediated cell motility and invasion followed by enhanced phosphorylation of LYRIC at serine 568. Finally, phosphorylated-LYRIC expression at serine 568 was significantly increased in metastatic tumors induced by DNP. Conclusion DNP regulates multiple signaling pathways through protein phosphorylation, including the phosphorylation of LYRIC at serine 568, and mediates NPC metastasis. These findings provide insights on the complexity and dynamics of DNP-facilitated metastasis, and may help to gain a better understanding of the mechanisms by clarifying NPC-induced metastasis. PMID:24708550

  12. 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

    2014-10-01

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS. PMID:25168779

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

    PubMed

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

    2011-08-25

    In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML. PMID:21730355

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

    PubMed

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

    2016-03-01

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

  15. 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

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

    PubMed

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

    2014-05-01

    The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT(2A) receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor. PMID:24637012

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

    PubMed Central

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

    2014-01-01

    The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2A receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor. PMID:24637012

  18. 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

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

    PubMed Central

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

    2009-01-01

    Summary 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 various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application 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 LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis. PMID:18785766

  20. 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.

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

    PubMed

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

    2016-02-01

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed. PMID:26689635

  2. In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth.

    PubMed

    Jiang, N; Hjorth-Jensen, K; Hekmat, O; Iglesias-Gato, D; Kruse, T; Wang, C; Wei, W; Ke, B; Yan, B; Niu, Y; Olsen, J V; Flores-Morales, A

    2015-05-21

    Prostate cancer remains a leading cause of cancer-related mortality worldwide owing to our inability to treat effectively castration-resistant tumors. To understand the signaling mechanisms sustaining castration-resistant growth, we implemented a mass spectrometry-based quantitative proteomic approach and use it to compare protein phosphorylation in orthotopic xenograft tumors grown in either intact or castrated mice. This investigation identified changes in phosphorylation of signaling proteins such as MEK, LYN, PRAS40, YAP1 and PAK2, indicating the concomitant activation of several oncogenic pathways in castration-resistant tumors, a notion that was confirmed by tumor transcriptome analysis. Further analysis demonstrated that the activation of mTORC1, PAK2 and the increased levels of YAP1 in castration-resistant tumors can be explained by the loss of androgen inhibitory actions. The analysis of clinical samples demonstrated elevated levels of PAK2 and YAP1 in castration-resistant tumors, whereas knockdown experiments in androgen-independent cells demonstrated that both YAP1 and PAK2 regulate cell colony formation and cell invasion activity. PAK2 also influenced cell proliferation and mitotic timing. Interestingly, these phenotypic changes occur in the absence of obvious alterations in the activity of AKT, MAPK or mTORC1 pathways, suggesting that PAK2 and YAP1 may represent novel targets for the treatment of castration-resistant prostate cancer. Pharmacologic inhibitors of PAK2 (PF-3758309) and YAP1 (Verteporfin) were able to inhibit the growth of androgen-independent PC3 xenografts. This work demonstrates the power of applying high-resolution mass spectrometry in the proteomic profiling of tumors grown in vivo for the identification of novel and clinically relevant regulatory proteins. PMID:25065596

  3. 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

  4. Dissection Dissected.

    ERIC Educational Resources Information Center

    Berman, William

    1984-01-01

    Discusses the role of dissection in science courses, examining essential lessons students can learn (such as developing an abiding respect for all forms of life, including the animal being dissected). Also presents a list of tips related to classroom dissection and comments on formaldehyde and formalin substitutes. (JN)

  5. Quantitative dissection of the simple repression inputoutput function

    PubMed Central

    Garcia, Hernan G.; Phillips, Rob

    2011-01-01

    We present a quantitative case study of transcriptional regulation in which we carry out a systematic dialogue between theory and measurement for an important and ubiquitous regulatory motif in bacteria, namely, that of simple repression. This architecture is realized by a single repressor binding site overlapping the promoter. From the theory point of view, this motif is described by a single gene regulation function based upon only a few parameters that are convenient theoretically and accessible experimentally. The usual approach is turned on its side by using the mathematical description of these regulatory motifs as a predictive tool to determine the number of repressors in a collection of strains with a large variation in repressor copy number. The predictions and corresponding measurements are carried out over a large dynamic range in both expression fold change (spanning nearly four orders of magnitude) and repressor copy number (spanning about two orders of magnitude). The predictions are tested by measuring the resulting level of gene expression and are then validated by using quantitative immunoblots. The key outcomes of this study include a systematic quantitative analysis of the limits and validity of the inputoutput relation for simple repression, a precise determination of the in vivo binding energies for DNArepressor interactions for several distinct repressor binding sites, and a repressor census for Lac repressor in Escherichia coli. PMID:21730194

  6. 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; Kcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlgelhofer, 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

  7. Dissection of a Synthesized Quantitative Trait to Characterize Transgene Interactions

    PubMed Central

    Nap, J. P.; Conner, A. J.; Mlynarova, L.; Stiekema, W. J.; Jansen, R. C.

    1997-01-01

    Six transgenic tobacco lines, each homozygous for the ?-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits. PMID:9286691

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

    PubMed

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

    2015-06-01

    Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70, heat-shock-related 70 kDa protein 2; IDHc, cytoplasmic isocitrate dehydrogenase [NADP+]; MAPK1, mitogen-activated protein kinase 1; MEK1, MAP kinase kinase 1; MDHc, cytoplasmic malate dehydrogenase; NFM, neurofilament medium polypeptide; NSF, N-ethylmaleimide-sensitive fusion protein; PHB, prohibitin; PINK1, PTEN-induced putative kinase 1; PPIaseA, peptidyl-prolyl cis-trans isomerase A; PSA2, proteasome subunit alpha type-2; TK, transketolase; VDAC-2, voltage-dependent anion-selective channel protein 2. PMID:25626353

  9. Dissecting Dissection.

    ERIC Educational Resources Information Center

    AV Magazine, 1996

    1996-01-01

    This journal features articles covering various aspects of dissection. "Biology--The Study of Life" (George Russell) offers students experiments that do not require using invasive procedures. "Animal Cruelty--Behind the Scenes" (Zoe Weil) describes sources of laboratory animals. "Doing without Dissection" (Juliana Texley) discusses objections over…

  10. Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri

    PubMed Central

    Le Bihan, Thierry; Hindle, Matthew; Martin, Sarah F.; Barrios-Llerena, Martin E.; Krahmer, Johanna; Kis, Katalin; Millar, Andrew J.; van Ooijen, Gerben

    2015-01-01

    Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild-type and CK2-overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2-responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. PMID:25930153

  11. Label-free quantitative analysis of the casein kinase 2-responsive phosphoproteome of the marine minimal model species Ostreococcus tauri.

    PubMed

    Le Bihan, Thierry; Hindle, Matthew; Martin, Sarah F; Barrios-Llerena, Martin E; Krahmer, Johanna; Kis, Katalin; Millar, Andrew J; van Ooijen, Gerben

    2015-12-01

    Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild-type and CK2-overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2-responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975). PMID:25930153

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. Quantitative 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

    2016-01-01

    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. PMID:26476206

  15. Quantitative Tools for Dissection of Hydrogen-Producing Metabolic Networks-Final Report

    SciTech Connect

    Rabinowitz, Joshua D.; Dismukes, G.Charles.; Rabitz, Herschel A.; Amador-Noguez, Daniel

    2012-10-19

    During this project we have pioneered the development of integrated experimental-computational technologies for the quantitative dissection of metabolism in hydrogen and biofuel producing microorganisms (i.e. C. acetobutylicum and various cyanobacteria species). The application of these new methodologies resulted in many significant advances in the understanding of the metabolic networks and metabolism of these organisms, and has provided new strategies to enhance their hydrogen or biofuel producing capabilities. As an example, using mass spectrometry, isotope tracers, and quantitative flux-modeling we mapped the metabolic network structure in C. acetobutylicum. This resulted in a comprehensive and quantitative understanding of central carbon metabolism that could not have been obtained using genomic data alone. We discovered that biofuel production in this bacterium, which only occurs during stationary phase, requires a global remodeling of central metabolism (involving large changes in metabolite concentrations and fluxes) that has the effect of redirecting resources (carbon and reducing power) from biomass production into solvent production. This new holistic, quantitative understanding of metabolism is now being used as the basis for metabolic engineering strategies to improve solvent production in this bacterium. In another example, making use of newly developed technologies for monitoring hydrogen and NAD(P)H levels in vivo, we dissected the metabolic pathways for photobiological hydrogen production by cyanobacteria Cyanothece sp. This investigation led to the identification of multiple targets for improving hydrogen production. Importantly, the quantitative tools and approaches that we have developed are broadly applicable and we are now using them to investigate other important biofuel producers, such as cellulolytic bacteria.

  16. Quantitative phosphoproteomics reveals the protein tyrosine kinase Pyk2 as a central effector of olfactory receptor signaling in prostate cancer cells.

    PubMed

    Wiese, Heike; Gelis, Lian; Wiese, Sebastian; Reichenbach, Christa; Jovancevic, Nikolina; Osterloh, Markus; Meyer, Helmut E; Neuhaus, Eva M; Hatt, Hanns H; Radziwill, Gerald; Warscheid, Bettina

    2015-06-01

    The prostate-specific G-protein-coupled receptor 1 (PSGR1) is an olfactory receptor specifically expressed in the prostate gland. PSGR1 expression is elevated both in benign prostatic hyperplasia tissue and in prostate cancer. Stimulation of PSGR1 by the odorant β-ionone leads to an increase in the intracellular Ca(2+) concentration, activation of mitogen-activated protein (MAP) kinases and a decrease in prostate cancer cell proliferation. To further extend our knowledge about PSGR1 signaling in prostate cancer cells, we performed a quantitative phosphoproteomics study using stable isotope labeling by amino acids in cell culture and mass spectrometry. We report 51 differentially regulated phosphorylation sites in 24 proteins with functions in cytoskeletal remodeling, signaling and ion transport. Activation of PSGR1 evoked an increase in intracellular pH mediated by the sodium/hydrogen exchanger NHE1. Furthermore, we report the protein tyrosine kinase Pyk2 as a central effector of PSGR1 signaling cascades in LNCaP cells. Our data show that phosphorylation of p38 MAP kinase is triggered by Pyk2. In addition, we confirmed dephosphorylation of the tumor suppressor protein N-myc downstream regulated gene 1 (NDRG1) at Ser330 downstream of Pyk2. Since NDRG1 impacts oncogenic signaling pathways interfering with tumor progression, we suggest that the Pyk2-NDRG1 axis is possibly involved in conveying the anti-proliferative effect of β-ionone in prostate cancer cells. This article is part of a Special Issue entitled: Medical Proteomics. PMID:25219547

  17. The Lottia gigantea shell matrix proteome: re-analysis including MaxQuant iBAQ quantitation and phosphoproteome analysis

    PubMed Central

    2014-01-01

    Background Although the importance of proteins of the biomineral organic matrix and their posttranslational modifications for biomineralization is generally recognized, the number of published matrix proteomes is still small. This is mostly due to the lack of comprehensive sequence databases, usually derived from genomic sequencing projects. However, in-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may also be the most interesting candidates for further functional analysis. Results Re-quantification of previously identified Lottia shell matrix proteins using the intensity-based absolute quantification (iBAQ) method as implemented in the MaxQuant identification and quantitation software showed that only 57 of the 382 accepted identifications constituted 98% of the total identified matrix proteome. This group of proteins did not contain obvious intracellular proteins, such as cytoskeletal components or ribosomal proteins, invariably identified as minor components of high-throughput biomineral matrix proteomes. Fourteen of these major proteins were phosphorylated to a variable extent. All together we identified 52 phospho sites in 20 of the 382 accepted proteins with high confidence. Conclusions We show that iBAQ quantitation may be a useful tool to narrow down the group of functional biomineral matrix protein candidates for further research in cell biology, genetics or materials research. Knowledge of posttranslational modifications in these major proteins could be a valuable addition to previously published proteomes. This is true especially for phosphorylation, because this modification was already shown to modify mineralization processes in some instances. PMID:25018669

  18. Comprehensive Quantitative Comparison of the Membrane Proteome, Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells*

    PubMed Central

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue; Swistowski, Andrzej; Palmisano, Giuseppe; Engholm-Keller, Kasper; Jakobsen, Lene; Zeng, Xianmin; Larsen, Martin Røssel

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process. PMID:24173317

  19. Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM).

    PubMed

    Adachi, Jun; Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

    Mass spectrometry-based phosphoproteomics has been rapidly spread based on the advancement of mass spectrometry and development of efficient enrichment techniques for phosphorylated proteins or peptides. Non-targeted approach has been employed in most of the studies for phosphoproteome analysis. However, targeted approach using selected/multiple reaction monitoring (SRM/MRM) is an indispensible technique used for the quantitation of known targets especially when we have many samples to quantitate phosphorylation events on proteins in biological or clinical samples. We herein describe the application of a large-scale phosphoproteome analysis and SRM-based quantitation for the systematic discovery and validation of biomarkers. PMID:26700043

  20. The phosphoproteomics data explosion.

    PubMed

    Lemeer, Simone; Heck, Albert J R

    2009-10-01

    There are likely more than 500000 potential phosphorylation sites in a cellular proteome. This dynamic phosphorylation is under tight control of a variety of kinases and phosphatases. In recent years significant progress has been made in the large-scale analysis of these in vitro and in vivo protein phosphorylation events. Much of this data have been deposited in public depositories, which have described so far about 25000 phosphorylation sites on about 7000 human proteins. However, the amount of phosphoproteomics data generated is now growing tremendously at a rate of about 10000 sites per reported study. The major advances made in global phosphoproteomics analyses originate from technological advances, whereby the largest contributions are from more selective phosphopeptide affinity enrichment technologies and from faster, and more sensitive mass spectrometers. PMID:19620020

  1. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    PubMed

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  2. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a proteinprotein interaction filter revealed a total of 14 kinasesubstrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion. PMID:24043427

  3. Stable isotope metabolic labeling-based quantitative phosphoproteomic analysis of Arabidopsis mutants reveals ethylene-regulated time-dependent phosphoproteins and putative substrates of constitutive triple response 1 kinase.

    PubMed

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-12-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on (15)N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein-protein interaction filter revealed a total of 14 kinase-substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion. PMID:24043427

  4. Clinical and Technical Phosphoproteomic Research

    PubMed Central

    2011-01-01

    An encouraging approach for the diagnosis and effective therapy of immunological pathologies, which would include cancer, is the identification of proteins and phosphorylated proteins. Disease proteomics, in particular, is a potentially useful method for this purpose. A key role is played by protein phosphorylation in the regulation of normal immunology disorders and targets for several new cancer drugs and drug candidates are cancer cells and protein kinases. Protein phosphorylation is a highly dynamic process. The functioning of new drugs is of major importance as is the selection of those patients who would respond best to a specific treatment regime. In all major aspects of cellular life signalling networks are key elements which play a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. A whole range of networks that are involved in the regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses is contained in the latter. It is so necessary to understand and monitor kinase signalling pathways in order to understand many immunology pathologies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as immunoproteomic techniques, phosphoenrichments and mass spectrometry (MS) is crucial for the identification and quantification of protein phosphorylation sites in order to advance in clinical research. Pharmacodynamic readouts of disease states and cellular drug responses in tumour samples will be provided as the field develops. We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide enrichment and quantitative techniques need to be combined to achieve good phosphopeptide recovery and good up- and-down phospho-regulation protein studies. PMID:21635771

  5. Challenges in plasma membrane phosphoproteomics

    PubMed Central

    Orsburn, Benjamin C; Stockwin, Luke H; Newton, Dianne L

    2011-01-01

    The response to extracellular stimuli often alters the phosphorylation state of plasma membrane-associated proteins. In this regard, generation of a comprehensive membrane phosphoproteome can significantly enhance signal transduction and drug mechanism studies. However, analysis of this subproteome is regarded as technically challenging, given the low abundance and insolubility of integral membrane proteins, combined with difficulties in isolating, ionizing and fragmenting phosphopeptides. In this article, we highlight recent advances in membrane and phosphoprotein enrichment techniques resulting in improved identification of these elusive peptides. We also describe the use of alternative fragmentation techniques, and assess their current and future value to the field of membrane phosphoproteomics. PMID:21819303

  6. Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.

    PubMed

    Wakabayashi, Masaki; Yoshihara, Hiroki; Masuda, Takeshi; Tsukahara, Mai; Sugiyama, Naoyuki; Ishihama, Yasushi

    2014-02-01

    Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC-MS sensitivity by miniaturizing nanoLC columns to 25 ?m inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 5.4 ?g of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases. PMID:24328109

  7. Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

    PubMed

    Bai, Chen; Tang, Siwei; Bai, Chen; Chen, Xian

    2014-04-01

    The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy. PMID:24363202

  8. The Interplay of Light and Oxygen in the Reactive Oxygen Stress Response of Chlamydomonas reinhardtii Dissected by Quantitative Mass Spectrometry*

    PubMed Central

    Barth, Johannes; Bergner, Sonja Verena; Jaeger, Daniel; Niehues, Anna; Schulze, Stefan; Scholz, Martin; Fufezan, Christian

    2014-01-01

    Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redoxII was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O2 and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles. PMID:24482124

  9. Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

    PubMed Central

    Sigala, Paul A.; Fafarman, Aaron T.; Schwans, Jason P.; Fried, Stephen D.; Fenn, Timothy D.; Caaveiro, Jose M. M.; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A.; Boxer, Steven G.; Herschlag, Daniel

    2013-01-01

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

  10. Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site.

    PubMed

    Sigala, Paul A; Fafarman, Aaron T; Schwans, Jason P; Fried, Stephen D; Fenn, Timothy D; Caaveiro, Jose M M; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A; Boxer, Steven G; Herschlag, Daniel

    2013-07-01

    Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

  11. Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry.

    PubMed

    Ostasiewicz, Pawe?; Zielinska, Dorota F; Mann, Matthias; Wi?niewski, Jacek R

    2010-07-01

    Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time-the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications. PMID:20469934

  12. Combining Quantitative Genetics Approaches with Regulatory Network Analysis to Dissect the Complex Metabolism of the Maize Kernel.

    PubMed

    Wen, Weiwei; Liu, Haijun; Zhou, Yang; Jin, Min; Yang, Ning; Li, Dong; Luo, Jie; Xiao, Yingjie; Pan, Qingchun; Tohge, Takayuki; Fernie, Alisdair R; Yan, Jianbing

    2016-01-01

    Metabolic quantitative trait locus (QTL) studies have allowed us to better understand the genetic architecture underlying naturally occurring plant metabolic variance. Here, we use two recombinant inbred line (RIL) populations to dissect the genetic architecture of natural variation of 155 metabolites measured in the mature maize (Zea mays) kernel. Overall, linkage mapping identified 882 metabolic QTLs in both RIL populations across two environments, with an average of 2.1 QTLs per metabolite. A large number of metabolic QTLs (more than 65%) were identified with moderate effects (r(2) = 2.1%-10%), while a small portion (less than 35%) showed major effects (r(2) > 10%). Epistatic interactions between these identified loci were detected for more than 30% of metabolites (with the proportion of phenotypic variance ranging from 1.6% to 37.8%), implying that genetic epistasis is not negligible in determining metabolic variation. In total, 57 QTLs were validated by our previous genome-wide association study on the same metabolites that provided clues for exploring the underlying genes. A gene regulatory network associated with the flavonoid metabolic pathway was constructed based on the transcriptional variations of 28,769 genes in kernels (15 d after pollination) of 368 maize inbred lines. A large number of genes (34 of 58) in this network overlapped with previously defined genes controlled by maize PERICARP COLOR1, while three of them were identified here within QTL intervals for multiple flavonoids. The deeply characterized RIL populations, elucidation of metabolic phenotypes, and identification of candidate genes lay the foundation for maize quality improvement. PMID:26556794

  13. Combining Quantitative Genetics Approaches with Regulatory Network Analysis to Dissect the Complex Metabolism of the Maize Kernel1[OPEN

    PubMed Central

    Wen, Weiwei; Liu, Haijun; Yang, Ning; Luo, Jie; Xiao, Yingjie; Pan, Qingchun; Tohge, Takayuki; Fernie, Alisdair R.; Yan, Jianbing

    2016-01-01

    Metabolic quantitative trait locus (QTL) studies have allowed us to better understand the genetic architecture underlying naturally occurring plant metabolic variance. Here, we use two recombinant inbred line (RIL) populations to dissect the genetic architecture of natural variation of 155 metabolites measured in the mature maize (Zea mays) kernel. Overall, linkage mapping identified 882 metabolic QTLs in both RIL populations across two environments, with an average of 2.1 QTLs per metabolite. A large number of metabolic QTLs (more than 65%) were identified with moderate effects (r2 = 2.1%–10%), while a small portion (less than 35%) showed major effects (r2 > 10%). Epistatic interactions between these identified loci were detected for more than 30% of metabolites (with the proportion of phenotypic variance ranging from 1.6% to 37.8%), implying that genetic epistasis is not negligible in determining metabolic variation. In total, 57 QTLs were validated by our previous genome-wide association study on the same metabolites that provided clues for exploring the underlying genes. A gene regulatory network associated with the flavonoid metabolic pathway was constructed based on the transcriptional variations of 28,769 genes in kernels (15 d after pollination) of 368 maize inbred lines. A large number of genes (34 of 58) in this network overlapped with previously defined genes controlled by maize PERICARP COLOR1, while three of them were identified here within QTL intervals for multiple flavonoids. The deeply characterized RIL populations, elucidation of metabolic phenotypes, and identification of candidate genes lay the foundation for maize quality improvement. PMID:26556794

  14. Phosphoproteomics and Lung Cancer Research

    PubMed Central

    Lpez, Elena; Cho, William C. S.

    2012-01-01

    Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed. PMID:23202899

  15. Phosphoproteomics: new insights into cellular signaling

    PubMed Central

    Mumby, Marc; Brekken, Deirdre

    2005-01-01

    Developments in the field of phosphoproteomics have been fueled by the need simultaneously to monitor many different phosphoproteins within the signaling networks that coordinate responses to changes in the cellular environment. This article presents a brief review of phosphoproteomics with an emphasis on the biological insights that have been derived so far. PMID:16168091

  16. The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

    PubMed Central

    Treeck, Moritz; Sanders, John L.; Gaji, Rajshekhar Y.; LaFavers, Kacie A.; Child, Matthew A.; Arrizabalaga, Gustavo; Elias, Joshua E.; Boothroyd, John C.

    2014-01-01

    Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress. PMID:24945436

  17. Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-05-01

    Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

  18. Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics.

    PubMed

    Xue, Liang; Arrington, Justine V; Tao, W Andy

    2016-01-01

    Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein kinases have become potential drug targets for treating a wide variety of diseases. Identification of kinase substrates is vital not only for dissecting signaling pathways, but also for understanding disease pathologies and identifying novel therapeutic targets. However, identification of bona fide kinase substrates has remained challenging, necessitating the development of new methods and techniques. The kinase assay linked phosphoproteomics (KALIP) approach integrates in vitro kinase assays with global phosphoproteomics experiments to identify the direct substrates of protein kinases. This strategy has demonstrated outstanding sensitivity and a low false-positive rate for kinase substrate screening. PMID:26584932

  19. Chemical visualization of phosphoproteomes on membrane.

    PubMed

    Iliuk, Anton; Liu, X Shawn; Xue, Liang; Liu, Xiaoqi; Tao, W Andy

    2012-09-01

    With new discoveries of important roles of phosphorylation on a daily basis, phospho-specific antibodies, as the primary tool for on-membrane detection of phosphoproteins, face enormous challenges. To address an urgent need for convenient and reliable analysis of phosphorylation events, we report a novel strategy for sensitive phosphorylation analysis in the Western blotting format. The chemical reagent, which we termed pIMAGO, is based on a multifunctionalized soluble nanopolymer and is capable of selectively binding to phosphorylated residues independent of amino acid microenvironment, thus offering great promise as a universal tool in biological analyses where the site of phosphorylation is not known or its specific antibody is not available. The specificity and sensitivity of the approach was first examined using a mixture of standard proteins. The method was then applied to monitor phosphorylation changes in in vitro kinase and phosphatase assays. Finally, to demonstrate the unique ability of pIMAGO to measure endogenous phosphorylation, we used it to visualize and determine the differences in phosphorylated proteins that interact with wild-type and kinase dead mutant of Polo-like kinase 1 during mitosis, the results of which were further confirmed by a quantitative phosphoproteomics experiment. PMID:22593177

  20. Global Analysis of Neuronal Phosphoproteome Regulation by Chondroitin Sulfate Proteoglycans

    PubMed Central

    Yu, Panpan; Pisitkun, Trairak; Wang, Guanghui; Wang, Rong; Katagiri, Yasuhiro; Gucek, Marjan; Knepper, Mark A.; Geller, Herbert M.

    2013-01-01

    Chondroitin sulfate proteoglycans (CSPGs) are major components of the extracellular matrix which mediate inhibition of axonal regeneration after injury to the central nervous system (CNS). Several neuronal receptors for CSPGs have recently been identified; however, the signaling pathways by which CSPGs restrict axonal growth are still largely unknown. In this study, we applied quantitative phosphoproteomics to investigate the global changes in protein phosphorylation induced by CSPGs in primary neurons. In combination with isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling, strong cation exchange chromatography (SCX) fractionation, immobilized metal affinity chromatography (IMAC) and LC-MS/MS, we identified and quantified 2214 unique phosphopeptides corresponding to 1118 phosphoproteins, with 118 changing significantly in abundance with CSPG treatment. The proteins that were regulated by CSPGs included key components of synaptic vesicle trafficking, axon guidance mediated by semaphorins, integrin signaling, cadherin signaling and EGF receptor signaling pathways. A significant number of the regulated proteins are cytoskeletal and related proteins that have been implicated in regulating neurite growth. Another highly represented protein category regulated by CSPGs is nucleic acid binding proteins involved in RNA post-transcriptional regulation. Together, by screening the overall phosphoproteome changes induced by CSPGs, this data expand our understanding of CSPG signaling, which provides new insights into development of strategies for overcoming CSPG inhibition and promoting axonal regeneration after CNS injury. PMID:23527152

  1. 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. PMID:18550893

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

    PubMed Central

    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 liquid chromatography-tandem mass spectrometry (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 non-phosphopeptide 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 5,243 phosphopeptides containing 3,715 non-redundant phosphosites on 1,455 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. PMID:25307156

  3. Debating Dissection.

    ERIC Educational Resources Information Center

    Orlans, F. Barbara

    1988-01-01

    Argues the pros, cons, and alternatives to animal dissection. Discusses several cases involving student objections and the enactment of a California state law which upholds the right of a student to conscientious objection to dissection involving the harmful or destructive use of animals. (RT)

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

    PubMed

    Polat, Ayse Nur; Karayel, zge; Giese, Sven H; Harmanda, B?ra; Sanal, Erdem; Hu, Chi-Kuo; Renard, Bernhard Y; zl, Nurhan

    2015-09-01

    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. PMID:26270265

  5. 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

  6. 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

  7. PhosFox: a bioinformatics tool for peptide-level processing of LC-MS/MS-based phosphoproteomic data

    PubMed Central

    2014-01-01

    Background It is possible to identify thousands of phosphopeptides and proteins in a single experiment with mass spectrometry-based phosphoproteomics. However, a current bottleneck is the downstream data analysis which is often laborious and requires a number of manual steps. Results Toward automating the analysis steps, we have developed and implemented a software, PhosFox, which enables peptide-level processing of phosphoproteomic data generated by multiple protein identification search algorithms, including Mascot, Sequest, and Paragon, as well as cross-comparison of their identification results. The software supports both qualitative and quantitative phosphoproteomics studies, as well as multiple between-group comparisons. Importantly, PhosFox detects uniquely phosphorylated peptides and proteins in one sample compared to another. It also distinguishes differences in phosphorylation sites between phosphorylated proteins in different samples. Using two case study examples, a qualitative phosphoproteome dataset from human keratinocytes and a quantitative phosphoproteome dataset from rat kidney inner medulla, we demonstrate here how PhosFox facilitates an efficient and in-depth phosphoproteome data analysis. PhosFox was implemented in the Perl programming language and it can be run on most common operating systems. Due to its flexible interface and open source distribution, the users can easily incorporate the program into their MS data analysis workflows and extend the program with new features. PhosFox source code, implementation and user instructions are freely available from https://bitbucket.org/phintsan/phosfox. Conclusions PhosFox facilitates efficient and more in-depth comparisons between phosphoproteins in casecontrol settings. The open source implementation is easily extendable to accommodate additional features for widespread application use cases. PMID:25028575

  8. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog

    PubMed Central

    Fortuin, Suereta; Tomazella, Gisele G.; Nagaraj, Nagarjuna; Sampson, Samantha L.; Gey van Pittius, Nicolaas C.; Soares, Nelson C.; Wiker, Harald G.; de Souza, Gustavo A.; Warren, Robin M.

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. PMID:25713560

  9. Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea

    PubMed Central

    Bajaj, Deepak; Saxena, Maneesha S.; Kujur, Alice; Das, Shouvik; Badoni, Saurabh; Tripathi, Shailesh; Upadhyaya, Hari D.; Gowda, C. L. L.; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K.; Parida, Swarup K.

    2015-01-01

    Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5′-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea. PMID:25504138

  10. Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea.

    PubMed

    Bajaj, Deepak; Saxena, Maneesha S; Kujur, Alice; Das, Shouvik; Badoni, Saurabh; Tripathi, Shailesh; Upadhyaya, Hari D; Gowda, C L L; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K; Parida, Swarup K

    2015-03-01

    Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea. PMID:25504138

  11. Mass Spectrometry-Based Quantitative Proteomics for Dissecting Multiplexed Redox Cysteine Modifications in Nitric Oxide-Protected Cardiomyocyte Under Hypoxia

    PubMed Central

    Pan, Kuan-Ting; Chen, Yi-Yun; Pu, Tsung-Hsien; Chao, Yu-Shu; Yang, Chun-Yi; Bomgarden, Ryan D.; Rogers, John C.

    2014-01-01

    Abstract Aims: Distinctive states of redox-dependent cysteine (Cys) modifications are known to regulate signaling homeostasis under various pathophysiological conditions, including myocardial injury or protection in response to ischemic stress. Recent evidence further implicates a dynamic interplay among these modified forms following changes in cellular redox environment. However, a precise delineation of multiplexed Cys modifications in a cellular context remains technically challenging. To this end, we have now developed a mass spectrometry (MS)-based quantitative approach using a set of novel iodoacetyl-based Cys-reactive isobaric tags (irreversible isobaric iodoacetyl Cys-reactive tandem mass tag [iodoTMT]) endowed with unique irreversible Cys-reactivities. Results: We have established a sequential iodoTMT-switch procedure coupled with efficient immunoenrichment and advanced shotgun liquid chromatography-MS/MS analysis. This workflow allows us to differentially quantify the multiple redox-modified forms of a Cys site in the original cellular context. In one single analysis, we have identified over 260 Cys sites showing quantitative differences in multiplexed redox modifications from the total lysates of H9c2 cardiomyocytes experiencing hypoxia in the absence and presence of S-nitrosoglutathione (GSNO), indicative of a distinct pattern of individual susceptibility to S-nitrosylation or S-glutathionylation. Among those most significantly affected are proteins functionally implicated in hypoxic damage from which we showed that GSNO would protect. Innovation: We demonstrate for the first time how quantitative analysis of various Cys-redox modifications occurring in biological samples can be performed precisely and simultaneously at proteomic levels. Conclusion: We have not only developed a new approach to map global Cys-redoxomic regulation in vivo, but also provided new evidences implicating Cys-redox modifications of key molecules in NO-mediated ischemic cardioprotection. Antioxid. Redox Signal. 20, 13651381. PMID:24152285

  12. Quantitative dissection and modeling of the NF-?B p100-p105 module reveals interdependent precursor proteolysis.

    PubMed

    Y?lmaz, Zekiye Buket; Kofahl, Bente; Beaudette, Patrick; Baum, Katharina; Ipenberg, Inbal; Weih, Falk; Wolf, Jana; Dittmar, Gunnar; Scheidereit, Claus

    2014-12-11

    The mechanisms that govern proteolytic maturation or complete destruction of the precursor proteins p100 and p105 are fundamental to homeostasis and activation of NF-?B; however, they remain poorly understood. Using mass-spectrometry-based quantitative analysis of noncanonical LT?R-induced signaling, we demonstrate that stimulation induces simultaneous processing of both p100 and p105. The precursors not only form hetero-oligomers but also interact with the ATPase VCP/p97, and their induced proteolysis strictly depends on the signal response domain (SRD) of p100, suggesting that the SRD-targeting proteolytic machinery acts in cis and in trans. Separation of cellular pools by isotope labeling revealed synchronous dynamics of p105 and p100 proteolysis. The generation of p50 and p52 from their precursors depends on functional VCP/p97. We have developed quantitative mathematical models that describe the dynamics of the system and predict that p100-p105 complexes are signal responsive. PMID:25482563

  13. 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 ? 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 for TSLP/TSLPR-associated diseases in humans. PMID:22345495

  14. Dissecting quantitative trait variation in the resequencing era: complementarity of bi-parental, multi-parental and association panels.

    PubMed

    Pascual, Laura; Albert, Elise; Sauvage, Christopher; Duangjit, Janejira; Bouchet, Jean-Paul; Bitton, Frdrique; Desplat, Nelly; Brunel, Dominique; Le Paslier, Marie-Christine; Ranc, Nicolas; Bruguier, Laure; Chauchard, Betty; Verschave, Philippe; Causse, Mathilde

    2016-01-01

    Quantitative trait loci (QTL) have been identified using traditional linkage mapping and positional cloning identified several QTLs. However linkage mapping is limited to the analysis of traits differing between two lines and the impact of the genetic background on QTL effect has been underlined. Genome-wide association studies (GWAs) were proposed to circumvent these limitations. In tomato, we have shown that GWAs is possible, using the admixed nature of cherry tomato genomes that reduces the impact of population structure. Nevertheless, GWAs success might be limited due to the low decay of linkage disequilibrium, which varies along the genome in this species. Multi-parent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage and GWAs by increasing the precision of QTL mapping. We have developed a MAGIC population by crossing eight tomato lines whose genomes were resequenced. We showed the potential of the MAGIC population when coupled with whole genome sequencing to detect candidate single nucleotide polymorphisms (SNPs) underlying the QTLs. QTLs for fruit quality traits were mapped and related to the variations detected at the genome sequence and expression levels. The advantages and limitations of the three types of population, in the context of the available genome sequence and resequencing facilities, are discussed. PMID:26566830

  15. DISSECTION OF RNO18 BLOOD PRESSURE AND SALT SENSITIVITY QUANTITATIVE TRAIT LOCI IN THE SPONTANEOUSLY HYPERTENSIVE RAT

    PubMed Central

    Johnson, Michelle D.; He, Liqun; Herman, Daniel; Wakimoto, Hiroko; Wallace, Caroline A.; Zidek, Vaclav; Mlejnek, Petr; Musilova, Alena; Simakova, Miroslava; Vorlicek, Jaroslav; Kren, Vladimir; Viklicky, Ondrej; Qi, Nathan R.; Wang, Jiaming; Seidman, Christine E.; Seidman, J.G.; Kurtz, Theodore W.; Aitman, Timothy J.; Pravenec, Michal

    2014-01-01

    Hypertension in humans and experimental models has a strong hereditary basis, but identification of causative genes remains challenging. Quantitative trait loci (QTLs) for hypertension and salt sensitivity have been reported on rat chromosome 18. We set out to genetically isolate and prioritise genes within the salt sensitivity and hypertension QTLs on the spontaneously hypertensive rat (SHR) chromosome 18, by developing and characterising a series of congenic strains derived from the SHR and normotensive Brown Norway (BN) rat strains. The SHR.BN-D18Rat113/D18Rat82 (SHR-18) congenic strain exhibits significantly lower blood pressure and is salt-resistant compared to SHR. Transplantation of kidneys from SHR-18 donors into SHR recipients is sufficient to attenuate increased blood pressure but not salt sensitivity. Derivation of congenic sublines allowed separation of salt sensitivity from hypertension QTL regions. Renal expression studies with microarray and Solexa-based sequencing in parental and congenic strains identified four differentially expressed genes within the hypertension QTL region, one of which is an unannotated transcript encoding a previously undescribed, small non-coding RNA. Sequencing selected biological candidate genes within the minimal congenic interval revealed a non-synonymous variant in SHR Transcription factor 4. The minimal congenic interval is syntenic to a region of human chromosome 18 where significant linkage to hypertension was observed in family-based linkage studies. These congenic lines provide reagents for identifying causative genes that underlie the chromosome 18 SHR QTLs for hypertension and salt sensitivity. Candidate genes identified in these studies merit further investigation as potentially causative hypertension genes in SHR and human hypertension. PMID:19620519

  16. Computational phosphoproteomics: From identification to localization

    PubMed Central

    Lee, Dave C H; Jones, Andrew R; Hubbard, Simon J

    2015-01-01

    Analysis of the phosphoproteome by MS has become a key technology for the characterization of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable side chain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or localize the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards. PMID:25475148

  17. Systems Analysis for Interpretation of Phosphoproteomics Data.

    PubMed

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

    2016-01-01

    Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. Systems level analysis provides the best means for extracting functional insights from such types of datasets, and this has primed a rapid development of bioinformatics tools and resources over the last decade. Many of these tools are specialized databases that can be mined for annotation and pathway enrichment, whereas others provide a platform to generate functional protein networks and explore the relations between proteins of interest. The use of these tools requires careful consideration with regard to the input data, and the interpretation demands a critical approach. This chapter provides a summary of the most appropriate tools for systems analysis of phosphoproteomics datasets, and the considerations that are critical for acquiring meaningful output. PMID:26584937

  18. Polyomino Dissections

    ERIC Educational Resources Information Center

    Hohn, Tiina; Liu, Andy

    2012-01-01

    One of Gardner's passions was to introduce puzzles into the classroom. From this point of view, polyomino dissections are an excellent topic. They require little background, provide training in geometric visualization, and mostly they are fun. In this article, we put together a large collection of such puzzles, introduce a new approach in solving

  19. Genetic dissection of milk yield traits and mastitis resistance quantitative trait loci on chromosome 20 in dairy cattle.

    PubMed

    Kadri, Naveen K; Guldbrandtsen, Bernt; Lund, Mogens S; Sahana, Goutam

    2015-12-01

    Intense selection to increase milk yield has had negative consequences for mastitis incidence in dairy cattle. Due to low heritability of mastitis resistance and an unfavorable genetic correlation with milk yield, a reduction in mastitis through traditional breeding has been difficult to achieve. Here, we examined quantitative trait loci (QTL) that segregate for clinical mastitis and milk yield on Bos taurus autosome 20 (BTA20) to determine whether both traits are affected by a single polymorphism (pleiotropy) or by multiple closely linked polymorphisms. In the latter but not the former situation, undesirable genetic correlation could potentially be broken by selecting animals that have favorable variants for both traits. First, we performed a within-breed association study using a haplotype-based method in Danish Holstein cattle (HOL). Next, we analyzed Nordic Red dairy cattle (RDC) and Danish Jersey cattle (JER) with the goal of determining whether these QTL identified in Holsteins were segregating across breeds. Genotypes for 12,566 animals (5,966 HOL, 5,458 RDC, and 1,142 JER) were determined by using the Illumina Bovine SNP50 BeadChip (50K; Illumina, San Diego, CA), which identifies 1,568 single nucleotide polymorphisms on BTA20. Data were combined, phased, and clustered into haplotype states, followed by within- and across-breed haplotype-based association analyses using a linear mixed model. Association signals for both clinical mastitis and milk yield peaked in the 26- to 40-Mb region on BTA20 in HOL. Single-variant association analyses were carried out in the QTL region using whole sequence level variants imputed from references of 2,036 HD genotypes (BovineHD BeadChip; Illumina) and 242 whole-genome sequences. The milk QTL were also segregating in RDC and JER on the BTA20-targeted region; however, an indication of differences in the causal factor(s) was observed across breeds. A previously reported F279Y mutation (rs385640152) within the growth hormone receptor gene showed strong association with milk, fat, and protein yields. In HOL, the highest peaks for milk yield and susceptibility to mastitis were separated by over 3.5 Mb (3.8 Mb by haplotype analysis, 3.6 Mb by single nucleotide polymorphism analysis), suggesting separate genetic variants for the traits. Further analysis yielded 2 candidate mutations for the mastitis QTL, at 33,642,072 bp (rs378947583) in an intronic region of the caspase recruitment domain protein 6 gene and 35,969,994 bp (rs133596506) in an intronic region of the leukemia-inhibitory factor receptor gene. These findings suggest that it may be possible to separate these beneficial and detrimental genetic factors through targeted selective breeding. PMID:26409972

  20. Phosphoproteomics by Mass Spectrometry: insights, implications, applications, and limitations

    PubMed Central

    Mayya, Viveka; Han, David K.

    2010-01-01

    Summary Phosphorylation of proteins is a predominant reversible post-translational modification. It is central to a wide variety of physiological responses and signaling mechanisms. Recent advances have allowed the global scope of phosphorylation to be addressed by mass spectrometry using phosphoproteomic approaches. In this perspective we discuss four aspects of phosphoproteomics; namely insights and implications from recently published phosphoproteomic studies, and applications and limitations of current phosphoproteomic strategies. As about 50,000 known phosphorylation sites do not yet have any ascribed function, we present our perspectives on a major function of protein phosphorylation that may be of predictive value in hypothesis based investigations. Finally we discuss strategies to measure stoichiometry of phosphorylation in a proteome-wide manner which is not provided by current phosphoproteomic approaches. PMID:19929607

  1. Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors

    PubMed Central

    2013-01-01

    Background Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma. Results Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (> 100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways. Conclusion This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network. PMID:23628362

  2. Sample Preparation for Phosphoproteomic Analysis of Circadian Time Series in Arabidopsis thaliana

    PubMed Central

    Krahmer, Johanna; Hindle, Matthew M.; Martin, Sarah F.; Le Bihan, Thierry; Millar, Andrew J.

    2015-01-01

    Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract. PMID:25662467

  3. Analysis of the Candida albicans Phosphoproteome

    PubMed Central

    Willger, S. D.; Liu, Z.; Olarte, R. A.; Adamo, M. E.; Myers, L. C.; Kettenbach, A. N.

    2015-01-01

    Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins. PMID:25750214

  4. 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

  5. Two Birds with One Stone: Parallel Quantification of Proteome and Phosphoproteome Using iTRAQ.

    PubMed

    Solari, Fiorella A; Kollipara, Laxmikanth; Sickmann, Albert; Zahedi, Ren P

    2016-01-01

    Altered and abnormal levels of proteins and their phosphorylation states are associated with many disorders. Detection and quantification of such perturbations may provide a better understanding of pathological conditions and help finding candidates for treatment or biomarkers. Over the years, isobaric mass tags for relative quantification of proteins and protein phosphorylation by mass spectrometry have become increasingly popular. One of the most commonly used isobaric chemical tags is iTRAQ (isobaric tag for relative and absolute quantitation). In a typical iTRAQ-8plex experiment, a multiplexed sample amounts for up to 800 ?g of peptides. Using state-of-the-art LC-MS approaches, only a fraction (~5 %) of such a sample is required to generate comprehensive quantitative data on the global proteome level, so that the bulk of the sample can be simultaneously used for quantitative phosphoproteomics. Here, we provide a simple and straightforward protocol to perform quantitative analyses of both proteome and phosphoproteome from the same sample using iTRAQ. PMID:26700039

  6. Dissected Surface

    NASA Technical Reports Server (NTRS)

    2005-01-01

    [figure removed for brevity, see original site] Context image for PIA03286 Dissected Surface

    Small channels dissect this region near Nectaris Fossae.

    Image information: VIS instrument. Latitude -22.9N, Longitude 17.4E. 17 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  7. Biology teachers' dissection practices and the influences that lead to their adoption: An exploratory research

    NASA Astrophysics Data System (ADS)

    Milano, Regina Nicole

    The lack of resolution in the on-going animal dissection debate inspired this mixed methods study to identify Connecticut secondary biology teachers' dissection practices and the influences that lead to their adoption. Qualitative findings indicate past experiences, managing objections to dissection, school culture, goals of biology teaching and ethics as major influences on dissection practices with 58.4% (n=7) of the sample dissecting and 41.6% not dissecting (n=5). Quantitative findings reveal gender, standards and curriculum, advantages of dissection and experiences as a student as major influences on dissection practices with 71.9% (n=92) of the sample dissecting and 28.1% (n=36) not dissecting. The study concludes that dissection policies are necessary and imminent in Connecticut school districts. Furthermore, it advises teacher-initiated, qualitative and quantitative assessments to expose disparities between student dissection perspectives and their own, prior to conducting dissection. Finally, it provides suggestions for addressing potential differences including administrative involvement.

  8. Investigation of phosphoproteome in RAGE signaling.

    PubMed

    Batkulwar, Kedar B; Bansode, Sneha B; Patil, Gouri V; Godbole, Rashmi K; Kazi, Rubina S; Chinnathambi, Subashchandrabose; Shanmugam, Dhanasekaran; Kulkarni, Mahesh J

    2015-01-01

    The receptor for advanced glycation end products (RAGE) is one of the most important proteins implicated in diabetes, cardiovascular diseases, neurodegenerative diseases, and cancer. It is a pattern recognition receptor by virtue of its ability to interact with multiple ligands, RAGE activates several signal transduction pathways through involvement of various kinases that phosphorylate their respective substrates. Only few substrates have been known to be phosphorylated in response to activation by RAGE (e.g., nuclear factor kappa B); however, it is possible that these kinases can phosphorylate multiple substrates depending upon their expression and localization, leading to altered cellular responses in different cell types and conditions. One such example is, glycogen synthase kinase 3 beta which is known to phosphorylate glycogen synthase, acts downstream to RAGE, and hyperphosphorylates microtubule-associated protein tau causing neuronal damage. Thus, it is important to understand the role of various RAGE-activated kinases and their substrates. Therefore, we have reviewed here the details of RAGE-activated kinases in response to different ligands and their respective phosphoproteome. Furthermore, we discuss the analysis of the data mined for known substrates of these kinases from the PhosphoSitePlus (http://www.phosphosite.org) database, and the role of some of the important substrates involved in cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases. In summary, this review provides information on RAGE-activated kinases and their phosphoproteome, which will be helpful in understanding the possible role of RAGE and its ligands in progression of diseases. PMID:25315903

  9. Toward defining the phosphoproteome of Xenopus laevis embryos

    PubMed Central

    McGivern, Jered V.; Swaney, Danielle L.; Coon, Joshua J.; Sheets, Michael D.

    2010-01-01

    Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. PMID:19384857

  10. Integrating Phosphoproteome and Transcriptome Reveals New Determinants of Macrophage Multinucleation*

    PubMed Central

    Rotival, Maxime; Ko, Jeong-Hun; Srivastava, Prashant K.; Kerloc'h, Audrey; Montoya, Alex; Mauro, Claudio; Faull, Peter; Cutillas, Pedro R.; Petretto, Enrico; Behmoaras, Jacques

    2015-01-01

    Macrophage multinucleation (MM) is essential for various biological processes such as osteoclast-mediated bone resorption and multinucleated giant cell-associated inflammatory reactions. Here we study the molecular pathways underlying multinucleation in the rat through an integrative approach combining MS-based quantitative phosphoproteomics (LC-MS/MS) and transcriptome (high-throughput RNA-sequencing) to identify new regulators of MM. We show that a strong metabolic shift toward HIF1-mediated glycolysis occurs at transcriptomic level during MM, together with modifications in phosphorylation of over 50 proteins including several ARF GTPase activators and polyphosphate inositol phosphatases. We use shortest-path analysis to link differential phosphorylation with the transcriptomic reprogramming of macrophages and identify LRRFIP1, SMARCA4, and DNMT1 as novel regulators of MM. We experimentally validate these predictions by showing that knock-down of these latter reduce macrophage multinucleation. These results provide a new framework for the combined analysis of transcriptional and post-translational changes during macrophage multinucleation, prioritizing essential genes, and revealing the sequential events leading to the multinucleation of macrophages. PMID:25532521

  11. Titchener's ? dissected.

    PubMed

    Landwehr, Klaus

    2015-08-01

    In three experiments, two independent samples of 12 observers each visually inspected modified versions of Titchener's ? from which the T-junctions had been deleted. For Experiment 1, the ?'s two lines had been replaced by dashed lines not meeting in a common point; for Experiment 2, the ? had been reduced to five dots, representing the original lines' end- and midpoints; and for Experiment 3 (in which the second sample of observers served), the ? had been dissected into two separate lines, differently spaced from each other. Observers haptically indicated the lengths of the two orthogonal lines of the modified ?s and verbally judged their relative lengths or the distances between the corresponding dots. The common perceptual illusions persisted in Experiments 1 and 2, but were markedly weakened in Experiment 3. Implications for a neurophysiological account of the illusions in terms of bottom-up, long-range interactions between orientation-sensitive mechanisms versus top-down activation of a figural schema are spelled out. PMID:25893471

  12. 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. PMID:25999967

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

    PubMed Central

    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. PMID:25999967

  14. The phosphoproteome of human Jurkat T cell clones upon costimulation with anti-CD3/anti-CD28 antibodies.

    PubMed

    Nguyen, Tien Dung; Carrascal, Montserrat; Vidal-Cortes, Oriol; Gallardo, Oscar; Casas, Vanessa; Gay, Marina; Phan, Van Chi; Abian, Joaquin

    2016-01-10

    Phosphorylation is a reversible post-translational modification, playing a vital role in protein function. In T cells, protein phosphorylation is the key mechanism regulating T cell receptor-driven signaling pathways. In order to gain insights into the phosphoproteome evolution of T cell activation, we performed a large-scale quantitative phosphoproteomics study of Jurkat E6.1 (wild type) and Jurkat gamma1 (Phospholipase gamma1 null) cell clones upon costimulation with anti-CD3 and anti-CD28 antibodies at times ranging from 15min to as long as 120min. In total, we identified 5585 phosphopeptides belonging to 2008 phosphoproteins from both cell clones. We detected 130 and 114 novel phosphopeptides in Jurkat E6.1 and Jurkat gamma1 clones, respectively. A significantly lower number of proteins containing regulated phosphorylation sites were identified in Jurkat gamma1 in comparison to Jurkat E6.1, reflecting the vital role of Phospholipase gamma1 in T cell signaling. Several new phosphorylation sites from lymphocyte-specific protein tyrosine kinase (Lck) were identified. Of these, serine-121 showed significant changes in JE6.1 while only small changes in the Jgamma1 clone. Our data may contribute to the current human T cell phosphoproteome and provide a better understanding on T cell receptor signaling. Data are available via ProteomeXchange with identifier PXD002871. PMID:26546556

  15. 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

  16. Specific Visualization and Identification of Phosphoproteome in Gels

    PubMed Central

    2015-01-01

    The applicability of gel-based proteomic strategies in phosphoproteomics has been largely limited by the lack of technologies for specific detection of phosphoproteins in gels. Here for the first time we report a strategy for simultaneous visualization and identification of phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies. PMID:24941108

  17. Dissecting Classroom Ethics.

    ERIC Educational Resources Information Center

    Allchin, Douglas

    1991-01-01

    Described are activities that lead to values clarification. Issues such as dissection, bioengineering, birth control, medical resources, and death are discussed. Included is a student questionnaire on the subject of dissection and the use of animals in laboratories. (KR)

  18. The Problems of Dissection.

    ERIC Educational Resources Information Center

    Davis, Pat

    1997-01-01

    Describes some problems of classroom dissection including the cruelty that animals destined for the laboratory suffer. Discusses the multilevel approach that the National Anti-Vivisection Society (NAVS) has developed to address the problems of animal dissection such as offering a dissection hotline, exhibiting at science teacher conferences, and…

  19. The Problems of Dissection.

    ERIC Educational Resources Information Center

    Davis, Pat

    1997-01-01

    Describes some problems of classroom dissection including the cruelty that animals destined for the laboratory suffer. Discusses the multilevel approach that the National Anti-Vivisection Society (NAVS) has developed to address the problems of animal dissection such as offering a dissection hotline, exhibiting at science teacher conferences, and

  20. Localization of Candidatus Liberibacter asiaticus in dissected organs of its psyllid vector Diaphorina citri using fluorescent in situ hybridization and quantitative PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vector interactions of huanglongbing (HLB) disease with its psyllid vectors, particularly at the organ and cellular levels, are poorly understood. We used fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR) for the localization of Candidatus Liberibacter asiaticus (CLas), associat...

  1. 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

  2. A chemical and phosphoproteomic characterization of dasatinib action in lung cancer

    PubMed Central

    Li, Jiannong; Rix, Uwe; Fang, Bin; Bai, Yun; Edwards, Arthur; Colinge, Jacques; Bennett, Keiryn L.; Gao, Jingchun; Song, Lanxi; Eschrich, Steven; Superti-Furga, Giulio; Koomen, John; Haura, Eric B.

    2010-01-01

    We describe a strategy to comprehend signaling pathways active in lung cancer cells and targeted by dasatinib employing chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SFK members (LYN, SRC, FYN, LCK, YES), non-receptor tyrosine kinases (FRK, BRK, ACK), and receptor tyrosine kinases (Ephrin receptors, DDR1, EGFR). Using quantitative phosphoproteomics we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug resistant gatekeeper mutants, we show that SFK kinases, particularly SRC and FYN, as well as EGFR are relevant targets for dasatinib action. The combined mass spectrometry based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and rationale combinatorial therapeutic strategies. PMID:20190765

  3. Phosphoproteomics reveals distinct modes of Mec1/ATR signaling during DNA replication.

    PubMed

    Bastos de Oliveira, Francisco Meirelles; Kim, Dongsung; Cussiol, Jos Renato; Das, Jishnu; Jeong, Min Cheol; Doerfler, Lillian; Schmidt, Kristina Hildegard; Yu, Haiyuan; Smolka, Marcus Bustamante

    2015-03-19

    The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their invivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in asystematic, unbiased, and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1's activation state induced by replication stress. This "replication-correlated" mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor and is distinguishable from Mec1's action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress. PMID:25752575

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

    PubMed

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

    2013-09-01

    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 comprehensive (phospho)proteomics resource for studying signaling from the point of view of both host and pathogen during a plant-pathogen interaction. PMID:23722186

  5. 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.; Jrgensen, 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 wheatS. 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, 143-3 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 comprehensive (phospho)proteomics resource for studying signaling from the point of view of both host and pathogen during a plantpathogen interaction. PMID:23722186

  6. Dissecting heart failure.

    PubMed

    Verma, Sameer; Gupta, Sameer; Guglin, Maya

    2014-06-01

    Dissection of ascending aorta is a medical emergency typically presenting with acute chest or back pain and hemodynamic instability. We are reporting a very unusual case of dissection of a large ascending aortic aneurysm presenting as a new onset heart failure. A 46-year-old man presented with gradually increasing dyspnea and orthopnea. His physical examination and laboratory findings were consistent with heart failure. The only unusual feature was a diastolic murmur, which prompted echocardiographic evaluation. Besides left ventricular dilatation, hypertrophy, and severe global hypokinesis, which were expected, we also found severely dilated aortic root with aortic regurgitation and a 8.69.7 cm ascending aortic aneurysm with dissection. The patient had a brother who died several years earlier from aortic dissection. Surgical treatment was successful. Type A aortic dissection may rarely present as heart failure. Aortic dissection at young age should prompt screening of first-degree relatives because genetic nature of the disease is very likely. PMID:24418447

  7. A consensus map of rapeseed (Brassica napus L.) based on diversity array technology markers: applications in genetic dissection of qualitative and quantitative traits

    PubMed Central

    2013-01-01

    Background Dense consensus genetic maps based on high-throughput genotyping platforms are valuable for making genetic gains in Brassica napus through quantitative trait locus identification, efficient predictive molecular breeding, and map-based gene cloning. This report describes the construction of the first B. napus consensus map consisting of a 1,359 anchored array based genotyping platform; Diversity Arrays Technology (DArT), and non-DArT markers from six populations originating from Australia, Canada, China and Europe. We aligned the B. napus DArT sequences with genomic scaffolds from Brassica rapa and Brassica oleracea, and identified DArT loci that showed linkage with qualitative and quantitative loci associated with agronomic traits. Results The integrated consensus map covered a total of 1,987.2?cM and represented all 19 chromosomes of the A and C genomes, with an average map density of one marker per 1.46?cM, corresponding to approximately 0.88 Mbp of the haploid genome. Through in silico physical mapping 2,457 out of 3,072 (80%) DArT clones were assigned to the genomic scaffolds of B. rapa (A genome) and B. oleracea (C genome). These were used to orientate the genetic consensus map with the chromosomal sequences. The DArT markers showed linkage with previously identified non-DArT markers associated with qualitative and quantitative trait loci for plant architecture, phenological components, seed and oil quality attributes, boron efficiency, sucrose transport, male sterility, and race-specific resistance to blackleg disease. Conclusions The DArT markers provide increased marker density across the B. napus genome. Most of the DArT markers represented on the current array were sequenced and aligned with the B. rapa and B. oleracea genomes, providing insight into the Brassica A and C genomes. This information can be utilised for comparative genomics and genomic evolution studies. In summary, this consensus map can be used to (i) integrate new generation markers such as SNP arrays and next generation sequencing data; (ii) anchor physical maps to facilitate assembly of B. napus genome sequences; and (iii) identify candidate genes underlying natural genetic variation for traits of interest. PMID:23617817

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

    PubMed Central

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

    2015-01-01

    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 0h and 24h after Xoo infection. 2367 and 2223 phosphosites on 1334 and 1297 representative proteins were identified in 0h and 24h 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.

  9. 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. PMID:26862573

  10. "Hands-Off" Dissection?

    ERIC Educational Resources Information Center

    Allchin, Douglas

    2005-01-01

    Computer programs and models are used to express respect for life by not sacrificing any animal but these alternatives might be deeply flawed. Alternatives to dissection are perverse alternatives that tend to preserve the features of inappropriate dissections like destructiveness, reductionism and objectification.

  11. Cellular Quantitative StructureActivity Relationship (Cell-QSAR): Conceptual Dissection of Receptor Binding and Intracellular Disposition in Antifilarial Activities of Selwood Antimycins

    PubMed Central

    2012-01-01

    We present the cellular quantitative structureactivity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drugs lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition. PMID:22468611

  12. Phosphoproteomics Study Based on In Vivo Inhibition Reveals Sites of Calmodulin‐Dependent Protein Kinase II Regulation in the Heart

    PubMed Central

    Scholten, Arjen; Preisinger, Christian; Corradini, Eleonora; Bourgonje, Vincent J.; Hennrich, Marco L.; van Veen, Toon A. B.; Swaminathan, Paari D.; Joiner, Mei‐Ling; Vos, Marc A.; Anderson, Mark E.; Heck, Albert J. R.

    2013-01-01

    Background The multifunctional Ca2+‐ and calmodulin‐dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is large‐scale analysis of phosphorylation events by mass spectrometry. However, current large‐scale phosphoproteomics approaches have proved inadequate for high‐fidelity identification of kinase‐specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue. Methods and Results To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial‐delimited expression of the specific peptide inhibitor of CaMKII (AC3‐I) or an inactive control (AC3‐C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of β‐adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3‐I and AC3‐C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high‐resolution LC‐MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3‐I‐mediated CaMKII inhibition. Conclusions Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling. PMID:23926118

  13. Global impact of Salmonella pathogenicity island 2-secreted effectors on the host phosphoproteome.

    PubMed

    Imami, Koshi; Bhavsar, Amit P; Yu, Hongbing; Brown, Nat F; Rogers, Lindsay D; Finlay, B Brett; Foster, Leonard J

    2013-06-01

    During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated. PMID:23459991

  14. Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response1[W][OPEN

    PubMed Central

    E. Stecker, Kelly; Minkoff, Benjamin B.; Sussman, Michael R.

    2014-01-01

    Elucidating how plants sense and respond to water loss is important for identifying genetic and chemical interventions that may help sustain crop yields in water-limiting environments. Currently, the molecular mechanisms involved in the initial perception and response to dehydration are not well understood. Modern mass spectrometric methods for quantifying changes in the phosphoproteome provide an opportunity to identify key phosphorylation events involved in this process. Here, we have used both untargeted and targeted isotope-assisted mass spectrometric methods of phosphopeptide quantitation to characterize proteins in Arabidopsis (Arabidopsis thaliana) whose degree of phosphorylation is rapidly altered by hyperosmotic treatment. Thus, protein phosphorylation events responsive to 5 min of 0.3 m mannitol treatment were first identified using 15N metabolic labeling and untargeted mass spectrometry with a high-resolution ion-trap instrument. The results from these discovery experiments were then validated using targeted Selected Reaction Monitoring mass spectrometry with a triple quadrupole. Targeted Selected Reaction Monitoring experiments were conducted with plants treated under nine different environmental perturbations to determine whether the phosphorylation changes were specific for osmosignaling or involved cross talk with other signaling pathways. The results indicate that regulatory proteins such as members of the mitogen-activated protein kinase family are specifically phosphorylated in response to osmotic stress. Proteins involved in 5′ messenger RNA decapping and phosphatidylinositol 3,5-bisphosphate synthesis were also identified as targets of dehydration-induced phosphoregulation. The results of these experiments demonstrate the utility of targeted phosphoproteomic analysis in understanding protein regulation networks and provide new insight into cellular processes involved in the osmotic stress response. PMID:24808101

  15. 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 spectrometrybased 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

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

    PubMed

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

    2016-05-01

    Reversible phosphorylation is one of the most important post-translational modifications in mammalian cells. Because this molecular switch is an important mechanism that diversifies and regulates proteins in cellular processes, knowledge about the extent and quantity of phosphorylation is very important to understand the complex cellular interplay. Although phosphoproteomics strategies are applied worldwide, they mainly include only molecular mass spectrometry (like MALDI or ESI)-based experiments. Although identification and relative quantification of phosphopeptides is straightforward with these techniques, absolute quantification is more complex and usually requires for specific isotopically phosphopeptide standards. However, the use of elemental mass spectrometry, and in particular inductively coupled plasma mass spectrometry (ICP-MS), in phosphoproteomics-based experiments, allow one to absolutely quantify phosphopeptides. Here, these phosphoproteomic applications with ICP-MS as elemental detector are reviewed. Pioneering work and recent developments in the field are both described. Additionally, the advantage of the parallel use of molecular and elemental mass spectrometry is stressed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:350-360, 2016. PMID:25139451

  17. Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

    PubMed Central

    Hoover-Plow, Jane; Sa, Qila; Huang, Menggui; Grondolsky, Jessica

    2013-01-01

    Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11. PMID:24147020

  18. Postpartum coronary artery dissection.

    PubMed Central

    Shaver, P J; Carrig, T F; Baker, W P

    1978-01-01

    A 27-year-old woman experienced anterior myocardial infarction three weeks after the delivery of her second child. Coronary arteriography subsequently showed primary dissection of the left coronary artery. This patient is believed to be the second reported survivor of angiographically proven peripartal left coronary artery dissection and the only such patient to achieve and maintain asymptomatic status for a prolonged period without operative intervention. Images PMID:626669

  19. Exploring G protein-coupled receptor signaling networks using SILAC-based phosphoproteomics.

    PubMed

    Williams, Grace R; Bethard, Jennifer R; Berkaw, Mary N; Nagel, Alexis K; Luttrell, Louis M; Ball, Lauren E

    2016-01-01

    The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry and bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation. PMID:26160508

  20. Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

    PubMed

    Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

    Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples. PMID:26584920

  1. Is dissection humane?

    PubMed

    Hasan, Tabinda

    2011-01-01

    Dissection is being jeopardized in the modern medical education. It has unrelentingly faced the lashes of time and has been the scapegoat for numerous convenient curricula reforms and subjective biases. The cadaver is unparallel in establishing core knowledge among the medical community and it needs to be appreciated in a new light in the "cyber anatomy" realm of today. This article elucidates the medical and ethical validity of continuing human body dissection in medicine which outweighs all the prejudices associated with it. PMID:23908746

  2. Is dissection humane?

    PubMed Central

    Hasan, Tabinda

    2011-01-01

    Dissection is being jeopardized in the modern medical education. It has unrelentingly faced the lashes of time and has been the scapegoat for numerous convenient curricula reforms and subjective biases. The cadaver is unparallel in establishing core knowledge among the medical community and it needs to be appreciated in a new light in the cyber anatomy realm of today. This article elucidates the medical and ethical validity of continuing human body dissection in medicine which outweighs all the prejudices associated with it. PMID:23908746

  3. 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. PMID:26584936

  4. Two Dimensional Gel Electrophoresis-Based Plant Phosphoproteomics.

    PubMed

    Han, Chao; Yang, Pingfang

    2016-01-01

    Phosphorylation is one of the most important reversible protein modifications and is involved in regulating signal transduction, subcellular localization and enzyme activity of target proteins. Phosphorylation or dephosphorylation of proteins is directly reflected in changed ratios of phosphoprotein abundance and total protein abundance. Two-dimensional gel electrophoresis (2-DE)-based proteomics allow quantification of both total protein abundance by Coomassie Brilliant Blue (CBB) staining and phosphoprotein abundance by fluorescence-based staining. Pro-Q diamond phosphoprotein stain (Pro-Q DPS) can bind to the phosphate moiety of the phospho-amino acid directly, regardless of the nature of the phospho-amino acid. Phosphoproteins can thus be detected using proper excitation light, quantified using image analysis software and subsequently be subjected to analysis by mass spectrometry. Here, we describe a protein phosphorylation status analysis method combining both CBB and Pro-Q DPS staining based on 2-DE gel-based phosphoproteomics, which has been widely applied to plant phosphoproteomics studies. PMID:26584928

  5. Breast Cancer Proteomic and Phosphoproteomic Data Released - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have released a dataset of proteins and phophorylated phosphopeptides identified through deep proteomic and phosphoproteomic analysis of breast tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).

  6. Dissecting Diversity Part II

    ERIC Educational Resources Information Center

    Matthews, Frank

    2005-01-01

    This article presents "Dissecting Diversity, Part II," the conclusion of a wide-ranging two-part roundtable discussion on diversity in higher education. The participants were as follows: Lezli Baskerville, J.D., President and CEO of the National Association for Equal Opportunity (NAFEO); Dr. Gerald E. Gipp, Executive Director of the American

  7. Dissecting Diversity Part II

    ERIC Educational Resources Information Center

    Matthews, Frank

    2005-01-01

    This article presents "Dissecting Diversity, Part II," the conclusion of a wide-ranging two-part roundtable discussion on diversity in higher education. The participants were as follows: Lezli Baskerville, J.D., President and CEO of the National Association for Equal Opportunity (NAFEO); Dr. Gerald E. Gipp, Executive Director of the American…

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

    PubMed Central

    Bonhomme, Ludovic; Valot, Benot; Tardieu, Franois; 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 plant water status, and their possible involvement in plant growth-related processes. PMID:22787273

  9. Comparative phosphoproteomics reveals evolutionary and functional conservation of phosphorylation across eukaryotes

    PubMed Central

    Boekhorst, Jos; van Breukelen, Bas; Heck, Albert JR; Snel, Berend

    2008-01-01

    Background Reversible phosphorylation of proteins is involved in a wide range of processes, ranging from signaling cascades to regulation of protein complex assembly. Little is known about the structure and evolution of phosphorylation networks. Recent high-throughput phosphoproteomics studies have resulted in the rapid accumulation of phosphopeptide datasets for many model organisms. Here, we exploit these novel data for the comparative analysis of phosphorylation events between different species of eukaryotes. Results Comparison of phosphoproteomics datasets of six eukaryotes yields an overlap ranging from approximately 700 sites for human and mouse (two large datasets of closely related species) to a single site for fish and yeast (distantly related as well as two of the smallest datasets). Some conserved events appear surprisingly old; those shared by plant and animals suggest conservation over the time scale of a billion years. In spite of the hypothesized incomprehensive nature of phosphoproteomics datasets and differences in experimental procedures, we show that the overlap between phosphoproteomes is greater than expected by chance and indicates increased functional relevance. Despite the dynamic nature of the evolution of phosphorylation, the relative overlap between the different datasets is identical to the phylogeny of the species studied. Conclusion This analysis provides a framework for the generation of biological insights by comparative analysis of high-throughput phosphoproteomics datasets. We expect the rapidly growing body of data from high-throughput mass spectrometry analysis to make comparative phosphoproteomics a powerful tool for elucidating the evolutionary and functional dynamics of reversible phosphorylation. PMID:18828897

  10. Alternatives To Dissection. Second Edition.

    ERIC Educational Resources Information Center

    DeRosa, Bill, Ed.; Winiarskyj, Lesia, Ed.

    This packet attempts to provide educationally sound alternatives to dissection in the classroom, thereby making it possible for teachers to eliminate dissection from the curriculum. This packet can also be used by educators who include dissection in their curricula but consider it important to respect the expression of students' ethical, moral, or

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

    PubMed

    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 chalcone isomerase and cytochrome P450 monooxygenase. A novel salt tolerance pathway involving chalcone metabolism, mostly mediated by phosphorylated MYB transcription factors, was proposed based on our findings. (The mass spectrometry raw data are available via ProteomeXchange with identifier PXD002856). PMID:26407991

  12. 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

    2014-12-01

    The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. Although the importance of post-translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C-like) through the [S-x-E] motif, which is supported by evidence that mRNA and protein expression of FAM20C-like protein kinase are both found in the highest level in the hypopharyngeal gland of nurse bees. Our data represent the first comprehensive RJ phosphorylation atlas, recording patterns of phosphorylated RJ protein abundance and antibacterial activity of some RJ proteins in two major managed honeybee species. These data constitute a firm basis for future research to better understand the biological roles of each RJ protein for honeybee biology and human health care. PMID:25265229

  13. Large Scale Phosphoproteome Profiles Comprehensive Features of Mouse Embryonic Stem Cells*

    PubMed Central

    Li, Qing-Run; Xing, Xiao-Bin; Chen, Tao-Tao; Li, Rong-Xia; Dai, Jie; Sheng, Quan-Hu; Xin, Shun-Mei; Zhu, Li-Li; Jin, Ying; Pei, Gang; Kang, Jiu-Hong; Li, Yi-Xue; Zeng, Rong

    2011-01-01

    Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells. PMID:21149613

  14. 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 ?3-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 ?3-adrenenoceptor signaling cascade that promotes thermogenesis in adipocytes. PMID:26525534

  15. Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions

    PubMed Central

    Courcelles, Mathieu; Frmin, Christophe; Voisin, Laure; Lemieux, Sbastien; Meloche, Sylvain; Thibault, Pierre

    2013-01-01

    The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases. PMID:23712012

  16. Phosphoproteome dynamics of Saccharomyces cerevisiae under heat shock and cold stress

    PubMed Central

    Kanshin, Evgeny; Kubiniok, Peter; Thattikota, Yogitha; D'Amours, Damien; Thibault, Pierre

    2015-01-01

    The ability of cells and organisms to survive and function through changes in temperature evolved from their specific adaptations to nonoptimal growth conditions. Responses to elevated temperatures have been studied in yeast and other model organisms using transcriptome profiling and provided valuable biological insights on molecular mechanisms involved in stress tolerance and adaptation to adverse environment. In contrast, little is known about rapid signaling events associated with changes in temperature. To gain a better understanding of global changes in protein phosphorylation in response to heat and cold, we developed a high temporal resolution phosphoproteomics protocol to study cell signaling in Saccharomyces cerevisiae. The method allowed for quantitative analysis of phosphodynamics on 2,777 phosphosites from 1,228 proteins. The correlation of kinetic profiles between kinases and their substrates provided a predictive tool to identify new putative substrates for kinases such as Cdc28 and PKA. Cell cycle analyses revealed that the increased phosphorylation of Cdc28 at its inhibitory site Y19 during heat shock is an adaptive response that delays cell cycle progression under stress conditions. The cellular responses to heat and cold were associated with extensive changes in phosphorylation on proteins implicated in transcription, protein folding and degradation, cell cycle regulation and morphogenesis. PMID:26040289

  17. Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage

    PubMed Central

    Morales, Miguel A.; Watanabe, Reiko; Dacher, Mariko; Chafey, Philippe; Osorio y Fortéa, José; Scott, David A.; Beverley, Stephen M.; Ommen, Gabi; Clos, Joachim; Hem, Sonia; Lenormand, Pascal; Rousselle, Jean-Claude; Namane, Abdelkader; Späth, Gerald F.

    2010-01-01

    Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1−/− null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases. PMID:20404152

  18. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

    PubMed Central

    Alam, Mahmood M.; Solyakov, Lev; Bottrill, Andrew R.; Flueck, Christian; Siddiqui, Faiza A.; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S.; Chitnis, Chetan E.; Doerig, Christian; Moon, Robert W.; Green, Judith L.; Holder, Anthony A.; Baker, David A.; Tobin, Andrew B.

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

  19. Targeted phosphoproteomics of insulin signaling using data-independent acquisition mass spectrometry.

    PubMed

    Parker, Benjamin L; Yang, Guang; Humphrey, Sean J; Chaudhuri, Rima; Ma, Xiuquan; Peterman, Scott; James, David E

    2015-06-01

    A major goal in signaling biology is the establishment of high-throughput quantitative methods for measuring changes in protein phosphorylation of entire signal transduction pathways across many different samples comprising temporal or dose data or patient samples. Data-independent acquisition (DIA) mass spectrometry (MS) methods, which involve tandem MS scans that are collected independently of precursor ion information and then are followed by targeted searching for known peptides, may achieve this goal. We applied DIA-MS to systematically quantify phosphorylation of components in the insulin signaling network in response to insulin as well as in stimulated cells exposed to a panel of kinase inhibitors targeting key downstream effectors in the network. We accurately quantified the effect of insulin on phosphorylation of 86 protein targets in the insulin signaling network using either stable isotope standards (SIS) or label-free quantification (LFQ) and mapped signal transmission through this network. By matching kinases to specific phosphorylation events (based on linear consensus motifs and temporal phosphorylation) to the quantitative phosphoproteomic data from cells exposed to inhibitors, we investigated predicted kinase-substrate relationships of AKT and mTOR in a targeted fashion. Furthermore, we applied this approach to show that AKT2-dependent phosphorylation of GAB2 promoted insulin signaling but inhibited epidermal growth factor (EGF) signaling in a manner dependent on 14-3-3 binding. Because DIA-MS can increase throughput and improve the reproducibility of peptide detection across multiple samples, this approach should facilitate more accurate, comprehensive, and quantitative assessment of signaling networks under various experimental conditions than are possible using other MS proteomic methods. PMID:26060331

  20. Aortic dissection during pregnancy.

    PubMed Central

    Pumphrey, C W; Fay, T; Weir, I

    1986-01-01

    Aortic dissection occurred in a nineteen year old woman during the thirty seventh week of pregnancy. Immediate elective delivery of a normal baby by caesarean section was followed by aortic root replacement 48 hours later. It was decided not to proceed immediately to operation on the aortic root because it was believed that the anticoagulation necessary for cardiopulmonary bypass might provoke dangerous haemorrhage from the raw placental site. Images Fig. 1 Fig. 2 PMID:3511928

  1. Temporal Phosphoproteome Dynamics Induced by an ATP Synthase Inhibitor Citreoviridin.

    PubMed

    Hu, Chia-Wei; Hsu, Chia-Lang; Wang, Yu-Chao; Ishihama, Yasushi; Ku, Wei-Chi; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2015-12-01

    Citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. However, the mechanism of citreoviridin triggering dynamic molecular responses in cancer cells remains unclear. Here, we performed temporal phosphoproteomics to elucidate the dynamic changes after citreoviridin treatment in cells and xenograft model. We identified a total of 829 phosphoproteins and demonstrated that citreoviridin treatment affects protein folding, cell cycle, and cytoskeleton function. Furthermore, response network constructed by mathematical modeling shows the relationship between the phosphorylated heat shock protein 90 ? and mitogen-activated protein kinase signaling pathway. This work describes that citreoviridin suppresses cancer cell growth and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 and provides perspectives in cancer therapeutic strategies. PMID:26503892

  2. Phosphoproteomic studies in Arabidopsis and tobacco male gametophytes.

    PubMed

    Fla, Jan; ?apkov, V?ra; Honys, David

    2014-04-01

    Mature pollen represents an extremely resistant quiescent structure surrounded by a tough cell wall. After its hydration on stigma papillary cells, pollen tube growth starts rapidly. Massive metabolic changes are likely to be accompanied by changes in protein phosphorylation. Protein phosphorylation belongs among the most rapid post-translational modifications. To date, only Arabidopsis thaliana and tobacco (Nicotiana tabacum) mature pollen have been subjected to phosphoproteomic studies in order to identify the phosphoproteins present. In the present mini-review, Arabidopsis and tobacco datasets were compared with each other. The representation of the O-phosphorylated amino acids was compared between these two datasets, and the putative pollen-specific or pollen-abundant phosphopeptides were highlighted. Finally, the phosphorylation sites common for both Arabidopsis and tobacco phosphoproteins are listed as well as the phosphorylation motifs identified. PMID:24646248

  3. Proteome and phosphoproteome of Africanized and European honeybee venoms.

    PubMed

    Resende, Virgnia Maria Ferreira; Vasilj, Andrej; Santos, Keity Souza; Palma, Mario Sergio; Shevchenko, Andrej

    2013-09-01

    Honey bee venom toxins trigger immunological, physiological, and neurological responses within victims. The high occurrence of bee attacks involving potentially fatal toxic and allergic reactions in humans and the prospect of developing novel pharmaceuticals make honey bee venom an attractive target for proteomic studies. Using label-free quantification, we compared the proteome and phosphoproteome of the venom of Africanized honeybees with that of two European subspecies, namely Apis mellifera ligustica and A. m. carnica. From the total of 51 proteins, 42 were common to all three subspecies. Remarkably, the toxins melittin and icarapin were phosphorylated. In all venoms, icarapin was phosphorylated at the (205) Ser residue, which is located in close proximity to its known antigenic site. Melittin, the major toxin of honeybee venoms, was phosphorylated in all venoms at the (10) Thr and (18) Ser residues. (18) Ser phosphorylated melittin-the major of its two phosphorylated forms-was less toxic compared to the native peptide. PMID:23798553

  4. Functional Divergence and Evolutionary Turnover in Mammalian Phosphoproteomes

    PubMed Central

    Freschi, Luca; Osseni, Mazid; Landry, Christian R.

    2014-01-01

    Protein phosphorylation is a key mechanism to regulate protein functions. However, the contribution of this protein modification to species divergence is still largely unknown. Here, we studied the evolution of mammalian phosphoregulation by comparing the human and mouse phosphoproteomes. We found that 84% of the positions that are phosphorylated in one species or the other are conserved at the residue level. Twenty percent of these conserved sites are phosphorylated in both species. This proportion is 2.5 times more than expected by chance alone, suggesting that purifying selection is preserving phosphoregulation. However, we show that the majority of the sites that are conserved at the residue level are differentially phosphorylated between species. These sites likely result from false-negative identifications due to incomplete experimental coverage, false-positive identifications and non-functional sites. In addition, our results suggest that at least 5% of them are likely to be true differentially phosphorylated sites and may thus contribute to the divergence in phosphorylation networks between mouse and humans and this, despite residue conservation between orthologous proteins. We also showed that evolutionary turnover of phosphosites at adjacent positions (in a distance range of up to 40 amino acids) in human or mouse leads to an over estimation of the divergence in phosphoregulation between these two species. These sites tend to be phosphorylated by the same kinases, supporting the hypothesis that they are functionally redundant. Our results support the hypothesis that the evolutionary turnover of phosphorylation sites contributes to the divergence in phosphorylation profiles while preserving phosphoregulation. Overall, our study provides advanced analyses of mammalian phosphoproteomes and a framework for the study of their contribution to phenotypic evolution. PMID:24465218

  5. Aortic dissection and dissecting aortic aneurysms.

    PubMed Central

    Crawford, E S; Svensson, L G; Coselli, J S; Safi, H J; Hess, K R

    1988-01-01

    Operation was employed in the treatment of 546 patients for complications of aortic dissection during the 32-year period of 1956-1988. Current concepts and operative techniques evolved during this period. Fortunately, about half the patients were treated during the latter 4 years, as modern therapy became standardized. The cumulative survival rate was 86% for all patients and 94% for those treated during recent years. Pathologic processes and requirements of operation became clearer by treating 174 patients who had had 198 previous operations by the time of referral. Reoperation was required for complications of operations now considered outdated, heart operations in patients with ascending aortic dilatation, and progressive dilatation of residual segments of the aorta. The 546 patients were followed, and a total of 838 operations were finally employed, resulting in total aortic replacement in 18, near total replacement in 41, entire thoracic aorta in 22, near total thoracic aorta in 33, and the entire thoracoabdominal aorta in 148 patients. Long-term survival in 439 patients after final operation was 66% and 44% at 5 and 10 years, respectively, despite the fact that the median age at first admission was 59. Operative treatment appears to be well-established for this disease. Images Figs. 12A-C. Figs. 12A-C. Figs. 1A and B. Figs. 2A and B. Fig. 3. Figs. 4A-D. Figs. 5A-C. Figs. 5A-C. Figs. 6A and B. Figs. 7A and B. Figs. 8A and B. Figs. 9A and B. Figs. 10A and B.,Figs. 11A and B. PMID:3421752

  6. Dissecting intracranial vertebral artery aneurysms.

    PubMed

    Bhattacharya, R N; Menon, G; Nair, S

    2001-12-01

    Dissecting aneurysms of the intracranial arteries are exceedingly rare vascular lesions that can produce acute cerebral or brain stem infarction in young healthy adults. They carry a high mortality rate. Two cases of dissecting vertebral artery aneurysms that presented with bleed, were successfully operated by trapping and excision of the dissecting segment. Both dissecting aneurysms were located distal to PICA origin. Both the patients developed post operative lower cranial nerve paresis and one developed lateral medullary syndrome, which improved subsequently. Dissecting aneurysms presenting with bleed should be surgically managed by trapping and excising the involved segment sparing the PICA origin or by interventional radiological techniques. Revascularisation procedures should be considered in addition to trapping of the main vertebral segment if PICA is involved in the trapped segment. The diagnostic and therapeutic difficulties associated with dissecting vertebral artery aneurysms and the controversies regarding their management have been reviewed. PMID:11799414

  7. Intraoperative aortic dissection.

    PubMed

    Singh, Ajmer; Mehta, Yatin

    2015-01-01

    Intraoperative aortic dissection is a rare but fatal complication of open heart surgery. By recognizing the population at risk and by using a gentle operative technique in such patients, the surgeon can usually avoid iatrogenic injury to the aorta. Intraoperative transesophageal echocardiography and epiaortic scanning are invaluable for prompt diagnosis and determination of the extent of the injury. Prevention lies in the strict control of blood pressure during cannulation/decannulation, construction of proximal anastomosis, or in avoiding manipulation of the aorta in high-risk patients. Immediate repair using interposition graft or Dacron patch graft is warranted to reduce the high mortality associated with this complication. PMID:26440240

  8. [Aortic dissection in pregnancy].

    PubMed

    Trudel, M; Koussa, M; Pontana, F; Deruelle, P; Debarge, V; Ducloy-Bouthors, A-S; Coulon, C; Subtil, D

    2015-05-01

    During pregnancy, the occurrence of aortic dissection is a rare event immediately threatening fetal and maternal prognosis. Its occurrence is more common in cases of connective tissue disease. But the absence risk factor shall not exclude or delay diagnosis. We must learn to think about it, because the prognosis is highly dependent on time management. The clinical presentation of this medical and surgical emergency varies, and pregnancy adds its own symptoms. We have to ask without hesitation that echocardiography or chest CT be performed since these diagnostic methods are both reliable and available. PMID:25908580

  9. Biomarkers in aortic dissection.

    PubMed

    Wen, Dan; Zhou, Xian-Liang; Li, Jian-Jun; Hui, Ru-Tai

    2011-04-11

    Aortic dissection (AD) is a severe cardiovascular disease with high mortality and morbidity, which is characterized by acute onset and rapid progress. Mechanically, it has been considered that circulating blood flows into the media of the aorta through the rupture of the intima forming true and false lumens. Generally, its pathologic process is considered as follows: initially, inflammatory reaction, inflammatory cells infiltration in aortic wall, and then apoptosis of vascular smooth muscle cells, degenerating of aortic media, elastin fracture, and degradation. At last, the ingredients of the aorta are destroyed and lead to aortic dilatation, aneurysm formation, dissection and rupture. Currently, several biomarkers in peripheral blood including C-reactive protein (CRP), matrix metalloproteinases (MMPs), soluble elastin fragments (sELAF), D-dimer, smooth muscle myosin heavy chain, calponin, N-terminal pro-brain natriuretic peptide (NT-proBNP), big endothelin-1 (Big ET-1), genetic markers and so on, have been demonstrated to play a major role in evaluation of AD, for example, making early diagnosis and classifying of AD. Additionally, those markers may also guide our treatment therapies and predict the prognosis. The aims of this review mainly focus on the clinical implications of the biomarkers in AD. PMID:21237193

  10. "Dissection" of a Hair Dryer

    ERIC Educational Resources Information Center

    Eisenstein, Stan; Simpson, Jeff

    2008-01-01

    The electrical design of the common hair dryer is based almost entirely on relatively simple principles learned in introductory physics classes. Just as biology students dissect a frog to see the principles of anatomy in action, physics students can "dissect" a hair dryer to see how principles of electricity are used in a real system. They can

  11. "Dissection" of a Hair Dryer

    ERIC Educational Resources Information Center

    Eisenstein, Stan; Simpson, Jeff

    2008-01-01

    The electrical design of the common hair dryer is based almost entirely on relatively simple principles learned in introductory physics classes. Just as biology students dissect a frog to see the principles of anatomy in action, physics students can "dissect" a hair dryer to see how principles of electricity are used in a real system. They can…

  12. Early Phosphoproteomic Changes in the Mouse Spleen During Deoxynivalenol-Induced Ribotoxic Stress

    PubMed Central

    Pestka, James J.

    2013-01-01

    The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DONs immunotoxic effects, stable isotope dimethyl labelingbased proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (? 30min) phosphoproteome changes in the spleens of mice orally exposed to 5mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate phosphatidylinositol 3-kinase/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells, and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins. PMID:23811945

  13. Automatic Dissection Of Plantlets

    NASA Astrophysics Data System (ADS)

    Batchelor, B. G.; Harris, I. P.; Marchant, J. A.; Tillett, R. D.

    1989-03-01

    Micropropagation is a technique used in horticulture for generating a monoclonal colony of plants. A tiny plantlet is cut into several parts, each of which is then replanted. At the moment, the cutting is performed manually. Automating this task would have significant economic benefits. A robot designed to dissect plants would need to be equipped with intelligent visual sensing. This article is concerned with the image acquisition and processing techniques which such a machine might use. A program, which can calculate where to cut a plant with an "open" structure, is presented. This is expressed in the ProVision language, which is described in another article presented at this conference. (Article 1002-65)

  14. The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle*

    PubMed Central

    Santamaria, Anna; Wang, Bin; Elowe, Sabine; Malik, Rainer; Zhang, Feng; Bauer, Manuel; Schmidt, Alexander; Silljé, Herman H. W.; Körner, Roman; Nigg, Erich A.

    2011-01-01

    Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle. PMID:20860994

  15. Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell-cell adhesion.

    PubMed

    Larive, R M; Urbach, S; Poncet, J; Jouin, P; Mascré, G; Sahuquet, A; Mangeat, P H; Coopman, P J; Bettache, N

    2009-06-18

    The spleen tyrosine kinase Syk has predominantly been studied in hematopoietic cells in which it is involved in immunoreceptor-mediated signaling. Recently, Syk expression was evidenced in numerous nonhematopoietic cells and shown to be involved in tumor formation and progression. The Syk downstream signaling effectors in nonhematopoietic cells remain, however, to be uncovered, and were investigated using MS-based quantitative phosphoproteomics. Two strategies, based on the inhibition of the Syk catalytic activity and on the loss of Syk expression were employed to identify phosphotyrosine-dependent complexes. Quantitative measurements were obtained on 350 proteins purified with phosphotyrosine affinity columns using the SILAC method. Forty-one proteins are dependent on both Syk expression and catalytic activity and were selected as signaling effectors. They are involved in a variety of biological processes such as signal transduction, cell-cell adhesion and cell polarization. We investigated the functional involvement of Syk in cell-cell adhesion and demonstrated the phosphorylation of E-cadherin and alpha-catenin. In addition, Syk is localized at cell-cell contacts, and Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Identification of the biochemical pathways regulated by Syk in human cancer cells will help to uncover its role in tumor formation and progression. PMID:19421152

  16. Brachial plexus endoscopic dissection and correlation with open dissection.

    PubMed

    Lafosse, T; Masmejean, E; Bihel, T; Lafosse, L

    2015-12-01

    Shoulder endoscopy is evolving and becoming extra-articular. More and more procedures are taking place in the area of the brachial plexus (BP). We carried out an anatomical study to describe the endoscopic anatomy of the BP and the technique used to dissect and expose the BP endoscopically. Thirteen fresh cadavers were dissected. We first performed an endoscopic dissection of the BP, using classical extra-articular shoulder arthroscopy portals. Through each portal, we dissected as many structures as possible and identified them. We then did an open dissection to corroborate the endoscopic findings and to look for damage to the neighboring structures. In the supraclavicular area, we were able to expose the C5, C6 and C7 roots, and the superior and middle trunks in 11 of 13 specimens through two transtrapezial portals by following the suprascapular nerve. The entire infraclavicular portion of the BP (except the medial cord and its branches) was exposed in 11 of 13 specimens. The approach to the infraclavicular portion of the BP led directly to the lateral and posterior cords, but the axillary artery hid the medial cord. The musculocutaneous nerve was the first nerve encountered when dissecting medially from the anterior aspect of the coracoid process. The axillary nerve was the first nerve encountered when following the anterior border of the subscapularis medially from the posterior aspect of the coracoid process. Knowledge of the endoscopic anatomy of the BP is mandatory to expose and protect this structure while performing advanced arthroscopic shoulder procedures. PMID:26585998

  17. [Endoscopic submucosal dissection].

    PubMed

    Hochberger, J; Khler, P; Kruse, E; Hppertz, J; Delvaux, M; Gay, G; Wedi, E

    2013-03-01

    Endoscopic submucosal dissection (ESD) was developed in Japan but has now also become permanently established in various centers in Europe. ESD is an endoscopic en bloc mucosal resection technique for the treatment of early cancers with a diameter >1 cm and also superficial precancerous lesions, which could only be removed unsatisfactorily in several fragments or with uncertain lateral safety margins using previous loop excision procedures. Using ESD a lesion is excised after circular marking and generous submucosal injection with a safety margin of approximately 5 mm and subsequently resected at the level of the submucosa with a 1-3 mm short diathermic knife. ESD requires high technical skills in interventional endoscopy and is more time-consuming than snare resection techniques. However, numerous studies have shown a clear superiority for ESD with respect to the R0 resection rate and the local recurrence rate. The present article gives a current review of the use of ESD in the upper and lower gastrointestinal tract and demonstrates perspectives of the procedure. PMID:23455659

  18. Biomedicine: an ontological dissection.

    PubMed

    Baronov, David

    2008-01-01

    Though ubiquitous across the medical social sciences literature, the term "biomedicine" as an analytical concept remains remarkably slippery. It is argued here that this imprecision is due in part to the fact that biomedicine is comprised of three interrelated ontological spheres, each of which frames biomedicine as a distinct subject of investigation. This suggests that, depending upon one's ontological commitment, the meaning of biomedicine will shift. From an empirical perspective, biomedicine takes on the appearance of a scientific enterprise and is defined as a derivative category of Western science more generally. From an interpretive perspective, biomedicine represents a symbolic-cultural expression whose adherence to the principles of scientific objectivity conceals an ideological agenda. From a conceptual perspective, biomedicine represents an expression of social power that reflects structures of power and privilege within capitalist society. No one perspective exists in isolation and so the image of biomedicine from any one presents an incomplete understanding. It is the mutually-conditioning interrelations between these ontological spheres that account for biomedicine's ongoing development. Thus, the ontological dissection of biomedicine that follows, with particular emphasis on the period of its formal crystallization in the latter nineteenth and early twentieth century, is intended to deepen our understanding of biomedicine as an analytical concept across the medical social sciences literature. PMID:18802784

  19. 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

  20. Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography.

    PubMed

    Loroch, Stefan; Zahedi, Ren Peiman; Sickmann, Albert

    2015-02-01

    In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 ?g of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ?7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10(7) down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti(4+), our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research. PMID:25405705

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

    PubMed Central

    Yang, Pengyi; Zheng, Xiaofeng; Jayaswal, Vivek; Hu, Guang; Yang, Jean Yee Hwa; Jothi, Raja

    2015-01-01

    Cell signaling underlies transcription/epigenetic control of a vast majority of cell-fate decisions. A key goal in cell signaling studies is to identify the set of kinases that underlie key signaling events. In a typical phosphoproteomics study, phosphorylation sites (substrates) of active kinases are quantified proteome-wide. By analyzing the activities of phosphorylation sites over a time-course, the temporal dynamics of signaling cascades can be elucidated. Since many substrates of a given kinase have similar temporal kinetics, clustering phosphorylation sites into distinctive clusters can facilitate identification of their respective kinases. Here we present a knowledge-based CLUster Evaluation (CLUE) approach for identifying the most informative partitioning of a given temporal phosphoproteomics data. Our approach utilizes prior knowledge, annotated kinase-substrate relationships mined from literature and curated databases, to first generate biologically meaningful partitioning of the phosphorylation sites and then determine key kinases associated with each cluster. We demonstrate the utility of the proposed approach on two time-series phosphoproteomics datasets and identify key kinases associated with human embryonic stem cell differentiation and insulin signaling pathway. The proposed approach will be a valuable resource in the identification and characterizing of signaling networks from phosphoproteomics data. PMID:26252020

  2. Computational Fluid Dynamics Analysis of Thoracic Aortic Dissection

    NASA Astrophysics Data System (ADS)

    Tang, Yik; Fan, Yi; Cheng, Stephen; Chow, Kwok

    2011-11-01

    Thoracic Aortic Dissection (TAD) is a cardiovascular disease with high mortality. An aortic dissection is formed when blood infiltrates the layers of the vascular wall, and a new artificial channel, the false lumen, is created. The expansion of the blood vessel due to the weakened wall enhances the risk of rupture. Computational fluid dynamics analysis is performed to study the hemodynamics of this pathological condition. Both idealized geometry and realistic patient configurations from computed tomography (CT) images are investigated. Physiological boundary conditions from in vivo measurements are employed. Flow configuration and biomechanical forces are studied. Quantitative analysis allows clinicians to assess the risk of rupture in making decision regarding surgical intervention.

  3. Experience with parametric binary dissection

    NASA Technical Reports Server (NTRS)

    Bokhari, Shahid H.

    1993-01-01

    Parametric Binary Dissection (PBD) is a new algorithm that can be used for partitioning graphs embedded in 2- or 3-dimensional space. It partitions explicitly on the basis of nodes + (lambda)x(edges cut), where lambda is the ratio of time to communicate over an edge to the time to compute at a node. The new algorithm is faster than the original binary dissection algorithm and attempts to obtain better partitions than the older algorithm, which only takes nodes into account. The performance of parametric dissection with plain binary dissection on 3 large unstructured 3-d meshes obtained from computational fluid dynamics and on 2 random graphs were compared. It was showm that the new algorithm can usually yield partitions that are substantially superior, but that its performance is heavily dependent on the input data.

  4. Wrangling phosphoproteomic data to elucidate cancer signaling pathways.

    PubMed

    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 packages are employed. PMID:23300999

  5. 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

  6. Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

    PubMed

    Kwon, Oh Kwang; Sim, JuHee; Kim, Sun Ju; Sung, Eunji; Kim, Jin Young; Jeong, Tae Cheon; Lee, Sangkyu

    2015-12-01

    Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation. PMID:26487105

  7. Carotid artery dissection following adenoidectomy.

    PubMed

    Garg, Arun; Singh, Yeeshu; Singh, Pankaj; Goel, Gaurav; Bhuyan, Susant

    2016-03-01

    Carotid dissection and cerebral infarction are extremely rare complications of adenoidectomy. We describe the case of seven year old girl, who suffered from left internal carotid artery dissection following adenoidectomy, leading to right hemiplegia with global aphasia. A CT angiogram confirmed a loop in contralateral right internal carotid artery. It is presumed that a similar loop also existed in left internal carotid artery, which possibly extended medially close to posterior pharyngeal wall and was injured during the course of surgery. PMID:26857324

  8. Quantitative analysis of phosphorylation-based protein signaling networks in the immune system by mass spectrometry.

    PubMed

    Nita-Lazar, Aleksandra

    2011-01-01

    Dynamic modification of cell proteins with phosphate is one of the key regulators of the cellular response to external stimuli. Phosphorylation-based signaling networks mediate cell proliferation, differentiation, and migration, and their dysregulation is the basis of multiple diseases. However, the transient nature of the regulatory protein phosphorylation and low site occupancy mean that only a fraction of the protein is phosphorylated at a given time, and it is a challenge to measure the degree and dynamics of phosphorylation using traditional biochemical means. Technological advances in the field of mass spectrometry (MS) made it possible to generate large sets of phosphoproteomics data, probing the phosphoproteome with great depth, sensitivity, and accuracy. Therefore, quantitative phosphoproteomics emerged as one of the essential components of the systems biology approach for profiling of complex biological networks. Nowadays, the challenge lies in validation of the information and in its integration into the comprehensive models of cell decision processes. This article reviews the role of phosphoproteomics in systems biology, the MS-based approach, and technical details of the methods. Recent examples of quantitative measurements and methodologies as well as applications to the studies of the immune system and infectious diseases are presented and discussed. PMID:20836078

  9. Interrogating cAMP-dependent Kinase Signaling in Jurkat T Cells via a Protein Kinase A Targeted Immune-precipitation Phosphoproteomics Approach*

    PubMed Central

    Giansanti, Piero; Stokes, Matthew P.; Silva, Jeffrey C.; Scholten, Arjen; Heck, Albert J. R.

    2013-01-01

    In the past decade, mass-spectrometry-based methods have emerged for the quantitative profiling of dynamic changes in protein phosphorylation, allowing the behavior of thousands of phosphorylation sites to be monitored in a single experiment. However, when one is interested in specific signaling pathways, such shotgun methodologies are not ideal because they lack selectivity and are not cost and time efficient with respect to instrument and data analysis time. Here we evaluate and explore a peptide-centric antibody generated to selectively enrich peptides containing the cAMP-dependent protein kinase (PKA) consensus motif. This targeted phosphoproteomic strategy is used to profile temporal quantitative changes of potential PKA substrates in Jurkat T lymphocytes upon prostaglandin E2 (PGE2) stimulation, which increases intracellular cAMP, activating PKA. Our method combines ultra-high-specificity motif-based immunoaffinity purification with cost-efficient stable isotope dimethyl labeling. We identified 655 phosphopeptides, of which 642 (i.e. 98%) contained the consensus motif [R/K][R/K/X]X[pS/pT]. When our data were compared with a large-scale Jurkat T-lymphocyte phosphoproteomics dataset containing more than 10,500 phosphosites, a minimal overlap of 0.2% was observed. This stresses the need for such targeted analyses when the interest is in a particular kinase. Our data provide a resource of likely substrates of PKA, and potentially some substrates of closely related kinases. Network analysis revealed that about half of the observed substrates have been implicated in cAMP-induced signaling. Still, the other half of the here-identified substrates have been less well characterized, representing a valuable resource for future research. PMID:23882029

  10. 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-dependence for translocation from the cytoplasm to the nucleus. These data provide new insights into the activation of ATM by oxidative stress through identification of novel substrates for ATM in the cytoplasm. PMID:26699800

  11. Combination of chemical genetics and phosphoproteomics for kinase signaling analysis enables confident identification of cellular downstream targets.

    PubMed

    Oppermann, Felix S; Grundner-Culemann, Kathrin; Kumar, Chanchal; Gruss, Oliver J; Jallepalli, Prasad V; Daub, Henrik

    2012-04-01

    Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates. PMID:22199227

  12. 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

  13. 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-? (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. PMID:26330541

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

    PubMed

    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

  15. Decreased expression of fibulin-4 in aortic wall of aortic dissection.

    PubMed

    Huawei, P; Qian, C; Chuan, T; Lei, L; Laing, W; Wenlong, X; Wenzhi, L

    2014-02-01

    In this research, we will examine the expression of Fibulin-4 in aortic wall to find out its role in aortic dissection development. The samples of aortic wall were obtained from 10 patients operated for acute ascending aortic dissection and five patients for chronic ascending aortic dissection. Another 15 pieces of samples from patients who had coronary artery bypass were as controls. The aortic samples were stained with aldehyde magenta dyeing to evaluate the arrangement of elastic fibers. The Fibulin-4 protein and mRNA expression were both determined by Western blot and realtime quantitative polymerase chain reaction. Compared with the control group, both in acute and chronic ascending aortic dissection, elastic fiber fragments increased and the expression of fibulin-4 protein significantly decreased (P= 0.045 < 0.05). The level of fibulin-4 mRNA decreased in acute ascending aortic dissection (P= 0.034 < 0.05), while it increased in chronic ascending aortic dissection (P=0.004 < 0.05). The increased amounts of elastic fiber fragments were negatively correlated with the expression of fibulin-4 mRNA in acute ascending aortic dissection. In conclusion, in aortic wall of ascending aortic dissection, the expression of fibulin-4 protein decreased and the expression of fibulin-4 mRNA was abnormal. Fibulin-4 may play an important role in the pathogenesis of aortic dissection. PMID:23518852

  16. Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation?

    PubMed Central

    Collins, Mark O.; Wright, James C.; Jones, Matthew; Rayner, Julian C.; Choudhary, Jyoti S.

    2014-01-01

    We present a workflow using an ETD-optimised version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for analysis of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analysed the Plasmodium falciparum schizont phosphoproteome using for the first time, a data-dependent neutral loss-triggered-ETD (DDNL) strategy and a conventional decision-tree method. At a posterior error probability threshold of 0.01, similar numbers of PSMs were identified using both methods with a 73% overlap in phosphopeptide identifications. The false discovery rate associated with spectral pairs where DDNL CID/ETD identified the same phosphopeptide was <1%. 72% of phosphorylation site assignments using turbo-SLoMo without any score filtering, were identical and 99.8% of these cases are associated with a false localisation rate of <5%. We show that DDNL acquisition is a useful approach for phosphoproteomics and results in an increased confidence in phosphopeptide identification without compromising sensitivity or duty cycle. Furthermore, the combination of Mascot Percolator and turbo-SLoMo represents a robust workflow for phosphoproteomic data analysis using CID and ETD fragmentation. Biological significance Protein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its analysis on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data analysis to unsure that sites are identified with high confidence for follow-up experiments to investigate their biological significance. The coverage and confidence of phosphoproteomic experiments can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data analysis pipeline for analysis of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation. PMID:24657495

  17. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

    PubMed Central

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul MK

    2015-01-01

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease. PMID:26471730

  18. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease. PMID:26471730

  19. [Pregnancy and coronary artery dissection].

    PubMed

    Martínez-Quintana, Efrén; Rodríguez-González, Fayna

    2015-01-01

    Acute myocardial infarction during pregnancy is associated with high maternal and fetal mortality. Coronary atherosclerosis is the most common cause due to an increase in the age of the patients and the association with cardiovascular risk factors such as smoking, hypertension, diabetes mellitus, preeclampsia, and the existence of family history of coronary disease. However, thrombosis, coronary dissection or coronary vasospasms are other causes that may justify it. We report the case of a 33 weeks pregnant first-time mother, without cardiovascular risk factors, who presented an acute coronary event in the context of atherosclerotic disease and coronary dissection after percutaneous coronary intervention. PMID:25795261

  20. [Tonsillar microsurgery by bipolar dissection].

    PubMed

    Snchez del Hoyo, A; Lacosta Nicols, J L; M Salm, J; Martnez Torre, I; Fernndez Martn, M J

    1995-01-01

    The 66 amygdalectomies done by the AA. under general anesthesia and tracheal intubation are considered in two groups. The first one was composed by 32 patients operated following a dissection and coagulation procedure with the aid of a bipolar forceps and under microscopical magnification. The second group, 34 cases, underwent the classical procedure with ligature of the bleeding vessels. The usage of the bipolar forceps procure lesser loss of blood as compared with the dissection-ligature procedure. On the contrary, the bipolar clip method showed and increased postoperative pain and also the lagging of the swallow function. Both techniques presented with very similar complications (bleeding, edema, loca infection). PMID:7573857

  1. A protocol on the use of titanium dioxide chromatography for phosphoproteomics.

    PubMed

    Pinkse, Martijn W H; Lemeer, Simone; Heck, Albert J R

    2011-01-01

    Over the past decade phosphoproteomics has become an emerging discipline within proteomics research, focusing on detection of the reversible modification of proteins by phosphorylation of serine, threonine, and tyrosine residues. For successful analysis, phosphopeptide enrichment is often a prerequisite due to their low stoichiometry, heterogeneity, and low abundance. The enrichment of phosphopeptides is often performed manually, which is inherently labor intensive and a major hindrance in large-scale analyses. Automation of the enrichment method would vastly improve reproducibility and thereby facilitate "high-throughput" phosphoproteomics research. Here, we describe the setup of a simple, robust, and automated online TiO(2)-based nanoscale chromatographic approach to selectively enrich and separate phosphorylated peptides from proteolytic digests of moderate and high complexity. PMID:21604125

  2. Back Cover: Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio).

    PubMed

    Kwon, Oh Kwang; Kim, Sun Ju; Lee, You-Mie; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Jin Young; Peng, Xiaojun; Cheng, Zhongyi; Zhao, Yingming; Lee, Sangkyu

    2016-01-01

    DOI: 10.1002/pmic.201500017 The 3500 non-redundant phosphorylation sites on 2166 phosphoproteins and 1564 quantified have been reported during the development of zebrafish embryos, which is the largest dataset for phosphoproteins in zebrafish. The GO enrichment of dynamic phosphoproteomics indicated the various regulatory processes involved in each development stage. For details, see the article by Oh Kwang Kwon et al. on page 136. PMID:26728308

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

    PubMed Central

    Spt, Philipp; Ma?ek, 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

  4. Animal Rights Activism Threatens Dissection.

    ERIC Educational Resources Information Center

    Holden, Constance

    1990-01-01

    Discussed is the movement against the use of dissections in science laboratories. Examples of protests across the United States are included. Compared is the plight of using animals in a biology classroom and the demise of the teaching of evolution in some areas. (KR)

  5. Dissection & Science Fairs. [Information Packet.

    ERIC Educational Resources Information Center

    National Anti-Vivisection Society, Chicago, IL.

    This collection of pamphlets and articles reprinted from other National Anti-Vivisection Society (NAVS) publications was compiled to address the issues of classroom laboratory dissection and the use of animals in science fair projects. Three of the pamphlets contained in this packet are student handbooks designed to help students of elementary,…

  6. Gallium localization in dissecting aortic aneurysm

    SciTech Connect

    Haden, H.T.; Lippman, H.R.

    1988-08-01

    Gallium concentration was demonstrated in a dissecting aneurysm of the aortic arch, imaged approximately 2 weeks after dissection. Concentration of gallium was apparently due to the inflammatory reaction associated with the organizing intramural hematoma.

  7. Dissect Your Squid and Eat It Too!

    ERIC Educational Resources Information Center

    McGinnis, Patricia

    2001-01-01

    Introduces a science lab activity in which students dissect fresh squids in groups of four and observe the anatomy. Parent volunteers cook the squid mantle for kids to taste. Includes directions for squid dissection. (YDS)

  8. Quantitative Phosphoproteomics of Alzheimer's Disease Reveals Crosstalk between Kinases and Small Heat Shock Proteins

    PubMed Central

    Duong, Duc M.; Gearing, Marla; Lah, James J.; Levey, Allan I.; Seyfried, Nicholas T

    2015-01-01

    Abnormal phosphorylation contributes to the formation of neurofibrillary tangles in Alzheimer Disease (AD), but may play other signaling roles during AD pathogenesis. In this study, we employed immobilized metal affinity chromatography (IMAC) followed by liquid chromatography-tandem mass spectrometry to identify phosphopeptides from 8 individual AD and 8 age-matched control postmortem human brain tissues. Using this approach, we identified 5,569 phosphopeptides in frontal cortex across all 16 cases in which phosphopeptides represented 80 percent of all peptide spectral counts collected following IMAC enrichment. Marker selection identified 253 significantly altered phosphopeptides by precursor intensity, changed by at least 1.75 fold relative to controls, with an empirical false discovery rate below 7 percent. Approximately 21 percent of all significantly altered phosphopeptides in AD tissue were derived from tau. Of the other 142 proteins hyperphosphorylated in AD, membrane, synapse, cell junction, and alternatively spliced proteins were overrepresented. Of these, we validated differential phosphorylation of heat-shock protein-beta-1 (HSPB1) and crystallin-alpha-B (CRYAB) as hyperphosphorylated by western blotting. We further identified a network of phosphorylated kinases, which co-enriched with phosphorylated small heat shock proteins. This supports a hypothesis that a number of kinases are regulating and/or regulated by the small heat shock protein folding network. PMID:25332170

  9. Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism*

    PubMed Central

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

    2014-01-01

    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 kinasesubstrate 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. PMID:24200585

  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. 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

  12. 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. PMID:23316220

  13. 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

    2015-06-01

    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. PMID:25926025

  14. Directed analysis of cyanobacterial membrane phosphoproteome using stained phosphoproteins and titanium-enriched phosphopeptides.

    PubMed

    Lee, Dong-Gi; Kwon, Joseph; Eom, Chi-Yong; Kang, Young-Moon; Roh, Seong Woon; Lee, Kyung-Bok; Choi, Jong-Soon

    2015-04-01

    Gel-free shotgun phosphoproteomics of unicellular cyanobacterium Synechocystis sp. PCC 6803 has not been reported up to now. The purpose of this study is to develop directed membrane phosphoproteomic method in Synechocystis sp. Total Synechocystis membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphoprotein-stained gel bands were selectively subjected to in-gel trypsin digestion. The phosphorylation sites of the resulting peptides were determined by assigning the neutral loss of [M-H(3)PO(4)] to Ser, Thr, and Tyr residues using nano-liquid chromatography 7 Tesla Fourier transform mass spectrometry. As an initial application, 111 proteins and 33 phosphoproteins were identified containing 11 integral membrane proteins. Identified four unknown phosphoproteins with transmembrane helices were suggested to be involved in membrane migration or transporters based on BLASTP search annotations. The overall distribution of hydrophobic amino acids in pTyr was lower in frequency than that of pSer or pThr. Positively charged amino acids were abundantly revealed in the surrounding amino acids centered on pTyr. A directed shotgun membrane phosphoproteomic strategy provided insight into understanding the fundamental regulatory processes underlying Ser, Thr, and Tyr phosphorylation in multi-layered membranous cyanobacteria. PMID:25845541

  15. Large-scale models of signal propagation in human cells derived from discovery phosphoproteomic data

    PubMed Central

    Terfve, Camille D. A.; Wilkes, Edmund H.; Casado, Pedro; Cutillas, Pedro R.; Saez-Rodriguez, Julio

    2015-01-01

    Mass spectrometry is widely used to probe the proteome and its modifications in an untargeted manner, with unrivalled coverage. Applied to phosphoproteomics, it has tremendous potential to interrogate phospho-signalling and its therapeutic implications. However, this task is complicated by issues of undersampling of the phosphoproteome and challenges stemming from its high-content but low-sample-throughput nature. Hence, methods using such data to reconstruct signalling networks have been limited to restricted data sets and insights (for example, groups of kinases likely to be active in a sample). We propose a new method to handle high-content discovery phosphoproteomics data on perturbation by putting it in the context of kinase/phosphatase-substrate knowledge, from which we derive and train logic models. We show, on a data set obtained through perturbations of cancer cells with small-molecule inhibitors, that this method can study the targets and effects of kinase inhibitors, and reconcile insights obtained from multiple data sets, a common issue with these data. PMID:26354681

  16. Morphological Variation in Leaf Dissection of Rheum palmatum Complex (Polygonaceae)

    PubMed Central

    Wang, Xu-Mei; Hou, Xiao-Qi; Zhang, Yu-Qu; Li, Yan

    2014-01-01

    Aims Rheum palmatum complex comprises all taxa within section Palmata in the genus Rheum, including R. officinale, R. palmatum, R. tanguticum, R. tanguticum var. liupanshanense and R. laciniatum. The identification of the taxa in section Palmata is based primarily on the degree of leaf blade dissection and the shape of the lobes; however, difficulties in species identification may arise from their significant variation. The aim of this study is to analyze the patterns of variation in leaf blade characteristics within and among populations through population-based sampling covering the entire distribution range of R. palmatum complex. Methods Samples were taken from 2340 leaves from 780 individuals and 44 populations representing the four species, and the degree of leaf blade dissection and the shape of the lobe were measured to yield a set of quantitative data. Furthermore, those data were statistically analyzed. Important Findings The statistical analysis showed that the degree of leaf blade dissection is continuous from lobed to parted, and the shape of the lobe is also continuous from broadly triangular to lanceolate both within and between populations. We suggested that taxa in section Palmata should be considered as one species. Based on the research on the R. palmatum complex, we considered that the quantitative characteristics were greatly influenced by the environment. Therefore, it is not reliable to delimitate the species according to the continuously quantitative vegetative characteristics. PMID:25349989

  17. Evaluation of Educator & Student Use of & Attitudes toward Dissection & Dissection Alternatives

    ERIC Educational Resources Information Center

    Osenkowski, Pamela; Green, Che; Tjaden, Anne; Cunniff, Peggy

    2015-01-01

    Animal dissection has been routinely practiced in American biology classrooms for decades. With technological advancements, more states adopting student choice measures, and increased awareness about ethical concerns surrounding dissection, many useful dissection alternatives have been developed. To understand the current use of animal dissection

  18. A Dissecting Competition for Medical Students

    ERIC Educational Resources Information Center

    Samalia, Latika; Stringer, Mark D.

    2012-01-01

    After repeated requests from medical students for more cadaver dissection opportunities, a voluntary dissecting "competition" was initiated for the third year medical students in 2006. This has been held annually on five occasions since, offering up to 30 dissection stations and accommodating an average of 53 students (range 40-66) per year,

  19. Student/Teacher Conflict Regarding Animal Dissection.

    ERIC Educational Resources Information Center

    Balcombe, Jonathan

    1997-01-01

    Outlines some misconceptions and misunderstandings that underlie most dissection conflicts with the aim of making the dissection issue less volatile and one that generates fewer difficulties for students, teachers, and administrators. Lists published surveys of students' attitudes toward dissection and other animal uses in education. Discusses the

  20. Hi-Tech Alternatives to Dissection.

    ERIC Educational Resources Information Center

    Strauss, Richard T.; Kinzie, Mable B.

    1991-01-01

    The debate on the educational value of dissection versus the value of animal life is examined. Interactive videodisc (IVD) technology is described in light of its potential for laboratory simulations. The design of the IVD-based dissection simulation, The Interactive Frog Dissection, is presented. (KR)

  1. A Dissecting Competition for Medical Students

    ERIC Educational Resources Information Center

    Samalia, Latika; Stringer, Mark D.

    2012-01-01

    After repeated requests from medical students for more cadaver dissection opportunities, a voluntary dissecting "competition" was initiated for the third year medical students in 2006. This has been held annually on five occasions since, offering up to 30 dissection stations and accommodating an average of 53 students (range 40-66) per year,…

  2. Cutting Edge Controversy: The Politics of Animal Dissection and Responses to Student Objection

    NASA Astrophysics Data System (ADS)

    Oakley, Jan

    2011-12-01

    This mixed methods study investigated the experiences and perspectives of former Ontario high school students and current Ontario science and biology teachers toward animal dissection, objection to dissection, and choice policies that grant students the right to opt out of dissection and use an alternative instead. Data was collected via a student questionnaire (n=311), a teacher questionnaire (n=153), and interviews with eight students and nine teachers. Quantitative and qualitative data analyses and reporting techniques were employed within a humane education and critical pedagogy framework to explore the experiences and perspectives of both groups.

  3. Genetic dissection of rice grain shape using a recombinant inbred line population derived from two contrasting parents and fine mapping a pleiotropic quantitative trait locus qGL7

    PubMed Central

    2010-01-01

    Background The three-dimensional shape of grain, measured as grain length, width, and thickness (GL, GW, and GT), is one of the most important components of grain appearance in rice. Determining the genetic basis of variations in grain shape could facilitate efficient improvements in grain appearance. In this study, an F7:8 recombinant inbred line population (RIL) derived from a cross between indica and japonica cultivars (Nanyangzhan and Chuan7) contrasting in grain size was used for quantitative trait locus (QTL) mapping. A genetic linkage map was constructed with 164 simple sequence repeat (SSR) markers. The major aim of this study was to detect a QTL for grain shape and to fine map a minor QTL, qGL7. Results Four QTLs for GL were detected on chromosomes 3 and 7, and 10 QTLs for GW and 9 QTLs for GT were identified on chromosomes 2, 3, 5, 7, 9 and 10, respectively. A total of 28 QTLs were identified, of which several are reported for the first time; four major QTLs and six minor QTLs for grain shape were also commonly detected in both years. The minor QTL, qGL7, exhibited pleiotropic effects on GL, GW, GT, 1000-grain weight (TGW), and spikelets per panicle (SPP) and was further validated in a near isogenic F2 population (NIL-F2). Finally, qGL7 was narrowed down to an interval between InDel marker RID711 and SSR marker RM6389, covering a 258-kb region in the Nipponbare genome, and cosegregated with InDel markers RID710 and RID76. Conclusion Materials with very different phenotypes were used to develop mapping populations to detect QTLs because of their complex genetic background. Progeny tests proved that the minor QTL, qGL7, could display a single mendelian characteristic. Therefore, we suggested that minor QTLs for traits with high heritability could be isolated using a map-based cloning strategy in a large NIL-F2 population. In addition, combinations of different QTLs produced diverse grain shapes, which provide the ability to breed more varieties of rice to satisfy consumer preferences. PMID:20184774

  4. Chemical dissection of endosomal pathways

    PubMed Central

    Drakakaki, Georgia; Robert, Stphanie; Raikhel, Natasha V

    2009-01-01

    Membrane trafficking and associated signal transduction pathways are critical for plant development and responses to environment. These transduction pathways, including those for brassinosteroids and auxins, require endocytosis to endosomes and recycling back to the plasma membrane. A major challenge toward understanding these processes and their biological roles has been the highly dynamic nature of endomembrane trafficking. To effectively study endocytosis and recycling, which occur in a time frame of minutes, bioactive chemicals provide a powerful and exacting tool. Pharmacological inhibitors such as Brefeldin A (BFA) and the newly identified Endosidin 1 (ES1) have been used to define endosome compartments. ES1 is a clear example of the ability of chemicals to dissect even distinct subpopulations of endosomes involved in trafficking and signal transduction. The ability to characterize and dissect such highly dynamic pathways in a temporal and spatial manner is possible only using pharmacological reagents which can act rapidly and reversibly. PMID:19704710

  5. Spontaneous dissection of coronary arteries.

    PubMed

    Patnaik, A N; Rao, D Seshagiri

    2005-01-01

    Spontaneous dissection of coronary arteries is an uncommon entity with varied presentation. It is commoner in young patients, specially females. We present three cases encountered by us in recent past. There were two males and the only female was in her post-partum period. All the three had diverse lines of management based on the angiographic picture, clinical background and myocardium at risk. PMID:16350685

  6. Phosphoproteomics Profiling of Human Skin Fibroblast Cells Reveals Pathways and Proteins Affected by Low Doses of Ionizing Radiation

    PubMed Central

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

    2010-01-01

    Background High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. Principal Findings We have identified 7117 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conclusions Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health. PMID:21152398

  7. On marathons and Sprints: an integrated quantitative proteomics and transcriptomics analysis of differences between slow and fast muscle fibers.

    PubMed

    Drexler, Hannes C A; Ruhs, Aaron; Konzer, Anne; Mendler, Luca; Bruckskotten, Mark; Looso, Mario; Günther, Stefan; Boettger, Thomas; Krüger, Marcus; Braun, Thomas

    2012-06-01

    Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and α-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented resolution. PMID:22210690

  8. Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling.

    PubMed

    Cerny, Martin; Dycka, Filip; Bobl'ov, Janette; Brzobohaty, Bretislav

    2011-01-01

    Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants. PMID:20974740

  9. 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. PMID:25998263

  10. Occurrence and detection of phosphopeptide isomers in large-scale phosphoproteomics experiments.

    PubMed

    Courcelles, Mathieu; Bridon, Gaëlle; Lemieux, Sébastien; Thibault, Pierre

    2012-07-01

    The past decade has been marked by the emergence of selective affinity media and sensitive mass spectrometry instrumentation that facilitated large-scale phosphoproteome analyses and expanded the repertoire of protein phosphorylation. Despite these remarkable advances, the precise location of the phosphorylation site still represents a sizable challenge in view of the labile nature of the phosphoester bond and the presence of neighboring phosphorylatable residues within the same peptide. This difficulty is exacerbated by the combinatorial distribution of phosphorylated residues giving rise to different phosphopeptide isomers. These peptides have similar physicochemical properties, and their separation by LC is often problematic. Few studies have described the frequency and distribution of phosphoisomers in large-scale phosphoproteomics experiments, and no convenient informatics tools currently exist to facilitate their detection. To address this analytical challenge, we developed two algorithms to detect separated and co-eluting phosphopeptide isomers and target their subsequent identification using an inclusion list in LC-MS/MS experiments. Using these algorithms, we determined that the proportion of isomers present in phosphoproteomics studies from mouse, rat, and fly cell extracts represents 3-6% of all identified phosphopeptides. While conventional analysis can identify chromatographically separated phosphopeptides, targeted LC-MS/MS analyses using inclusion lists provided complementary identification and expanded the number of phosphopeptide isomers by at least 52%. Interestingly, these analyses revealed that the occurrence of phosphopeptides isomers can also correlate with the presence of extended phosphorylatable amino acids that can act as a "phosphorylation switch" to bind complementary domains such as those present in SR proteins and ribonucleoprotein complexes. PMID:22668510

  11. Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

    PubMed Central

    Černý, Martin; Dyčka, Filip; Bobál'ová, Janette; Brzobohatý, Břetislav

    2011-01-01

    Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants. PMID:20974740

  12. Optimal Diagnostic Imaging of Aortic Dissection

    PubMed Central

    Wilbers, Christopher R.H.; Carrol, Clark L.; Hnilica, Mark A.

    1990-01-01

    Without prompt diagnosis and treatment, aortic dissection is rapidly fatal. While standard chest radiography may give clues to the diagnosis of aortic dissection, suspected dissection can be confirmed by only 4 imaging techniques: aortography, echocardiography, computed tomography, and magnetic resonance imaging. The following review discusses each of these methods. It also explains why aortography, the previous diagnostic benchmark, has been replaced by newer techniques and why magnetic resonance imaging has become the diagnostic method of choice for imaging aortic dissection. (Texas Heart Institute Journal 1990;17:271-8) Images PMID:15227519

  13. A mill based instrument and software system for dissecting slide-mounted tissue that provides digital guidance and documentation

    PubMed Central

    2013-01-01

    Background Dissection of specific Areas Of Interest (AOIs) of slide-mounted tumor samples is often used to enrich for cancer cells in order to generate better signal to noise ratios in subsequent biochemical characterization. Most clinical laboratories utilize manual dissection for practical reasons and to avoid the expense and difficulties of laser microdissection systems. Unfortunately, manual methods often lack resolution and process documentation. The goal of this project was to design a dissection system for slide-mounted tissue with better precision than manual methods that also provides digital image guidance and electronic process documentation. Methods An instrument that is essentially a micro tissue mill was developed. It employs a specialized disposable mill bit that simultaneously dispenses liquid, cuts tissue from the slide surface, and aspirates the liquid along with the displaced tissue fragments. A software package was also developed that is capable of transferring digitally annotated AOIs between images of serially cut tissue sections to guide dissection and generate an electronic record of the process. Results The performance of this “meso” dissection system was tested using post dissection visual examination for resolution and accuracy, fluorescence based DNA quantitation for recovery efficiency, and dissection of closely situated mouse-human tissue sections followed by PCR amplification for purity determination. The minimum resolution is a dissected circle smaller than 200 microns in diameter, edge dissection accuracy is tighter than 100 microns, recovery efficiency appears greater than 95%, and recovery purity is greater than 99% relative to a different tissue located 100 microns from the dissection boundary. The system can dissect from both paraffinized and deparaffinized FFPE tissue sections that are mounted on plain glass slides, and it is compatible with DNA, RNA, and protein isolation. Conclusions The mesodissection system is an effective alternative to manual dissection methods and is applicable for biomarker analysis of anatomical pathology samples, where enrichment of AOIs from the tissue section is helpful, but pure cell populations are not required. PMID:24188417

  14. Evaluation of Educator & Student Use of & Attitudes toward Dissection & Dissection Alternatives

    ERIC Educational Resources Information Center

    Osenkowski, Pamela; Green, Che; Tjaden, Anne; Cunniff, Peggy

    2015-01-01

    Animal dissection has been routinely practiced in American biology classrooms for decades. With technological advancements, more states adopting student choice measures, and increased awareness about ethical concerns surrounding dissection, many useful dissection alternatives have been developed. To understand the current use of animal dissection…

  15. 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

  16. An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome.

    PubMed

    Zappacosta, Francesca; Scott, Gilbert F; Huddleston, Michael J; Annan, Roland S

    2015-02-01

    While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications. Here we present refinements to our overall protocol that make it simpler and more efficient, while they provide greater coverage of the phosphoproteome. We introduce filter-aided sample prep (FASP) for cell lysis and trypsin digestion. Following HILIC separation, fractions are IMAC enriched using a 96-well filter plate. Finally, enriched samples are analyzed using an LC-MS strategy optimized for the fractionation scheme. The optimized protocol improves protein recovery, simplifies phosphopeptide enrichment, and optimizes instrument time, while it maintains deep coverage of the phosphoproteome. By using the refined protocol, we identified more than 16,000 unique phosphosites from rat liver in a single experiment, which used approximately 1 day of instrument time. All together, we present evidence for 24,485 rat liver phosphosites that represents the deepest coverage of a tissue phosphoproteome to date. PMID:25575281

  17. Phosphoproteome mapping of cardiomyocyte mitochondria in a rat model of heart failure.

    PubMed

    Giorgianni, Francesco; Usman Khan, M; Weber, Karl T; Gerling, Ivan C; Beranova-Giorgianni, Sarka

    2014-04-01

    Mitochondria are complex organelles essential to cardiomyocyte survival. Protein phosphorylation is emerging as a key regulator of mitochondrial function. In the study reported here, we analyzed subsarcolemmal (SSM) mitochondria harvested from rats who have received 4weeks of aldosterone/salt treatment to simulate the neurohormonal profile of human congestive heart failure. Our objective was to obtain an initial qualitative inventory of the phosphoproteins in this biologic system. SSM mitochondria were harvested, and the phosphoproteome was analyzed with a gel-free bioanalytical platform. Mitochondrial proteins were digested with trypsin, and the digests were enriched for phosphopeptides with immobilized metal ion affinity chromatography. The phosphopeptides were analyzed by ion trap liquid chromatography-tandem mass spectrometry, and the phosphoproteins identified via database searches. Based on MS/MS and MS(3) data, we characterized a set of 42 phosphopeptides that encompassed 39 phosphorylation sites. These peptides mapped to 26 proteins, for example, long-chain specific acyl-CoA dehydrogenase, Complex III subunit 6, and mitochondrial import receptor TOM70. Collectively, the characterized phosphoproteins belong to diverse functional modules, including bioenergetic pathways, protein import machinery, and calcium handling. The phosphoprotein panel discovered in this study provides a foundation for future differential phosphoproteome profiling toward an integrated understanding of the role of mitochondrial phosphorylation in heart failure. PMID:24395194

  18. Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase Substrates and Phosphorylation Networks1[W

    PubMed Central

    Reiland, Sonja; Messerli, Galle; Baerenfaller, Katja; Gerrits, Bertran; Endler, Anne; Grossmann, Jonas; Gruissem, Wilhelm; Baginsky, Sacha

    2009-01-01

    We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as well as two new motifs at the carboxyl terminus of ribosomal proteins. Using the phosphorylation motifs as a footprint for the activity of a specific kinase class, we connected the phosphoproteins with their putative kinases and constructed a chloroplast CKII phosphorylation network. The network topology suggests that CKII is a central regulator of different chloroplast functions. To provide insights into the dynamic regulation of protein phosphorylation, we analyzed the phosphoproteome at the end of day and end of night. The results revealed only minor changes in chloroplast kinase activities and phosphorylation site utilization. A notable exception was ATP synthase ?-subunit, which is found phosphorylated at CKII phosphorylation sites preferentially in the dark. We propose that ATP synthase is regulated in cooperation with 14-3-3 proteins by CKII-mediated phosphorylation of ATP synthase ?-subunit in the dark. PMID:19376835

  19. Analysis of the Phosphoproteome of Chlamydomonas reinhardtii Provides New Insights into Various Cellular Pathways

    PubMed Central

    Wagner, Volker; Gener, Gunther; Heiland, Ines; Kaminski, Marc; Hawat, Susan; Scheffler, Kai; Mittag, Maria

    2006-01-01

    The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases. PMID:16524901

  20. Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways.

    PubMed

    Wagner, Volker; Gessner, Gunther; Heiland, Ines; Kaminski, Marc; Hawat, Susan; Scheffler, Kai; Mittag, Maria

    2006-03-01

    The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases. PMID:16524901

  1. Phosphoproteome Dynamics in Onset and Maintenance of Oncogene-induced Senescence*

    PubMed Central

    de Graaf, Erik L.; Kaplon, Joanna; Zhou, Houjiang; Heck, Albert J. R.; Peeper, Daniel S.; Altelaar, A. F. Maarten

    2014-01-01

    Expression of the BRAFV600E oncoprotein is known to cause benign lesions, such as melanocytic nevi (moles). Despite the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called oncogene-induced senescence. Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor-suppression mechanism, we performed a mass-spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells and in cells with abrogated senescence. Proteome analysis of senescent cells revealed the up-regulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti4+-IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK, and CDK-retinoblastoma pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAFV600E-expressing senescent cells provides molecular clues as to how oncogene-induced senescence is initiated, maintained, or evaded, serving as a comprehensive proteomic basis for functional validation. PMID:24961811

  2. Phosphoproteomic Analysis Using the WW and FHA Domains as Biological Filters.

    PubMed

    Hasanuzzaman Shohag, Md; Nishioka, Tomoki; Uddin Ahammad, Rijwan; Nakamuta, Shinichi; Yura, Yoshimitsu; Hamaguchi, Tomonari; Kaibuchi, Kozo; Amano, Mutsuki

    2015-01-01

    Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3? to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pin1-WW, CHEK2-FHA, and DLG1-GK. Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis. PMID:26119529

  3. The Global Phosphoproteome of Chlamydomonas reinhardtii Reveals Complex Organellar Phosphorylation in the Flagella and Thylakoid Membrane *

    PubMed Central

    Wang, Hongxia; Gau, Brian; Slade, William O.; Juergens, Matthew; Li, Ping; Hicks, Leslie M.

    2014-01-01

    Chlamydomonas reinhardtii is the most intensively-studied and well-developed model for investigation of a wide-range of microalgal processes ranging from basic development through understanding triacylglycerol production. Although proteomic technologies permit interrogation of these processes at the protein level and efforts to date indicate phosphorylation-based regulation of proteins in C. reinhardtii is essential for its underlying biology, characterization of the C. reinhardtii phosphoproteome has been limited. Herein, we report the richest exploration of the C. reinhardtii proteome to date. Complementary enrichment strategies were used to detect 4588 phosphoproteins distributed among every cellular component in C. reinhardtii. Additionally, we report 18,160 unique phosphopeptides at <1% false discovery rate, which comprise 15,862 unique phosphosites - 98% of which are novel. Given that an estimated 30% of proteins in a eukaryotic cell are subject to phosphorylation, we report the majority of the phosphoproteome (23%) of C. reinhardtii. Proteins in key biological pathways were phosphorylated, including photosynthesis, pigment production, carbon assimilation, glycolysis, and protein and carbohydrate metabolism, and it is noteworthy that hyperphosphorylation was observed in flagellar proteins. This rich data set is available via ProteomeXchange (ID: PXD000783) and will significantly enhance understanding of a range of regulatory mechanisms controlling a variety of cellular process and will serve as a critical resource for the microalgal community. PMID:24917610

  4. Changes of proteome and phosphoproteome trigger embryo-larva transition of honeybee worker (Apis mellifera ligustica).

    PubMed

    Gala, Alemayehu; Fang, Yu; Woltedji, Dereje; Zhang, Lan; Han, Bin; Feng, Mao; Li, Jianke

    2013-01-14

    The development of the last day embryo to the first instar larva is an essential process in the honeybee life cycle. However, the molecular mechanism of this life transition is still unknown. The proteome and phosphoproteome of last day embryos (72 h) and first instar larvae (24h, post hatching) were analyzed using 2-DE, multiplex fluorescent staining, mass spectrometry, bioinformatics, and qRT-PCR. Sixty-five proteins and 34 phosphoproteins changed their expression across the shift of embryos to larvae. The embryo stronger expression of proteins related to energy metabolism, development and amino acid metabolism suggests its high metabolic energy demand during active embryogenesis. While, the newly hatched larvae escalated the expression of proteins related to cytoskeleton, biosynthesis, protein folding, fatty acid and oxidative metabolism, particularly the higher phosphorylation of cytoskeleton and biosynthesis indicates their roles to ensure the fast growing larvae. These differences in protein expression level illustrate that specific protein functions are restricted to particular developmental stage. Our data suggest the essential changes of proteome and phosphoproteome to trigger the transition of embryo to larvae. This unravels the molecular event behind the first life cycle transition of honeybees and is potentially helpful for future reverse genetic studies in this model insect. PMID:23088927

  5. Phosphoproteomic Analysis of Protein Phosphorylation Networks in the Hypopharyngeal Gland of Honeybee Workers (Apis mellifera ligustica).

    PubMed

    Qi, Yuping; Fan, Pei; Hao, Yue; Han, Bin; Fang, Yu; Feng, Mao; Cui, Ziyou; Li, Jianke

    2015-11-01

    The hypopharyngeal gland (HG) in honeybee workers changes functions according to physiological age in the bee colony from producing royal jelly (RJ) in nurse bees to digestive enzymes in foragers. The same set of secretory cells expresses different genes or proteins to create these age-dependent changes; however, it is unknown precisely how the phosphorylation network regulates physiological differences across the development of the adult worker HG. We employed high-accuracy mass-spectrometry-based proteomics to survey phosphoproteome changes in the newly emerged, nurse, and forager bees. Overall, 941, 1322, and 1196 phosphorylation sites matching 1007, 1353, and 1199 phosphopeptides from 549, 720, and 698 phosphoproteins were identified in the three ages of the HG, respectively. Specialized, interconnected phosphorylation networks within each age were found by comparing protein abundance and phosphorylation levels. This illustrates that many proteins are regulated by phosphorylation independent of their expression levels. Furthermore, proteins in key biological processes and pathways were dynamically phosphorylated with age development, including the centrosome cycle, mitotic spindle elongation, macromolecular complex disassembly, and ribosome, indicating that phosphorylation tunes protein activity to optimize cellular behavior of the HG over time. Moreover, complementary protein and phosphoprotein expression is required to support the unique physiology of secretory activity in the HG. This reported data set of the honeybee phosphoproteome significantly improves our understanding of a range of regulatory mechanisms controlling a variety of cellular processes and will serve as a valuable resource for those studying the honeybee and other insects. PMID:26384081

  6. Biomarkers in descending thoracic aortic dissection.

    PubMed

    Shalhub, Sherene; Dua, Anahita; Brooks, Jared

    2014-12-01

    The clinical application of serum biomarkers (d-dimer, C-reactive protein) to predict the natural history of descending thoracic aortic dissection remains elusive. In this review, our current understanding of biomarkers in descending thoracic aortic dissection detection, predicting complications, and aiding in patient management is discussed. PMID:26073830

  7. Quick Dissection of the Segmental Bronchi

    ERIC Educational Resources Information Center

    Nakajima, Yuji

    2010-01-01

    Knowledge of the three-dimensional anatomy of the bronchopulmonary segments is essential for respiratory medicine. This report describes a quick guide for dissecting the segmental bronchi in formaldehyde-fixed human material. All segmental bronchi are easy to dissect, and thus, this exercise will help medical students to better understand the

  8. Headache in Intracranial and Cervical Artery Dissections.

    PubMed

    Sheikh, Huma U

    2016-01-01

    Dissection refers to a tear in the wall of an artery, with the two main types being intracranial or extracranial. Dissections tend to occur most commonly in the young, sometimes secondary to trauma involving the neck. To confirm a dissection, some type of vessel imaging is necessary, including magnetic resonance angiography (MRA), computed tomography angiography (CTA), or angiography. The most common presentation of a dissection (especially extracranial) is pain, usually head and neck pain along with a Horner's syndrome. Patients may also present with ischemic symptoms, including transient ischemic attack (TIA) or stroke, which may also be a complication of a dissection. Although headache is a common presentation, there is little research into phenotype or long-term outcomes. There are a number of case reports detailing the phenotypes of headaches that may be present in dissection, including a migraine-like or hemicrania-like headache. Dissections are usually treated with some type of anti-platelet or anti-coagulation, although there are only a few randomized controlled trials. In a new acute headache, dissection is an important diagnosis to keep in mind. PMID:26757710

  9. Keeping Dissection Alive for Medical Students

    ERIC Educational Resources Information Center

    Chambers, James; Emlyn-Jones, Daniel

    2009-01-01

    Traditional dissection teaching is being reduced in a number of medical schools, particularly in the United Kingdom. In response to this, 12 medical students from Warwick University, UK, traveled to the Island of Grenada for an intensive extracurricular dissection course at St. George's University. This course not only benefited the host

  10. Objecting To Dissection: A College Student's Handbook.

    ERIC Educational Resources Information Center

    National Anti-Vivisection Society, Chicago, IL.

    In a number of states, students from kindergarten through high school have won the right to refuse to dissect or kill animals and the right to substitute an alternative project. This booklet was designed to help college science students take an ethical stand by refusing to participate in dissection exercises. The booklet begins with an overview of

  11. Aortic Dissection Type A in Alpine Skiers

    PubMed Central

    Schachner, Thomas; Fischler, Nikolaus; Dumfarth, Julia; Bonaros, Nikolaos; Krapf, Christoph; Schobersberger, Wolfgang; Grimm, Michael

    2013-01-01

    Patients and Methods. 140 patients with aortic dissection type A were admitted for cardiac surgery. Seventy-seven patients experienced their dissection in the winter season (from November to April). We analyzed cases of ascending aortic dissection associated with alpine skiing. Results. In 17 patients we found skiing-related aortic dissections. Skiers were taller (180 (172–200) cm versus 175 (157–191) cm, P = 0.008) and heavier (90 (68–125) kg versus 80 (45–110) kg, P = 0.002) than nonskiers. An extension of aortic dissection into the aortic arch, the descending thoracic aorta, and the abdominal aorta was found in 91%, 74%, and 69%, respectively, with no significant difference between skiers and nonskiers. Skiers experienced RCA ostium dissection requiring CABG in 17.6% while this was true for 5% of nonskiers (P = 0.086). Hospital mortality of skiers was 6% versus 13% in nonskiers (P = 0.399). The skiers live at an altitude of 170 (0–853) m.a.s.l. and experience their dissection at 1602 (1185–3105; P < 0.001) m.a.s.l. In 82% symptom start was during recreational skiing without any trauma. Conclusion. Skiing associated aortic dissection type A is usually nontraumatic. The persons affected live at low altitudes and practice an outdoor sport at unusual high altitude at cold temperatures. Postoperative outcome is good. PMID:23971024

  12. Beyond Dissection: Innovative Tools for Biology Education.

    ERIC Educational Resources Information Center

    Larson, Sandra, Ed.

    This catalog lists resources available for classroom use in teaching about anatomy and physiology which are alternatives to dissection. The entries are provided under three main categories: (1) Whole Animal Dissection/Vivisection; (2) Animal Organ or System Anatomy and Physiology; and (3) Other, including animal behavior, biotechnology,…

  13. Objecting To Dissection: A College Student's Handbook.

    ERIC Educational Resources Information Center

    National Anti-Vivisection Society, Chicago, IL.

    In a number of states, students from kindergarten through high school have won the right to refuse to dissect or kill animals and the right to substitute an alternative project. This booklet was designed to help college science students take an ethical stand by refusing to participate in dissection exercises. The booklet begins with an overview of…

  14. [Internal carotid artery dissection after Heimlich maneuver].

    PubMed

    Rakotoharinandrasana, H; Petit, E; Dumas, P; Vandermarcq, P; Gil, R; Neau, J-Ph

    2003-01-01

    We report a case of cervical artery dissection following a Heimlich maneuver. Cervical artery dissections are at the present time well known and are sometimes associated with trivial traumas. However, to our knowledge, this complication of such maneuver was never reported in the literature. Pathophysiological mechanisms are discussed. PMID:12738019

  15. Spontaneous Coronary Artery Dissection with Cardiac Tamponade

    PubMed Central

    Lundstrom, Robert J.

    2015-01-01

    Spontaneous coronary artery dissection is a rare cause of acute coronary syndrome. Clinical presentation ranges from chest pain alone to ST-segment-elevation myocardial infarction, ventricular fibrillation, and sudden death. The treatment of patients with spontaneous coronary artery dissection is challenging because the disease pathophysiology is unclear, optimal treatment is unknown, and short- and long-term prognostic data are minimal. We report the case of a 70-year-old woman who presented with an acute ST-segment-elevation myocardial infarction secondary to a spontaneous dissection of the left anterior descending coronary artery. She was treated conservatively. Cardiac tamponade developed 16 hours after presentation. Repeat coronary angiography revealed extension of the dissection. Medical therapy was continued after the hemopericardium was aspirated. The patient remained asymptomatic 3 years after hospital discharge. To our knowledge, this is the first reported case of spontaneous coronary artery dissection in association with cardiac tamponade that was treated conservatively and had a successful outcome. PMID:26504447

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

    PubMed

    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(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 ? were further confirmed by Mn(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. PMID:25448045

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

    PubMed Central

    Domanski, Dominik; Helbing, Caren C

    2007-01-01

    Background Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone. PMID:17683616

  18. Videoendoscopic single-port axillary dissection

    PubMed Central

    Uras, Cihan; Aytac, Erman; Aydogan, Fatih

    2011-01-01

    Videoendoscopy is newly used in breast and axillary surgery. Single-port surgery is one of the newest methods of minimally invasive surgery. This report describes the first case of videoendoscopic single-port axillary dissection. In histopathological evaluation, 24 lymph nodes were identified and one node was infiltrated by the cancer cells. Videoendoscopic single-port axillary dissection is a precise and improvable technique. Single-port videoendoscopic axillary dissection could be more feasible with individual tools that will be designed for minimally invasive breast surgery. PMID:22022116

  19. The coming of age of phosphoproteomics--from large data sets to inference of protein functions.

    PubMed

    Roux, Philippe P; Thibault, Pierre

    2013-12-01

    Protein phosphorylation is one of the most common post-translational modifications used in signal transduction to control cell growth, proliferation, and survival in response to both intracellular and extracellular stimuli. This modification is finely coordinated by a network of kinases and phosphatases that recognize unique sequence motifs and/or mediate their functions through scaffold and adaptor proteins. Detailed information on the nature of kinase substrates and site-specific phosphoregulation is required in order for one to better understand their pathophysiological roles. Recent advances in affinity chromatography and mass spectrometry (MS) sensitivity have enabled the large-scale identification and profiling of protein phosphorylation, but appropriate follow-up experiments are required in order to ascertain the functional significance of identified phosphorylation sites. In this review, we present meaningful technical details for MS-based phosphoproteomic analyses and describe important considerations for the selection of model systems and the functional characterization of identified phosphorylation sites. PMID:24037665

  20. Activation of aortic endothelial cells by oxidized phospholipids: a phosphoproteomic analysis

    PubMed Central

    Zimman, Alejandro; Chen, Sharon S.; Komisopoulou, Evangelia; Titz, Bjoern; Martnez-Pinna, Roxana; Kafi, Aarya; Berliner, Judith A.; Graeber, Thomas G.

    2010-01-01

    Previous studies have shown that oxidized products of the phospholipid PAPC (Ox-PAPC) are strong activators of aortic endothelial cells and play an important role in atherosclerosis and other inflammatory diseases. We and others have demonstrated that Ox-PAPC activates specific signaling pathways and regulates a large number of genes. Using a phosphoproteomic approach based on phosphopeptide enrichment and mass spectrometry analysis, we identified candidate changes in Ox-PAPC-induced protein phosphorylation of 228 proteins. Functional annotation of these proteins showed an enrichment of the regulation of cytoskeleton, junctional components, and tyrosine kinases, all of which may contribute to the phenotypic and molecular changes observed in endothelial cells treated with Ox-PAPC. Many changes in protein phosphorylation induced by Ox-PAPC are reported here for the first time and provide new insights into the mechanism of activation by oxidized lipids, including phosphorylation-based signal transduction. PMID:20307106

  1. Development of the affinity materials for phosphorylated proteins/peptides enrichment in phosphoproteomics analysis.

    PubMed

    Wang, Zhi-Gang; Lv, Nan; Bi, Wen-Zhi; Zhang, Ji-Lin; Ni, Jia-Zuan

    2015-04-29

    Reversible protein phosphorylation is a key event in numerous biological processes. Mass spectrometry (MS) is the most powerful analysis tool in modern phosphoproteomics. However, the direct MS analysis of phosphorylated proteins/peptides is still a big challenge because of the low abundance and insufficient ionization of phosphorylated proteins/peptides as well as the suppression effects of nontargets. Enrichment of phosphorylated proteins/peptides by affinity materials from complex biosamples is the most widely used strategy to enhance the MS detection. The demand of efficiently enriching phosphorylated proteins/peptides has spawned diverse affinity materials based on different enrichment principles (e.g., electronic attraction, chelating). In this review, we summarize the recent development of various affinity materials for phosphorylated proteins/peptides enrichment. We will highlight the design and fabrication of these affinity materials, discuss the enrichment mechanisms involved in different affinity materials, and suggest the future challenges and research directions in this field. PMID:25845677

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

    PubMed Central

    Rampitsch, Christof; Bykova, Natalia V.

    2012-01-01

    This review examines why a knowledge of plant protein phosphorylation events is important in devising strategies to protect crops from both biotic and abiotic stresses, and why proteomics should be included when studying stress pathways. Most of the achievements in elucidating phospho-signaling pathways in biotic and abiotic stress are reported from model systems: while these are discussed, this review attempts mainly to focus on work done with crops, with examples of achievements reported from rice, maize, wheat, grape, Brassica, tomato, and soy bean after cold acclimation, hormonal and oxidative hydrogen peroxide treatment, salt stress, mechanical wounding, or pathogen challenge. The challenges that remain to transfer this information into a format that can be used to protect crops against biotic and abiotic stresses are enormous. The tremendous increase in the speed and ease of DNA sequencing is poised to reveal the whole genomes of many crop species in the near future, which will facilitate phosphoproteomics and phosphogenomics research. PMID:22783265

  3. Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation.

    PubMed

    Lin, Miao-Hsia; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-09-15

    Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. PMID:26373674

  4. 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; Gelp, Emilio; Abian, Joaquin

    2010-02-01

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines. PMID:19941383

  5. Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages

    PubMed Central

    Chamberlain, Michael Dean; Wells, Laura A.; Lisovsky, Alexandra; Guo, Hongbo; Isserlin, Ruth; Talior-Volodarsky, Ilana; Mahou, Redouan; Emili, Andrew; Sefton, Michael V.

    2015-01-01

    An unbiased phosphoproteomic method was used to identify biomaterial-associated changes in the phosphorylation patterns of macrophage-like cells. The phosphorylation differences between differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with a vascular regenerative methacrylic acid (MAA) copolymer or a control methyl methacrylate (MM) copolymer were determined by MS. There were 1,470 peptides (corresponding to 729 proteins) that were differentially phosphorylated in dTHP1 cells treated with the two materials with a greater cellular response to MAA treatment. In addition to identifying pathways (such as integrin signaling and cytoskeletal arrangement) that are well known to change with cell–material interaction, previously unidentified pathways, such as apoptosis and mRNA splicing, were also discovered. PMID:26261332

  6. Application of metal-chelate affinity chromatography to the study of the phosphoproteome.

    PubMed

    Imam-Sghiouar, N; Joubert-Caron, R; Caron, M

    2005-02-01

    With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe(3+) was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed. PMID:15645166

  7. 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.

  8. GPM Dissects Typhoon Hagupit - Duration: 38 seconds.

    NASA Video Gallery

    NASA/JAXA's GPM Dissects Typhoon Hagupit Animation revealing a swath of NASA/JAXA's Global Precipitation Measurement (GPM) mission's Core Observatory GMI precipitation rates over Typhoon Hagupit. A...

  9. Animal Rights Groups Target High School Dissection.

    ERIC Educational Resources Information Center

    Trotter, Andrew

    1992-01-01

    Two groups leading the charge against dissection are People for the Ethical Treatment of Animals (PETA) and the Student Action Corps for Animals (SACA). Protests by student and community members remain the movement's strongest weapon. (MLF)

  10. Phosphoproteome Profiling of Human Skin Fibroplast Cells in Response to Low- and High-Dose Irradiation

    SciTech Connect

    Yang, Feng; Stenoien, David L.; Strittmatter, Eric F.; Wang, Jeng-Han; Ding, Lianghao; Lipton, Mary S.; Monroe, Matthew E.; Nicora, Carrie D.; Gritsenko, Marina A.; Tang, Keqi; Fang, Ruihua; Adkins, Joshua N.; Camp, David G.; Chen, David J.; Smith, Richard D.

    2006-05-01

    The biological effect of low-dose radiation is currently not well understood. A hallmark of the response to radiation is the phosphorylation of proteins involved in DNA repair, DNA damage signaling, and cell cycle checkpoint control, which is important in prompt cellular response. The objective of the work presented here was to explore the phosphoproteome of normal human skin fibroblast (HSF) cells to reveal differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques Trizol extract of proteins, methylation of the enzyme digest (peptides), enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry were combined for analysis of the HSF cell phosphoproteome following low- and high-doses of irradiation. More than 95% of the peptides identified after IMAC enrichment were phosphopeptides. Among the 493 unique phosphopeptides, 232 were singly phosphorylated, 220 were doubly phosphorylated, and 41 were triply phosphorylated, indicating the overall effectiveness of the IMAC technique to enrich both singly and multiple phosphorylated peptides. Over 700 phosphorylation sites were assigned to a total of 346 proteins, many of which are known or proposed to be highly relevant to a plethora of fundamental biological processes. The profile for proteins identified from the low-dose (2cGy) irradiated HSF cells was shown to be different from the profile obtained for proteins irradiated at the high-dose (4 Gy). This type of fundamental information regarding radiation-response to cellular events at the molecular level provides a mechanistic basis for identifying relevant molecular markers that can be used in future to better evaluate human health risks at low doses of irradiation and to develop low dose radiation counter measurements.

  11. 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

  12. Multiplexed Detection of O-GlcNAcome, Phosphoproteome, and Whole Proteome within the Same Gel

    PubMed Central

    Cieniewski-Bernard, Caroline; Dupont, Erwan; Deracinois, Barbara; Lambert, Matthias; Bastide, Bruno

    2014-01-01

    The cellular diversity of proteins results in part from their post-translational modifications. Among all of them, the O-GlcNAcylation is an atypical glycosylation, more similar to phosphorylation than classical glycosylations. Highly dynamic, reversible, and exclusively localized on cytosolic, nuclear, and mitochondrial proteins, O-GlcNAcylation is known to regulate almost all if not all cellular processes. Fundamental for the cell life, O-GlcNAcylation abnormalities are involved in the etiology of several inherited diseases. Assessing to O-GlcNAcylation pattern will permit to get relevant data about the role of O-GlcNAcylation in cell physiology. To get understanding about the role of O-GlcNAcylation, as also considering its interplay with phosphorylation, the O-GlcNAc profiling remains a real challenge for the community of proteomists/glycoproteomists. The development of multiplexed proteomics based on fluorescent detection of proteins permits to go further in the understanding of the proteome complexity. We propose herein a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. In particular, we investigated the phosphoproteome through the ProQ Diamond staining, while the whole proteome was visualized through Sypro Ruby staining, or after the labeling of proteins with a T-Dye fluorophore. The O-GlcNAcome was revealed by the way of the Click chemistry and the azidealkyne cycloaddition of a fluorophore on GlcNAc moieties. This method permits, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome. PMID:25389416

  13. Plasma phosphoproteome and differential plasma phosphoproteins with opisthorchis viverrini-related cholangiocarcinoma.

    PubMed

    Kotawong, Kanawut; Thitapakorn, Veerachai; Roytrakul, Sittiruk; Phaonakrop, Narumon; Viyanant, Vithoon; Na-Bangchang, Kesara

    2015-01-01

    This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis. PMID:25735322

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

    PubMed Central

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

    2015-01-01

    Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LCMS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing. PMID:25690035

  15. Phosphoproteomic analysis of the highly-metastatic hepatocellular carcinoma cell line, MHCC97-H.

    PubMed

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

    2015-01-01

    Invasion and metastasis of hepatocellular carcinoma (HCC) is a major cause for lethal liver cancer. Signaling pathways associated with cancer progression are frequently reconfigured by aberrant phosphorylation of key proteins. To capture the key phosphorylation events in HCC metastasis, we established a methodology by an off-line high-pH HPLC separation strategy combined with multi-step IMAC and LC-MS/MS to study the phosphoproteome of a metastatic HCC cell line, MHCC97-H (high metastasis). In total, 6593 phosphopeptides with 6420 phosphorylation sites (p-sites) of 2930 phosphoproteins were identified. Statistical analysis of gene ontology (GO) categories for the identified phosphoproteins showed that several of the biological processes, such as transcriptional regulation, mRNA processing and RNA splicing, were over-represented. Further analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations demonstrated that phosphoproteins in multiple pathways, such as spliceosome, the insulin signaling pathway and the cell cycle, were significantly enriched. In particular, we compared our dataset with a previously published phosphoproteome in a normal liver sample, and the results revealed that a number of proteins in the spliceosome pathway, such as U2 small nuclear RNA Auxiliary Factor 2 (U2AF2), Eukaryotic Initiation Factor 4A-III (EIF4A3), Cell Division Cycle 5-Like (CDC5L) and Survival Motor Neuron Domain Containing 1 (SMNDC1), were exclusively identified as phosphoproteins only in the MHCC97-H cell line. These results indicated that the phosphorylation of spliceosome proteins may participate in the metastasis of HCC by regulating mRNA processing and RNA splicing. PMID:25690035

  16. Identification of kinase inhibitor targets in the lung cancer microenvironment by chemical and phosphoproteomics

    PubMed Central

    Gridling, Manuela; Ficarro, Scott B.; Breitwieser, Florian P.; Song, Lanxi; Parapatics, Katja; Colinge, Jacques; Haura, Eric B.; Marto, Jarrod A.; Superti-Furga, Giulio; Bennett, Keiryn L.; Rix, Uwe

    2014-01-01

    A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. Whereas these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib) and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes and signaling pathways that are simultaneously engaged by multi-targeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments. PMID:25189542

  17. 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; Jrgensen, Claus

    2016-01-01

    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. PMID:26861043

  18. 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 study provides a global landscape of phosphoregulation in the testis, and should prove to be of value in future studies of spermatogenesis. PMID:25293948

  19. Thrombolytic therapy in spontaneous coronary artery dissection.

    PubMed

    Behnam, R; Tillinghast, S

    1991-07-01

    A 42-year-old female with no cardiac risk factors had an acute anterolateral myocardial infarction treated with intravenous (IV) tissue plasminogen activator (t-PA). Selective coronary cineangiography a week later revealed extensive dissection of the left anterior descending coronary artery (LAD) and its second diagonal branch. Sixteen months later, she is asymptomatic. This is the fifth reported case of spontaneous coronary artery dissection treated with thrombolytic therapy during the acute event with uneventful recovery. PMID:1747972

  20. Postoperative hypertension after radical neck dissection.

    PubMed

    Celikkanat, S; Akyol, M U; Ko, C; Oler, S; Ensari, S; Turgut, S; Ozdem, C

    1997-07-01

    Postoperative hypertension after radical neck dissection was detected in 20.2% of 109 neck dissections in our department between 1989 and 1993. It was probably caused by carotid sinus denervation and appeared after the vasodilation generated by anesthesia had subsided. If postoperative hypertension was encountered after the first operation, the risk of such hypertension after surgery on the contralateral side significantly increased. PMID:9230330

  1. Peripartum presentation of an acute aortic dissection.

    PubMed

    Lewis, S; Ryder, I; Lovell, A T

    2005-04-01

    We report the case of an acute type A aortic dissection occurring in a 35-year-old parturient. The initial diagnosis was missed; a subsequent emergency Caesarean section 3 weeks after presentation was followed by the development of left ventricular failure and pulmonary oedema in the early postoperative period. Echocardiography confirmed the diagnosis of aortic dissection and the patient underwent a successful surgical repair. PMID:15640303

  2. Phosphoproteomic analysis reveals an intrinsic pathway for histone deacetylase 7 regulation that controls cytotoxic T lymphocyte function

    PubMed Central

    Navarro, Maria N.; Goebel, Jurgen; Feijoo-Carnero, Carmen; Morrice, Nick; Cantrell, Doreen A.

    2011-01-01

    The present study reports an unbiased analysis of cytotoxic T cell serine-threonine phosphoproteome using high resolution mass spectrometry. Approximately 2,000 phosphorylations were identified in CTLs of which approximately 450 were controlled by TCR signaling. A significantly overrepresented group of molecules identified were transcription activators, co-repressors and chromatin regulators. A focus on chromatin regulators revealed that CTLs have high expression of histone deacetylase HDAC7 but continually phosphorylate and export this transcriptional repressor from the nucleus. HDAC7 dephosphorylation results in its nuclear accumulation and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. The screening of CTL phosphoproteome thus reveals intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program. PMID:21399638

  3. Molecular Dissection of Phage Endolysin

    PubMed Central

    Pohane, Amol Arunrao; Joshi, Himanshu; Jain, Vikas

    2014-01-01

    Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages. PMID:24627486

  4. The Effect of Animal Dissections on Student Acquisition of Knowledge of and Attitudes toward the Animals Dissected.

    ERIC Educational Resources Information Center

    McCollum, Terry L.

    A conflict exists over the use of animals in the classroom. One aspect of this use involved the dissection of animals. Animal protection advocates report that dissections constitute abuse of the animals dissected. The advocates state that what is learned by dissection could be more effectively learned by other means. Some science educators state

  5. Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.).

    PubMed

    Yang, Zhong-Bao; Eticha, Dejene; Fhrs, Hendrik; Heintz, Dimitri; Ayoub, Daniel; Van Dorsselaer, Alain; Schlingmann, Barbara; Rao, Idupulapati Madhusudana; Braun, Hans-Peter; Horst, Walter Johannes

    2013-12-01

    Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure. PMID:24123251

  6. Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

    PubMed Central

    Horst, Walter Johannes

    2013-01-01

    Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure. PMID:24123251

  7. In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers*

    PubMed Central

    Iliuk, Anton B.; Martin, Victoria A.; Alicie, Bethany M.; Geahlen, Robert L.; Tao, W. Andy

    2010-01-01

    The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro- and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. PMID:20562096

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

    PubMed Central

    2012-01-01

    Background Genome-wide statistics established that long intrinsically disordered regions (over 30 residues) are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. Results Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation). It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. Conclusions The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed. PMID:23110452

  9. Predicting flow in aortic dissection: comparison of computational model with PC-MRI velocity measurements.

    PubMed

    Cheng, Z; Juli, C; Wood, N B; Gibbs, R G J; Xu, X Y

    2014-09-01

    Aortic dissection is a life-threatening process in which the weakened wall develops a tear, causing separation of wall layers. The dissected layers separate the original true aortic lumen and a newly created false lumen. If untreated, the condition can be fatal. Flow rate in the false lumen is a key feature for false lumen patency, which has been regarded as one of the most important predictors of adverse early and later outcomes. Detailed flow analysis in the dissected aorta may assist vascular surgeons in making treatment decisions, but computational models to simulate flow in aortic dissections often involve several assumptions. The purpose of this study is to assess the computational models adopted in previous studies by comparison with in vivo velocity data obtained by means of phase-contrast magnetic resonance imaging (PC-MRI). Aortic dissection geometry was reconstructed from computed tomography (CT) images, while PC-MRI velocity data were used to define inflow conditions and to provide distal velocity components for comparison with the simulation results. The computational fluid dynamics (CFD) simulation incorporated a laminar-turbulent transition model, which is necessary for adequate flow simulation in aortic conditions. Velocity contours from PC-MRI and CFD in the two lumens at the distal plane were compared at four representative time points in the pulse cycle. The computational model successfully captured the complex regions of flow reversal and recirculation qualitatively, although quantitative differences exist. With a rigid wall assumption and exclusion of arch branches, the CFD model over-predicted the false lumen flow rate by 25% at peak systole. Nevertheless, an overall good agreement was achieved, confirming the physiological relevance and validity of the computational model for type B aortic dissection with a relatively stiff dissection flap. PMID:25070022

  10. Student injuries in the dissecting room.

    PubMed

    Cornwall, Jon; Davies, Tilman M; Lees, David

    2013-01-01

    Cadaver dissection is the first opportunity for many students to practice handling human tissue and is their first exposure to the occupational hazards involved with this task. Few studies examine dissection room injuries to ascertain the dangers associated with dissecting. We performed a retrospective cohort analysis of dissection room injuries from four student cohorts over an eleven-year period (2001-2011), including second-year medical students, third-year medical students, second-year dental students, and third-year science students. Injury data included activity causing injury, object responsible, and injury site. A total of 163 injuries during 70,039 hours of dissection were recorded, with 66 in third-year medical students, 42 in second-year medical students, 36 in third-year science students, and 16 in second-year dental students. The overall rate was 2.87 injuries per 1,000 dissection hours, with second-year medical students most frequently injured (5.5 injuries per 1,000 hours); third-year medical students were least frequently injured (1.3 injuries per 1,000 hours). A significant difference in injury rates between student groups indicated a higher than expected injury rate to second-year medical students and lower than expected rates to third-year medical students. Injury rates increased for most groups between 2001-2006 and 2007-2011 periods. Most injuries (79%) were from scalpel cuts to the finger or thumb. This study provides injury rates for dissection room injuries to students, indicating differences in injury frequency between cohorts and an increase in injury rate over time. As scalpel cuts were the most likely injury mechanism, targeting scalpel handling with preventative strategies may reduce future injury risk. PMID:23536433

  11. Science Teachers and the Dissection Debate: Perspectives on Animal Dissection and Alternatives

    ERIC Educational Resources Information Center

    Oakley, Jan

    2012-01-01

    This study investigated Ontario science and biology teachers' practices and attitudes toward animal dissection and dissection alternatives. The data was collected through a mixed methods approach involving online surveys (n = 153) and subsequent telephone interviews (n = 9) with secondary school science and biology teachers. The findings indicate…

  12. Preventable Sternocleidomastoid Muscular Atrophy after Neck Dissection

    PubMed Central

    Yamamoto, Nao; Sawai, Natsuko Yoshimura; Ishimoto, Shunsuke; Ogura, Hide; Aikawa, Tomonao; Kogo, Mikihiko

    2015-01-01

    Background: Modified radical neck dissection (mRND) [preserving the sternocleidomastoid muscle (SCM) and the spinal accessory nerve] and supraomohyoid neck dissection have become common surgical procedures for treating head and neck cancer. Postoperative severe asymmetry of the neck and severe atrophy of the SCM, however, have been demonstrated. Methods: Using computed tomographic images, cross-sectional areas of the SCMs were measured in 99 patients with carcinoma of the oral cavity who underwent unilateral mRND or supraomohyoid neck dissection. An asymmetry index was used. Results: Innervation to the SCM was preserved in 91 patients. The spinal accessory nerve and the innervation were sacrificed in 3 patients; the innervation was repaired in 5 patients. Sacrifice of innervation to the SCM resulted in extremely severe asymmetry. Repair of the innervation prevented severe asymmetry in 40%. Preservation of the innervation prevented severe asymmetry in 75% at the middle portion of the neck and in 56% at the lower portion after mRND. Conclusion: Preserving innervation to the SCM and gentle handling of the nerve during neck dissection could prevent severe asymmetry after neck dissection. PMID:26495217

  13. Internal carotid artery dissections: duplex ultrasound imaging.

    PubMed

    Gardner, D J; Gosink, B B; Kallman, C E

    1991-11-01

    Duplex ultrasound findings in seven patients (eight vessels) demonstrating extracranial internal carotid artery (ICA) dissections are presented. The two-dimensional (2D) sonographic findings of ICA dissection consisted of three categories: (1) normal, (2) luminal flap with or without thrombus formation, and (3) hypoechoic thrombus with or without lumen narrowing. The Doppler ultrasound waveforms were variable. In the common carotid artery (CCA) these included (1) normal, (2) resistive, damped, or biphasic CCA waveforms, and (3) positive temporal artery tap in the mid-CCA. In the ICA, the appearances included (1) normal, (2) damped, resistive, or biphasic waveforms, (3) absent flow, and (4) high velocity flow. Although these appearances are nonspecific, the finding of some of these duplex ultrasound waveforms in the appropriate clinical setting suggests a diagnosis of extracranial internal carotid artery dissection. PMID:1811077

  14. Bowel "dissection" in microvillus inclusion disease.

    PubMed

    Chiang, Ming-Chou; Hsu, Jen-Fu; Hsueh, Chuen; Chao, Hsun-Chin; Wang, Tzu-Hao; Chen, Chih-Ping; Lai, Ming-Wei

    2015-04-01

    A preterm male neonate was diagnosed as having microvillus inclusion disease based on the characteristics of histological and ultrastructural findings. The peripheral blood sample also revealed MYO5B mutation. He had been on long-term parenteral nutrition. However, a bowel segment was seen in the baby's diaper during hospitalization when he was 5 months old. Serial abdominal ultrasound demonstrated progressive dissection of the bowel wall with detached mucosa in real-time. Small intestinal epithelia were seen on the histology of the detached bowel segment. He died 2 weeks after the episode; postmortem autopsy showed diffuse detachment of mucosa of small bowels without perforation. This is the first report of an infant with microvillus inclusion disease that presented with bowel "dissection". Weakened adhesion and integrity of intestinal epithelial cells caused by MYO5B mutation was speculated to result in the dissection and detachment of the epithelia of the gastrointestinal tract. PMID:23608388

  15. The nature of dissection: Exploring student conceptions

    NASA Astrophysics Data System (ADS)

    York, Katharine

    The model of conceptual change in science describes the process of learning as a complete restructuring of knowledge, when learners discover or are shown more plausible, intelligent alternatives to existing conceptions. Emotions have been acknowledged as part of a learner's conceptual ecology, but the effects of emotions on learning have yet to be described. This research was conducted to examine the role that emotions have on learning for thirteen high school students, as they dissected cats in a Human Anatomy and Physiology class. The project also investigated whether a student's emotional reactions may be used to develop a sense of connectedness with the nonhuman world, which is defined as ecological literacy. This study utilized a grounded theory approach, in which student responses to interviews were the primary source of data. Interviews were transcribed, and responses were coded according to a constant comparative method of analysis. Responses were compared with the four conditions necessary for conceptual change to occur, and also to five principles of ecological literacy. Students who had negative reactions to dissection participated less in the activity, and demonstrated less conceptual change. Two female students showed the strongest emotional reactions to dissection, and also the lowest amount of conceptual change. One male student also had strong negative reactions to death, and showed no conceptual change. The dissection experiences of the students in this study did not generally reflect ecological principles. The two students whose emotional reactions to dissection were the most negative demonstrated the highest degree of ecological literacy. These results provide empirical evidence of the effects that emotions have on learning, and also supports the opinions of educators who do not favor dissection, because it does not teach students to respect all forms of life.

  16. Improving the Phosphoproteome Coverage for Limited Sample Amounts Using TiO2-SIMAC-HILIC (TiSH) Phosphopeptide Enrichment and Fractionation.

    PubMed

    Engholm-Keller, Kasper; Larsen, Martin R

    2016-01-01

    Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein. PMID:26584925

  17. Spontaneous vertebral artery dissection: Posterior circulation stroke.

    PubMed

    Deshmukh, Manoj; Wadhwa, Anju; Rajdeo, Ravi

    2015-01-01

    Spontaneous vertebral artery dissection (VAD) is relatively rare but an important cause of posterior circulation stroke. A 46-year-male complaining of sudden onset headache, neck pain with right-sided neuro deficit in the form of hemiparesis was evaluated by contrast magnetic resonance imaging and dual-energy computed tomography (CT) and brain neck angiography which revealed a short segment extracranial left-sided VAD, associated with acute infarct in the left occipital region. The patient was managed conservatively and followed up for 6 months. Follow-up CT angiography after a period of 6 months revealed the near complete resolution of the arterial dissection in left vertebral artery. PMID:26692700

  18. Coronary Thrombosis without Dissection following Blunt Trauma

    PubMed Central

    Sibel, Michael; Thomas, Peter; Burt, Francis; Cipolla, James; Puleo, Peter; Baker, Keith

    2016-01-01

    Blunt trauma to the chest resulting in coronary thrombosis and ST elevation myocardial infarction (STEMI) is a rare but well-described occurrence in adults. Angiography in such cases has generally disclosed complete epicardial coronary occlusion with thrombus, indistinguishable from the findings commonly found in spontaneous plaque rupture due to atherosclerotic disease. In all previously reported cases in which coronary interrogation with intravascular ultrasound (IVUS) was performed in association with acute revascularization, coronary artery dissection was implicated as the etiology of coronary thrombosis. We present the first case report of blunt trauma-associated coronary thrombosis without underlying atherosclerosis or coronary dissection, as documented by IVUS imaging. PMID:27006836

  19. Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis.

    PubMed

    Chen, Yanmei; Hoehenwarter, Wolfgang

    2015-12-01

    Salinity and oxidative stress are major factors affecting and limiting the productivity of agricultural crops. The molecular and biochemical processes governing the plant response to abiotic stress have often been researched in a reductionist manner. Here, we report a systemic approach combining metabolic labeling and phosphoproteomics to capture early signaling events with quantitative metabolome analysis and enzyme activity assays to determine the effects of salt and oxidative stress on plant physiology. K(+) and Na(+) transporters showed coordinated changes in their phosphorylation pattern, indicating the importance of dynamic ion homeostasis for adaptation to salt stress. Unique phosphorylation sites were found for Arabidopsis (Arabidopsis thaliana) SNF1 kinase homolog10 and 11, indicating their central roles in the stress-regulated responses. Seven Sucrose Non-fermenting1-Related Protein Kinase2 kinases showed varying levels of phosphorylation at multiple serine/threonine residues in their kinase domain upon stress, showing temporally distinct modulation of the various isoforms. Salinity and oxidative stress also lead to changes in protein phosphorylation of proteins central to photosynthesis, in particular the kinase State Transition Protein7 required for state transition and light-harvesting II complex proteins. Furthermore, stress-induced changes of the phosphorylation of enzymes of central metabolism were observed. The phosphorylation patterns of these proteins were concurrent with changes in enzyme activity. This was reflected by altered levels of metabolites, such as the sugars sucrose and fructose, glycolysis intermediates, and amino acids. Together, our study provides evidence for a link between early signaling in the salt and oxidative stress response that regulates the state transition of photosynthesis and the rearrangement of primary metabolism. PMID:26471895

  20. 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; Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 ; Oyama, Masaaki; Kozuka-Hata, Hiroko; Ishikawa, Kosuke; Inoue, Takafumi; Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 ; Muta, Tatsushi; Semba, Kentaro; Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 ; Inoue, Jun-ichiro; Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639

    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.

  1. Thoracoscopic left mediastinal lymph node dissection.

    PubMed

    Nagashima, Takuya

    2016-01-01

    In Japan, the use of video-assisted thoracoscopic surgery (VATS) for primary lung cancer is increasing. However, it is not easy to perform mediastinal lymph node dissection using VATS as effectively as it is performed using thoracotomy. Herein, I have presented two techniques for subcarinal lymph node dissection from the left thoracic cavity: one involves the retraction of the lower bronchus towards the visceral and cranial side before inferior pulmonary vein resection to secure the view of the right lower portion. The other involves the separation of lymph nodes from the right main bronchus before separation from the left bronchus, to prevent the lymph nodes from falling down and interrupting the right-side view. Moreover, I have also described a technique that facilitates left upper mediastinal lymph node dissection. It involves traction of a thoracic cardiac branch from the recurrent laryngeal nerve allowing visualization of the bottom of the #4L lymph node, so that it can be dissected easily. There has been no observation of recurrent nerve paralysis using this procedure. PMID:26855946

  2. Trace Element Changes in Thoracic Aortic Dissection.

    PubMed

    Edvinsson, Marie; Ilbäck, Nils-Gunnar; Frisk, Peter; Thelin, Stefan; Nyström-Rosander, Christina

    2016-02-01

    Thoracic aortic dissection is a life-threatening condition with an incompletely understood pathogenesis. Trace elements are essential for the functioning of different processes in the body, including the immune system and associated responses to infection/inflammation. Because inflammation may be part of the pathogenesis of thoracic aortic dissection, we investigated whether trace element changes associated with inflammation occur in serum and tissue samples during the disease. The study included 21 patients undergoing surgery for thoracic aortic dissection, 10 forensic autopsy specimens for tissue controls and 23 healthy blood donors for serum controls. Levels of magnesium (Mg), calcium (Ca), vanadium (V), manganese (Mn), iron (Fe), cobalt (Co), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd) and mercury (Hg) were measured in the aortic tissue and serum by inductively coupled plasma-mass spectrometry (ICP-MS). In the serum, Ca, V, Cu and Zn decreased, whereas Fe increased. In the tissue, Cu and Zn decreased and Fe tended to increase. The Cu/Zn ratio in the serum, a marker of infection/inflammation, did not change in the patients. Concerning trace element changes in the serum and tissue, our data do not support the hypothesis that inflammation is involved in the pathogenesis of thoracic aortic dissection. PMID:26152852

  3. Thoracoscopic left mediastinal lymph node dissection

    PubMed Central

    2016-01-01

    In Japan, the use of video-assisted thoracoscopic surgery (VATS) for primary lung cancer is increasing. However, it is not easy to perform mediastinal lymph node dissection using VATS as effectively as it is performed using thoracotomy. Herein, I have presented two techniques for subcarinal lymph node dissection from the left thoracic cavity: one involves the retraction of the lower bronchus towards the visceral and cranial side before inferior pulmonary vein resection to secure the view of the right lower portion. The other involves the separation of lymph nodes from the right main bronchus before separation from the left bronchus, to prevent the lymph nodes from falling down and interrupting the right-side view. Moreover, I have also described a technique that facilitates left upper mediastinal lymph node dissection. It involves traction of a thoracic cardiac branch from the recurrent laryngeal nerve allowing visualization of the bottom of the #4L lymph node, so that it can be dissected easily. There has been no observation of recurrent nerve paralysis using this procedure. PMID:26855946

  4. The etiology of cervical artery dissection

    PubMed Central

    Haneline, Michael T.; Rosner, Anthony L.

    2007-01-01

    Abstract The etiology of cervical artery dissection (CAD) is unclear, although a number of risk factors have been reported to be associated with the condition. On rare occasions, patients experience CAD after cervical spine manipulation, making knowledge about the cervical arteries, the predisposing factors, and the pathogenesis of the condition of interest to chiropractors. This commentary reports on the relevant anatomy of the cervical arteries, developmental features of CAD, epidemiology of the condition, and mechanisms of dissection. The analysis of CAD risk factors is confusing, however, because many people are exposed to mechanical events and known pathophysiological associations without ever experiencing dissection. No cause-and-effect relationship has been established between cervical spine manipulation and CAD, but it seems that cervical manipulation may be capable of triggering dissection in a susceptible patient or contributing to the evolution of an already existing CAD. Despite the many risk factors that have been proposed as possible causes of CAD, it is still unknown which of them actually predispose patients to CAD after cervical spine manipulation. PMID:19674705

  5. Global Distribution of Dissected Duricrust on Mars

    NASA Technical Reports Server (NTRS)

    Mustard, J. F.; Cooper, C. D.

    2000-01-01

    Evidence for dissected duricrust was identified in high resolution MOC images. Analysis of all available images was used to map the global distribution of this terrain. It is apparently restricted to two latitude bands: 30-60 deg. N and 30-60 deg. S.

  6. Squid Dissection: From Pen to Ink.

    ERIC Educational Resources Information Center

    Brown, Cindy; Kisiel, Jim

    2003-01-01

    Introduces students to dissection, which is an important part of scientific discovery. Students not only gain an understanding of the anatomy of a squid, but also develop a sense of responsibility and respect for the animal that they are using as a learning tool. (Author/SOE)

  7. Intraoperative aortic dissection in pediatric heart surgery.

    PubMed

    Hibino, Narutoshi; Harada, Yorikazu; Hiramatsu, Takeshi; Yasukochi, Satoshi; Satomi, Gengi

    2006-06-01

    Intraoperative aortic dissection occurred in a 3-year-old-boy undergoing repair of an atrial septal defect. Transesophageal echocardiography was useful for the diagnosis, and conservative medical treatment under close observation was feasible in this case which involved a limited intimal tear. PMID:16714685

  8. Cow's Eye Dissection in the Physics Lab.

    ERIC Educational Resources Information Center

    Lapp, David R.; Keenan, James E.

    1991-01-01

    Proposes the science demonstration of dissecting a cow's eye to integrate biology and physics in the study of optics and lenses. Reviews the anatomy of the eye, describes the visual process and covers topics as index of refraction of the cornea, microscopic receptors, the lens, and the retina. (MDH)

  9. Genetics Home Reference: Familial thoracic aortic aneurysm and dissection

    MedlinePLUS

    ... OMIM Genetic disorder catalog Conditions > Familial thoracic aortic aneurysm and dissection (often shortened to familial TAAD ) On ... 2015 What is familial TAAD? Familial thoracic aortic aneurysm and dissection (familial TAAD) involves problems with the ...

  10. Genetics Home Reference: Familial thoracic aortic aneurysm and dissection

    MedlinePLUS

    ... literature OMIM Genetic disorder catalog Conditions > Familial thoracic aortic aneurysm and dissection (often shortened to familial TAAD ) On ... January 2015 What is familial TAAD? Familial thoracic aortic aneurysm and dissection (familial TAAD) involves problems with the ...

  11. Conservative Management of Chronic Aortic Dissection with Underlying Aortic Aneurysm

    PubMed Central

    Yusuf Beebeejaun, Mohammad; Malec, Aleksandra; Gupta, Ravi

    2013-01-01

    Aortic dissection is one of the most common aortic emergencies affecting around 2000 Americans each year. It usually presents in the acute state but in a small percentage of patients aortic dissections go unnoticed and these patients survive without any adequate therapy. With recent advances in medical care and diagnostic technologies, aortic dissection can be successfully managed through surgical or medical options, consequently increasing the related survival rate. However, little is known about the optimal long-term management of patients suffering from chronic aortic dissection. The purpose of the present report is to review aortic dissection, namely its pathology and the current diagnostic tools available, and to discuss the management options for chronic aortic dissection. We report a patient in which chronic aortic dissection presented with recurring episodes of vomiting and also discuss the management plan of our patient who had a chronic aortic dissection as well as an underlying aortic aneurysm. PMID:24179638

  12. Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach.

    PubMed

    Luo, Rong; Zhou, Chunjing; Lin, Jiaojiao; Yang, Dehao; Shi, Yaojun; Cheng, Guofeng

    2012-01-01

    Schistosome is the causative agent of human schistosomiasis and related animal disease. Reversible protein phosphorylation plays a key role in signaling processing that are vital for a cell and organism. However, it remains to be undercharacterized in schistosomes. In the present study, we characterized in vivo protein phosphorylation events in different developmental stages (schistosomula and adult worms) of Schistosoma japonicum by using microvolume immobilized metal-ion affinity chromatography (IMAC) pipette tips coupled to nanoLC-ESI-MS/MS. In total, 127 distinct phosphorylation sites were identified in 92 proteins in S. japonicum. A comparison of the phosphopeptides identified between the schistosomula and the adult worms revealed 30 phosphoproteins co-detected in both of the two worms. These proteins included several signal molecules and enzymes such as 14-3-3 protein, cysteine string protein, heat shock protein 90, epidermal growth factor receptor pathway substrate 8, proliferation-associated protein 2G4, peptidyl-prolyl isomerase G, phosphofructokinase and thymidylate kinase. Additionally, the phosphorylation sites were examined for phosphorylation specific motif and evolutionarily conservation. The study represents the first attempt to determine in vivo protein phosphorylation in S. japonicum by using a phosphoproteomic approach. The results by providing an inventory of phosphorylated proteins may facilitate to further understand the mechanisms involved in schistosome development and growth, and then may result in the development of novel vaccine candidates and drug targets for schistosomiasis control. PMID:22036931

  13. Defining the phospho-adhesome through the phosphoproteomic analysis of integrin signalling

    PubMed Central

    Robertson, Joseph; Jacquemet, Guillaume; Byron, Adam; Jones, Matthew C.; Warwood, Stacey; Selley, Julian N.; Knight, David; Humphries, Jonathan D.; Humphries, Martin J.

    2015-01-01

    Cell–extracellular matrix (ECM) adhesion is a fundamental requirement for multicellular existence due to roles in positioning, proliferation and differentiation. Phosphorylation plays a major role in adhesion signalling; however, a full understanding of the phosphorylation events that occur at sites of adhesion is lacking. Here we report a proteomic and phosphoproteomic analysis of adhesion complexes isolated from cells spread on fibronectin. We identify 1,174 proteins, 499 of which are phosphorylated (1,109 phosphorylation sites), including both well-characterized and novel adhesion-regulated phosphorylation events. Immunoblotting suggests that two classes of phosphorylated residues are found at adhesion sites—those induced by adhesion and those constitutively phosphorylated but recruited in response to adhesion. Kinase prediction analysis identifies novel kinases with putative roles in adhesion signalling including CDK1, inhibition of which reduces adhesion complex formation. This phospho-adhesome data set constitutes a valuable resource to improve our understanding of the signalling mechanisms through which cell–ECM interactions control cell behaviour. PMID:25677187

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

    PubMed Central

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

    2015-01-01

    Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology. PMID:26401955

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

    PubMed Central

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

    2016-01-01

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

  16. Phosphoproteomic analysis of kinase-deficient mice reveals multiple TAK1 targets in osteoclast differentiation.

    PubMed

    Sumiya, Eriko; Negishi-Koga, Takako; Nagai, Yusuke; Suematsu, Ayako; Suda, Tomomi; Shinohara, Masahiro; Sato, Kojiro; Sanjo, Hideki; Akira, Shizuo; Takayanagi, Hiroshi

    2015-08-01

    TAK1 (encoded by Map3k7) is a mitogen-activated protein kinase kinase kinase (MAP3K), which activates the transcription factors AP-1 and NF-?B in response to receptor activator of NF-?B ligand (RANKL) stimulation, thus constituting a key regulator of osteoclast differentiation. Here we report the functional relevance of the kinase activity of TAK1 in the late stage of osteoclast differentiation invivo using Ctsk-Cre mice and TAK1 mutant mice in which the TAK1 kinase domain was flanked by loxP. The Map3k7(flox/kd)Ctsk(Cre/+) mice displayed a severe osteopetrotic phenotype due to a marked decrease in osteoclast number. RANKL-induced activation of MAPK and NF-?B was impaired in the late stage of osteoclast differentiation. The absence of suppressive effect of an administered NF-?B inhibitor on the late stage of osteoclastogenesis led us to investigate unknown TAK1 targets in osteoclast differentiation. We performed a phosphoproteomic analysis of RANKL-stimulated osteoclast precursor cells from Map3k7(flox/kd)Ctsk(Cre/+) mice, revealing multiple targets regulated by TAK1 during osteoclastogenesis. Thus, TAK1 functions as a critical regulator of the phosophorylation status of various cellular proteins that govern osteoclastogenesis. PMID:26102028

  17. Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

    PubMed Central

    Nakayasu, Ernesto S.; Gaynor, Matthew R.; Sobreira, Tiago J.P.; Ross, Jeremy A.; Almeida, Igor C.

    2009-01-01

    Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America and has become a public health concern in the United States and areas of Europe. The possibility that kinase inhibitors represent novel anti-parasitic agents is currently being explored. However, fundamental understanding of the cell signaling networks requires the detailed analysis of the involved phosphorylated proteins. Here, we have performed a comprehensive mass spectrometry (MS)-based phosphorylation mapping of phosphoproteins from T. cruzi epimastigote forms. Our liquid chromatography (LC)-tandem MS (MS/MS, MS/MS/MS, and multistage activation) analysis has identified 237 phosphopeptides from 119 distinct proteins. Furthermore, 220 phosphorylation sites were unambiguously mapped: 148 on serine, 57 on threonine, and 8 on tyrosine. In addition, immunoprecipitation and Western blotting analysis confirmed the presence of at least seven tyrosine-phosphorylated proteins in T. cruzi. The identified phosphoproteins were subjected to Gene Ontology, InterPro, and BLAST analysis, and categorized based on their role in cell structure, motility, transportation, metabolism, pathogenesis, DNA/RNA/protein turnover, and signaling. Taken together our phosphoproteomic data provide new insights into the molecular mechanisms governed by protein kinases and phosphatases in T. cruzi. We discuss the potential roles of the identified phosphoproteins in parasite physiology and drug development. PMID:19579231

  18. Probing the phosphoproteome of HeLa cells using nanocast metal oxide microspheres for phosphopeptide enrichment.

    PubMed

    Leitner, Alexander; Sturm, Martin; Hudecz, Otto; Mazanek, Michael; Smått, Jan-Henrik; Lindén, Mika; Lindner, Wolfgang; Mechtler, Karl

    2010-04-01

    Metal oxide affinity chromatography (MOAC) has become a prominent method to enrich phosphopeptides prior to their analysis by liquid chromatography-mass spectrometry. To overcome limitations in material design, we have previously reported the use of nanocasting as a means to generate metal oxide spheres with tailored properties. Here, we report on the application of two oxides, tin dioxide (stannia) and titanium dioxide (titania), for the analysis of the HeLa phosphoproteome. In combination with nanoflow LC-MS/MS analysis on a linear ion trap-Fourier transform ion cyclotron resonance instrument, we identified 619 phosphopeptides using the new stannia material, and 896 phosphopeptides using titania prepared in house. We also compared the newly developed materials to commercial titania material using an established enrichment protocol. Both titania materials yielded a comparable total number of phosphopeptides, but the overlap of the two data sets was less than one-third. Although fewer peptides were identified using stannia, the complementarity of SnO(2)-based MOAC could be shown as more than 140 phosphopeptides were exclusively identified by this material. PMID:20201521

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

    PubMed

    Lemeer, Simone; Pinkse, Martijn W H; Mohammed, Shabaz; van Breukelen, Bas; den Hertog, Jeroen; Slijper, Monique; Heck, Albert J R

    2008-04-01

    In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation, and growth. Therefore the large-scale identification of signaling proteins and their post-translational modifications is crucial to understand the proteome biology of the developing zebrafish embryo. Here, we used an automated, robust, and sensitive online TiO 2-based LC-MS/MS setup to enrich for phosphorylated peptides from 1 day old zebrafish embryos. We identified, with high confidence, 1067 endogenous phosphorylation sites in a sample taken from 60 embryos (approximately 180 microg), 321 from 10 embryos, and 47 phosphorylation sites from a single embryo, illustrating the sensitivity of the method. This data set, representing by far the largest for zebrafish, was further exploited by searching for serine/threonine or tyrosine kinase motifs using Scansite. For one-third of the identified phosphopeptides a potential kinase motif could be predicted, where it appeared that Cdk5 kinase, p38MAPK, PKA, and Casein Kinase 2 substrates were the most predominant motifs present, underpinning the importance of these kinases in signaling pathways in embryonic development. The phosphopeptide data set was further interrogated using alignments with phosphopeptides identified in recent large-scale phosphoproteomics screens in human and mouse samples. These alignments revealed conservation of phosphorylation sites in several proteins suggesting preserved function in embryonic development. PMID:18307296

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

    PubMed

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

    2015-01-01

    Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology. PMID:26401955

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

    PubMed

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

    2016-02-19

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

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

    SciTech Connect

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

    2014-11-01

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

  3. Phosphoproteomics Identifies Driver Tyrosine Kinases in Sarcoma Cell Lines and Tumors

    PubMed Central

    Bai, Yun; Li, Jiannong; Fang, Bin; Edwards, Arthur; Zhang, Guolin; Bui, Marilyn; Eschrich, Steven; Altiok, Soner; Koomen, John; Haura, Eric B.

    2015-01-01

    Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival. PMID:22461510

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

    PubMed

    Bennetzen, Martin V; Mario, Guillermo; Pultz, Dennis; Morselli, Eugenia; Frgeman, Nils J; Kroemer, Guido; Andersen, Jens S

    2012-05-01

    Macroautophagy is a self-cannibalistic process that enables cells to adapt to various stresses and maintain energy homeostasis. Additionally, autophagy is an important route for turnover of misfolded proteins and damaged organelles, with important implications in cancer, neurodegenerative diseases and aging. Resveratrol and spermidine are able to induce autophagy by affecting deacetylases and acetylases, respectively, and have been found to extend the life-span of model organisms. With the aim to reveal the signaling networks involved in this drug-induced autophagic response, we quantified resveratrol and spermidine-induced changes in the phosphoproteome using SILAC and mass spectrometry. The data were subsequently analyzed using the NetworKIN algorithm to extract key features of the autophagy-responsive kinase-substrate network. We found that two distinct sequence motifs were highly responsive to resveratrol and spermidine and that key proteins modulating the acetylation, phosphorylation, methylation and ubiquitination status were affected by changes in phosphorylation during the autophagic response. Essential parts of the apoptotic signaling network were subjected to post-translational modifications during the drug-induced autophagy response, suggesting potential crosstalk and balancing between autophagy and apoptosis. Additionally, we predicted cellular signaling networks affected by resveratrol and spermidine using a computational framework. Altogether, these results point to a profound crosstalk between distinct networks of post-translational modifications and provide a resource for future analysis of autophagy and cell death. PMID:22517431

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

    PubMed

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

    2014-11-01

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi. PMID:24881580

  6. FAIMS and Phosphoproteomics of Fibroblast Growth Factor Signaling: Enhanced Identification of Multiply Phosphorylated Peptides.

    PubMed

    Zhao, Hongyan; Cunningham, Debbie L; Creese, Andrew J; Heath, John K; Cooper, Helen J

    2015-12-01

    We have applied liquid chromatography high-field asymmetric waveform ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) to the investigation of site-specific phosphorylation in fibroblast growth factor (FGF) signaling. We have combined a SILAC approach with chemical inhibition by SU5402 (an FGF receptor tyrosine kinase inhibitor) and dasatinib (a Src family kinase inhibitor). The results show that incorporation of FAIMS within the workflow results in (a) an increase in the relative proportion of phosphothreonine and phosphotyrosine sites identified, (b) an increase in phosphopeptide identifications from precursors with charge states ? +3 (with an associated increase in peptide length), and (c) an increase in the identification of multiply phosphorylated peptides. Approximately 20% of the phosphorylation sites identified via the FAIMS workflow had not been reported previously, and over 80% of those were from multiply phosphorylated peptides. Moreover, FAIMS provided access to a distinct set of phosphorylation sites regulated in response to SU5402 and dasatinib. The enhanced identification of multiply phosphorylated peptides was particularly striking in the case of sites regulated by SU5402. In addition to providing a compelling example of the complementarity of FAIMS in phosphoproteomics, the results provide a valuable resource of phosphorylation sites for further investigation of FGF signaling and trafficking. PMID:26503514

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

    PubMed

    Dash, Swagatika; Chandramouli, Kondethimma H; Zhang, Yu; Qian, Pei-Yuan

    2012-01-01

    Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds. PMID:22519465

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

    PubMed Central

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

    2014-01-01

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC–MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi. PMID:24881580

  9. Dissection as Instructional Techniques in Secondary Science: Choice and Alternatives.

    ERIC Educational Resources Information Center

    Bowd, Alan D.

    1993-01-01

    Examines the role of dissection in the teaching of secondary biology and environmental science, within the context of the development of attitudes toward animals. It was found that most students were required to perform dissections, several experience negative and stable emotional reactions, and teachers employed limited alternatives to dissection

  10. 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-MS/MS phosphoproteome investigation. The results of iPhos-facilitated targeted LC-MS/MS analysis convey more thorough and confident phosphopeptide identification than the results of pure DDA-based analysis. PMID:25521246

  11. Preservation of the optic radiations based on comparative analysis of diffusion tensor imaging tractography and anatomical dissection

    PubMed Central

    Nooij, Roland P.; Hoving, Eelco W.; van Hulzen, Arjen L. J.; Cornelissen, Frans W.; Renken, Remco J.

    2015-01-01

    Background: Visualization of the precise course of the visual pathways is relevant to prevent damage that may inflict visual field deficits during neurosurgical resections. In particular the optic radiations (OR) are susceptible to such damage during neurosurgery. Cortical pathways can be mapped in vivo, by using Diffusion Tensor Imaging (DTI). Visualization of these pathways would be potentially helpful to prevent neurosurgical visual morbidity. In this study an anatomical dissection of the visual pathways was compared to DTI fiber tractography (DTI-FT) data of four human brains. The feasibility of a definition of a Safety Zone is investigated. Methods: Four adult brains were dissected using Klingler's fiber dissection method, which allowed preparation of the OR. Measurements before and after dissection were used to establish distances from the cortex to the OR. DTI-scans were also obtained from these brains to determine the same distances. Results: Measurements from specific landmark points on the cortex to the lateral border of the OR were performed in four brains. Analysis through DTI tractography corresponded with the dissection results. Based on the combined results of both dissection and DTI-FT, we defined a quantitative surgical Safety Zone with respect to various anatomical landmarks (in particular the ventricle system). Conclusion: We conclude that there is a good correlation between the visualizations of the optic pathways based on dissection and DTI. Furthermore, we conclude that defining a neurosurgical Safety Zone which could preserve the integrity of the OR during surgery, based on the combination of DTI-FT images and dissection is feasible. PMID:26300739

  12. Which Comes First: The Use of Computer Simulation of Frog Dissection or Conventional Dissection as Academic Exercise?

    ERIC Educational Resources Information Center

    Akpan, Joseph; Strayer, Jeremy

    2010-01-01

    Science educators and school administrators are reexamining the educational value of animal dissection in the nation's schools and are focusing on simulation as an instructional alternative. One implication of the debate is that simulations can lead to equivalent learning to hands-on dissection. The second implication is whether dissection is…

  13. Which Comes First: The Use of Computer Simulation of Frog Dissection or Conventional Dissection as Academic Exercise?

    ERIC Educational Resources Information Center

    Akpan, Joseph; Strayer, Jeremy

    2010-01-01

    Science educators and school administrators are reexamining the educational value of animal dissection in the nation's schools and are focusing on simulation as an instructional alternative. One implication of the debate is that simulations can lead to equivalent learning to hands-on dissection. The second implication is whether dissection is

  14. Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer.

    PubMed

    Searleman, Adam C; Iliuk, Anton B; Collier, Timothy S; Chodosh, Lewis A; Tao, W Andy; Bose, Ron

    2014-12-01

    Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer-based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2-driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC-MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC-MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well-known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA-approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens. PMID:24723360

  15. Endoscopic submucosal dissection for gastrointestinal neoplasms

    PubMed Central

    Kakushima, Naomi; Fujishiro, Mitsuhiro

    2008-01-01

    Endoscopic submucosal dissection (ESD) is an advanced technique of therapeutic endoscopy for superficial gastrointestinal neoplasms. Three steps characterize it: injecting fluid into the submucosa to elevate the lesion, cutting the surrounding mucosa of the lesion, and dissecting the submucosa beneath the lesion. The ESD technique has rapidly permeated in Japan for treatment of early gastric cancer, due to its excellent results of en-bloc resection compared to endoscopic mucosal resection (EMR). Although there is still room for improvement to lessen its technical difficulty, ESD has recently been applied to esophageal and colorectal neoplasms. Favorable short-term results have been reported, but the application of ESD should be well considered by three aspects: (1) the possibility of nodal metastases of the lesion, (2) technical difficulty such as location, ulceration and operators skill, and (3) organ characteristics. PMID:18494043

  16. Therapeutic node dissections in malignant melanoma.

    PubMed

    Karakousis, C P

    1998-06-01

    In the absence of distant disease, therapeutic node dissections in malignant melanoma, i.e., dissections of regional nodal basins for palpable suspicious or biopsy-proven positive nodes, offer the chance of cure. The 5-year survival rates after therapeutic lymphadenectomy closely correlate with expected cure rates. Although they varied greatly in the literature, from 19% to 38%, the currently obtainable survival rates are in the upper ranges of this spectrum because patients now are closely followed-up and operated for early palpable nodal disease. Properly done, these procedures carry a low morbidity, but they should be done thoroughly to completely eradicate regional disease and avoid recurrences in the same nodal basin to achieve the maximum survival that is surgically attainable. PMID:9588722

  17. Spontaneous Coronary Artery Dissection: A Case Report

    PubMed Central

    Mokhberi, Vahid; Bagheri, Babak; Navidi, Seyfollah; Amini, Seyed Mohammad

    2015-01-01

    Spontaneous coronary artery dissection (SCAD) is a rare and important cause of acute coronary syndrome and sudden cardiac death. Various etiologies are thought to be responsible for this condition, among which underlying atherosclerosis seems to be the most common. SCAD is predominant in women and is usually diagnosed via coronary artery angiography. Therapeutic interventions include medical therapy, percutaneous coronary artery intervention, and surgery based on lesion characteristics. We describe a 36-year-old woman with SCAD presenting with acute chest pain to Fatemeh-Zahra Hospital, Sari, Iran. The patient had no current atherosclerosis risk factors and had given birth 6 months previously. Coronary angiography was performed due to the persistence of the chest pain after initial management, and a spontaneous dissection of the left anterior descending artery was observed. She underwent coronary artery bypass graft and was discharged in good condition. PMID:26697091

  18. Recurrent tamponade and aortic dissection in syphilis.

    PubMed

    Stansal, Audrey; Mirault, Tristan; Rossi, Aude; Dupin, Nicolas; Bruneval, Patrick; Bel, Alain; Azarine, Arshid; Minozzi, Catherine; Deman, Anne Laure; Messas, Emmanuel

    2013-11-01

    Syphilitic cardiovascular disease has been described since the 19th century, mainly on autopsy series. Major clinical manifestations are aortic aneurysm, aortic insufficiency, and coronary ostial stenosis. The diagnosis of syphilitic cardiovascular disease is based mainly on positive serologic tests and overt clinical manifestations. We present here a rare and unusual clinical presentation of a tertiary syphilis with recurrent tamponade and type B aortic dissection, whose positive diagnosis was made by polymerase chain reaction on pericardial fluid analysis. PMID:24182507

  19. Emergency vectorcardiographic study of acute aortic dissection.

    PubMed

    Carson, Wangden; Tseng, Yung-Zu; Chu, Shu-Hsun

    2003-05-01

    There has been no report in the literature of emergency vectorcardiographic study in patients with acute aortic dissection. A total of 135 consecutive patients with suspected coronary artery disease was studied. On admission to the intensive-care unit through the emergency service, each patient was given electrocardiographic and vectorcardiographic examinations, which were repeated at intervals of 24 hours. Fourteen patients (9 type I, 2 type II, and 3 type III according to DeBakey's classification) had acute aortic dissection indicated by clinical symptoms and proven by angiography. The electrocardiograms all revealed abnormal T-wave inversion in limb or precordial leads except 3 patients. Nine (64%) of the 14 patients had abnormal length/width (L/W) ratios of the T-loop in 2 planes of the emergency vectorcardiogram. Two (22%) among these 9 patients died of ventricular tachyarrhythmia. The inscription direction of the T-loop showed abnormality in 4 (29%) of the 14 patients; 2 (50%) among these 4 patients died of ventricular tachyarrhythmia. The de novo occurrence of sustained ventricular tachyarrhythmia after emergency cardiac surgery in patients with acute aortic dissection has been documented in this report. Furthermore, patients with acute aortic dissection who have (1) an abnormal sense of inscription direction of the T-loop in at least 1 of either the horizontal or left sagittal plane, and (2) an abnormal L/W ratio in at least 2 planes in the preoperational emergency vectorcardiogram, have altered ventricular repolarization and thus are at high risk of postoperationally unexpected ventricular tachyarrhythmia. This information is not available from the electrocardiogram. PMID:12811709

  20. Quantitative genetics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The majority of economically important traits targeted for cotton improvement are quantitatively inherited. In this chapter, the current state of cotton quantitative genetics is described and separated into four components. These components include: 1) traditional quantitative inheritance analysis, ...

  1. A case of carotid artery dissection after surgical repair of aortic dissection successfully treated with endovascular therapy using intravascular ultrasound.

    PubMed

    Katoh, Hiromasa; Nozue, Tsuyoshi; Nagamine, Hiroshi; Kawase, Yushi; Michishita, Ichiro

    2014-07-01

    A 71-year-old woman developed the dissection from right brachiocephalic trunk (BCT) to internal carotid artery 6days after the operation of ascending aortic dissection. Since no improvement of symptoms was observed despite conservative therapy, we performed endovascular therapy (EVT). Although a balloon-expandable stent was implanted in the BCT to seal the entry point of the dissection, the true lumen of the carotid artery was still compressed. Thus, we covered the dissected lesion completely with multiple stents, and her neurological findings improved. This case demonstrates that EVT using multiple stents is an effective strategy to manage dissection of supra-aortic branches. PMID:24142487

  2. Advanced endoscopic submucosal dissection with traction

    PubMed Central

    Imaeda, Hiroyuki; Hosoe, Naoki; Kashiwagi, Kazuhiro; Ohmori, Tai; Yahagi, Naohisa; Kanai, Takanori; Ogata, Haruhiko

    2014-01-01

    Endoscopic submucosal dissection (ESD) has been established as a standard treatment for early stage gastric cancer (EGC) in Japan and has spread worldwide. ESD has been used not only for EGC but also for early esophageal and colonic cancers. However, ESD is associated with several adverse events, such as bleeding and perforation, which requires more skill. Adequate tissue tension and clear visibility of the tissue to be dissected are important for effective and safe dissection. Many ESD methods using traction have been developed, such as clip-with-line method, percutaneous traction method, sinker-assisted method, magnetic anchor method, external forceps method, internal-traction method, double-channel-scope method, outerroute method, double-scope method, endoscopic-surgical-platform, and robot-assisted method. Each method has both advantages and disadvantages. Robotic endoscopy, enabling ESD with a traction method, will become more common due to advances in technology. In the near future, simple, noninvasive, and effective ESD using traction is expected to be developed and become established as a worldwide standard treatment for superficial gastrointestinal neoplasias. PMID:25031787

  3. Building mental models by dissecting physical models.

    PubMed

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to ensure focused learning; models that are too constrained require less supervision, but can be constructed mechanically, with little to no conceptual engagement. We propose "model-dissection" as an alternative to "model-building," whereby instructors could make efficient use of supervisory resources, while simultaneously promoting focused learning. We report empirical results from a study conducted with biology undergraduate students, where we demonstrate that asking them to "dissect" out specific conceptual structures from an already built 3D physical model leads to a significant improvement in performance than asking them to build the 3D model from simpler components. Using questionnaires to measure understanding both before and after model-based interventions for two cohorts of students, we find that both the "builders" and the "dissectors" improve in the post-test, but it is the latter group who show statistically significant improvement. These results, in addition to the intrinsic time-efficiency of "model dissection," suggest that it could be a valuable pedagogical tool. © 2015 by The International Union of Biochemistry and Molecular Biology, 44:7-11, 2016. PMID:26712513

  4. Future directions of duodenal endoscopic submucosal dissection

    PubMed Central

    Matsumoto, Satohiro; Miyatani, Hiroyuki; Yoshida, Yukio

    2015-01-01

    Endoscopic therapies for lesions of the duodenum are technically more difficult than those for lesions of the other parts of the gastrointestinal tract due to the anatomical features of the duodenum, and the incidence rate of complications such as perforation and bleeding is also higher. These aforementioned trends were especially noticeable for the case of duodenal endoscopic submucosal dissection (ESD). The indication for ESD of duodenal tumors should be determined by assessment of the histopathology, macroscopic morphology, and diameter of the tumors. The three types of candidate lesions for endoscopic therapy are adenoma, carcinoma, and neuroendocrine tumors. For applying endoscopic therapies to duodenal lesions, accurate preoperative histopathological diagnosis is necessary. The most important technical issue in duodenal ESD is the submucosal dissection process. In duodenal ESD, a short needle-type knife is suitable for the mucosal incision and submucosal dissection processes, and the Small-caliber-tip Transparent hood is an important tool. After endoscopic therapies, the wound should be closed by clipping in order to prevent complications such as secondary hemorrhage and delayed perforation. At present, the criteria for selection between ESD and EMR vary among institutions. The indications for ESD should be carefully considered. Duodenal ESD should have limitations, such as the need for its being performed by experts with abundant experience in performing the procedure. PMID:25901218

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

    PubMed

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

    2009-12-01

    Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1alpha) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on pathways, the cornerstone for identifying mechanisms of drug's efficacy. PMID:19997482

  6. 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.

  7. Phosphoproteome and transcriptome analysis of the neuronal response to a CDK5 inhibitor.

    PubMed

    Gillardon, Frank; Steinlein, Peter; Bürger, Erich; Hildebrandt, Tobias; Gerner, Christopher

    2005-04-01

    In Alzheimer's disease and amyotrophic lateral sclerosis deregulation of cyclin-dependent kinase 5 (CDK5) causes hyperphosphorylation of tau and neurofilament proteins, respectively, leading to neuronal cell death. We have demonstrated recently that pharmacological inhibition of CDK5 protects neurons under various stressful conditions (Weishaupt J. H., et al., Molec. Cell. Neurosci. 2003, 24, 489-502). To get an overview on the cellular mechanisms of action we analyzed global changes in protein phosphorylation in cultured cerebellar granule neurons by [(32)P]orthophosphate labeling after administration of a CDK5 inhibitor. Since CDK5 has recently been shown to phosphorylate and inactivate transcription factor MEF2, we included gene expression profiling using cDNA microarrays. By two-dimensional gel electrophoresis and matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry we identified several phosphoproteins that were modulated by compound administration. Among them syndapin I which is involved in vesicle recycling, and dynein light intermediate chain 2 which represents a regulatory subunit of the dynein protein complex. These findings are consistent with the known physiological function of CDK5 in synaptic signaling and axonal transport. Moreover, we detected phosphoproteins acting in neuronal surival and/or neurite outgrowth, such as cofilin and collapsin response mediator protein. Subsequent testing in cell cultures revealed that the CDK5 inhibitor blocked mitochondrial translocation of pro-apoptotic cofilin in cerebellar granule neurons and enhanced neurite outgrowth in dorsal root ganglia. Numerous genes exhibiting MEF2 consensus binding sequences were modulated by CDK5 inhibitor treatment. Among them some that may contribute to neurite elongation or neuronal survival, but also several genes functioning in synaptic transmission. Taken together, phosphoproteome and transcriptome analysis indicate that the compound promotes both neuronal survival and neurite outgrowth, but also may affect synaptic function in cultured neurons. PMID:15712243

  8. Proteomic and phosphoproteomic analyses reveal extensive phosphorylation of regulatory proteins in developing rice anthers.

    PubMed

    Ye, Juanying; Zhang, Zaibao; Long, Haifei; Zhang, Zhimin; Hong, Yue; Zhang, Xumin; You, Chenjiang; Liang, Wanqi; Ma, Hong; Lu, Pingli

    2015-11-01

    Anther development, particularly around the time of meiosis, is extremely crucial for plant sexual reproduction. Meanwhile, cell-to-cell communication between somatic (especial tapetum) cells and meiocytes are important for both somatic anther development and meiosis. To investigate possible molecular mechanisms modulating protein activities during anther development, we applied high-resolution mass spectrometry-based proteomic and phosphoproteomic analyses for developing rice (Oryza sativa) anthers around the time of meiosis (RAM). In total, we identified 4984 proteins and 3203 phosphoproteins with 8973 unique phosphorylation sites (p-sites). Among those detected here, 1544 phosphoproteins are currently absent in the Plant Protein Phosphorylation DataBase (P(3) DB), substantially enriching plant phosphorylation information. Mapman enrichment analysis showed that 'DNA repair','transcription regulation' and 'signaling' related proteins were overrepresented in the phosphorylated proteins. Ten genetically identified rice meiotic proteins were detected to be phosphorylated at a total of 25 p-sites; moreover more than 400 meiotically expressed proteins were revealed to be phosphorylated and their phosphorylation sites were precisely assigned. 163 putative secretory proteins, possibly functioning in cell-to-cell communication, are also phosphorylated. Furthermore, we showed that DNA synthesis, RNA splicing and RNA-directed DNA methylation pathways are extensively affected by phosphorylation. In addition, our data support 46 kinase-substrate pairs predicted by the rice Kinase-Protein Interaction Map, with SnRK1 substrates highly enriched. Taken together, our data revealed extensive protein phosphorylation during anther development, suggesting an important post-translational modification affecting protein activity. PMID:26360816

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

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-01

    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. PMID:25316100

  10. Phosphoproteomic Approach to Characterize Protein Mono- and Poly(ADP-ribosyl)ation Sites from Cells

    PubMed Central

    2015-01-01

    Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome. PMID:24920161

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

    PubMed Central

    2014-01-01

    Background Selaginella (Selaginella moellendorffii) is a lycophyte which diverged from other vascular plants approximately 410 million years ago. As the first reported non-seed vascular plant genome, Selaginella genome data allow comparative analysis of genetic changes that may be associated with land plant evolution. Proteomics investigations on this lycophyte model have not been extensively reported. Phosphorylation represents the most common post-translational modifications and it is a ubiquitous regulatory mechanism controlling the functional expression of proteins inside living organisms. Results In this study, polyethylene glycol fractionation and immobilized metal ion affinity chromatography were employed to isolate phosphopeptides from wild-growing Selaginella. Using liquid chromatography-tandem mass spectrometry analysis, 1593 unique phosphopeptides spanning 1104 non-redundant phosphosites with confirmed localization on 716 phosphoproteins were identified. Analysis of the Selaginella dataset revealed features that are consistent with other plant phosphoproteomes, such as the relative proportions of phosphorylated Ser, Thr, and Tyr residues, the highest occurrence of phosphosites in the C-terminal regions of proteins, and the localization of phosphorylation events outside protein domains. In addition, a total of 97 highly conserved phosphosites in evolutionary conserved proteins were identified, indicating the conservation of phosphorylation-dependent regulatory mechanisms in phylogenetically distinct plant species. On the other hand, close examination of proteins involved in photosynthesis revealed phosphorylation events which may be unique to Selaginella evolution. Furthermore, phosphorylation motif analyses identified Pro-directed, acidic, and basic signatures which are recognized by typical protein kinases in plants. A group of Selaginella-specific phosphoproteins were found to be enriched in the Pro-directed motif class. Conclusions Our work provides the first large-scale atlas of phosphoproteins in Selaginella which occupies a unique position in the evolution of terrestrial plants. Future research into the functional roles of Selaginella-specific phosphorylation events in photosynthesis and other processes may offer insight into the molecular mechanisms leading to the distinct evolution of lycophytes. PMID:24628833

  12. Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection*

    PubMed Central

    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

    2013-01-01

    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 with 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, 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 tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that 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 control infection by this pathogen. PMID:23970565

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

    PubMed Central

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

    2009-01-01

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

  14. 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 spectrometrybased 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

  15. Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis

    PubMed Central

    Grimaud, Florent; Rogniaux, Hélène; James, Martha G.; Myers, Alan M.; Planchot, Véronique

    2008-01-01

    In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein–protein interactions. PMID:18653693

  16. Peer-Assisted Learning in a Gross Anatomy Dissection Course

    PubMed Central

    Han, Eui-Ryoung; Chung, Eun-Kyung; Nam, Kwang-Il

    2015-01-01

    Peer-assisted learning encourages students to participate more actively in the dissection process and promotes thoughtful dissection. We implemented peer-assisted dissection in 2012 and compared its effects on students’ self-assessments of learning and their academic achievement with those of faculty-led dissection. All subjects performed dissections after a lecture about upper-limb gross anatomy. Experimental group (n = 134) dissected a cadaver while guided by peer tutors who had prepared for the dissection in advance, and control group (n = 71) dissected a cadaver after the introduction by a faculty via prosection. Self-assessment scores regarding the learning objectives related to upper limbs were significantly higher in experimental group than in control group. Additionally, experimental group received significantly higher academic scores than did control group. The students in peer-assisted learning perceived themselves as having a better understanding of course content and achieved better academic results compared with those who participated in faculty-led dissection. Peer-assisted dissection contributed to self-perception and to the ability to retain and explain anatomical knowledge. PMID:26565616

  17. Background music in the dissection laboratory: impact on stress associated with the dissection experience.

    PubMed

    Anyanwu, Emeka G

    2015-06-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on stress associated with the dissection experience. After 8 wk of normal dissection without BM, various genres of BM were introduced into the cadaver dissection sessions of 260 medical and dental students for 3 wk. Feedback on the impact of BM on students in the CDL and students' attitude were accessed using a questionnaire. Psychological stress assessment was done using Psychological Stress Measure 9. Two batches of 30 students each were made to dissect same areas of the body for 2 h, one batch with BM playing and the other batch without. The same examination was given to both groups at the end. Over 90% of the participants expressed a desire to incorporate BM into the CDL; 87% of the sampled population that expressed love for music also reported BM to be a very useful tool that could be used to enhance learning conditions in the CDL. A strong positive relationship was established between love for music and its perception as a tool for learning in the CDL (P < 0.001). Students that studied under the influence of BM had significantly higher scores (P < 0.001) in the overall examination result. BM reduced the level of stress associated with the dissection experience by ∼33%. PMID:26031725

  18. The Harmonic Scalpel versus Conventional Hemostasis for Neck Dissection: A Meta-Analysis of the Randomized Controlled Trials

    PubMed Central

    Ren, Zhen-Hu; Xu, Jian-Lin; Fan, Teng-Fei; Ji, Tong; Wu, Han-Jiang; Zhang, Chen-Ping

    2015-01-01

    Objective Neck dissection is the most definitive and effective treatment for head and neck cancer. This systematic review aims to compare the efficacy and surgical outcomes of neck dissection between the harmonic scalpel and conventional surgical techniques and conduct a quantitative meta-analysis of the randomized trials. Methods Randomized controlled trials (RCTs) were identified from the major electronic databases (MEDLINE, EMBASE and Cochrane Library) using the keywords harmonic scalpel and neck dissection, and a quantitative meta-analysis was conducted. The operative time and intraoperative bleeding were the primary outcome measures, and other parameters assessed included the drainage fluid volume and length of hospital stay. Results Seven trials that met the inclusion criteria included 406 neck dissection cases (201 in the harmonic scalpel group). Compared with conventional surgical techniques, the HS group had an operative time that was significantly reduced by 29.3 minutes [mean difference: -29.29; 95% CI = (-44.26, -14.32); P=0.0001], a reduction in intraoperative bleeding by 141.1 milliliters [mean difference: -141.13; 95% CI = (-314.99, 32.73); P=0.11], and a reduction in drainage fluid volume by 64.9 milliliters [mean difference: -64.86; 95% CI = (-110.40, -19.32); P=0.005] , but it is not significant after removal of studies driving heterogeneity. There was no significant difference in the length of the hospital stay [mean difference: -0.21; 95% CI = (-0.48, 0.07); P=0.14]. Conclusion This systematic review showed that using the harmonic scalpel for neck dissection significantly reduces the operative time and drainage fluid volume and that it is not associated with an increased length of hospital stay or perioperative complications. Therefore, the harmonic scalpel method is safe and effective for neck dissection. However, the statistical heterogeneity was high. Further studies are required to substantiate our findings. PMID:26161897

  19. Metabolic Signature of Electrosurgical Liver Dissection

    PubMed Central

    von Schnfels, Witigo; von Kampen, Oliver; Patsenker, Eleonora; Stickel, Felix; Schniewind, Bodo; Hinz, Sebastian; Ahrens, Markus; Balschun, Katharina; Egberts, Jan-Hendrik; Richter, Klaus; Landrock, Andreas; Sipos, Bence; Will, Olga; Huebbe, Patrizia; Schreiber, Stefan; Nothnagel, Michael; Rcken, Christoph; Rimbach, Gerald; Becker, Thomas

    2013-01-01

    Background and Aims High frequency electrosurgery has a key role in the broadening application of liver surgery. Its molecular signature, i.e. the metabolites evolving from electrocauterization which may inhibit hepatic wound healing, have not been systematically studied. Methods Human liver samples were thus obtained during surgery before and after electrosurgical dissection and subjected to a two-stage metabolomic screening experiment (discovery sample: N?=?18, replication sample: N?=?20) using gas chromatography/mass spectrometry. Results In a set of 208 chemically defined metabolites, electrosurgical dissection lead to a distinct metabolic signature resulting in a separation in the first two dimensions of a principal components analysis. Six metabolites including glycolic acid, azelaic acid, 2-n-pentylfuran, dihydroactinidiolide, 2-butenal and n-pentanal were consistently increased after electrosurgery meeting the discovery (p<2.010?4) and the replication thresholds (p<3.510?3). Azelaic acid, a lipid peroxidation product from the fragmentation of abundant sn-2 linoleoyl residues, was most abundant and increased 8.1-fold after electrosurgical liver dissection (preplication?=?1.610?4). The corresponding phospholipid hexadecyl azelaoyl glycerophosphocholine inhibited wound healing and tissue remodelling in scratch- and proliferation assays of hepatic stellate cells and cholangiocytes, and caused apoptosis dose-dependently in vitro, which may explain in part the tissue damage due to electrosurgery. Conclusion Hepatic electrosurgery generates a metabolic signature with characteristic lipid peroxidation products. Among these, azelaic acid shows a dose-dependent toxicity in liver cells and inhibits wound healing. These observations potentially pave the way for pharmacological intervention prior liver surgery to modify the metabolic response and prevent postoperative complications. PMID:24058442

  20. Spontaneous coronary artery dissectionA review

    PubMed Central

    Yip, Amelia

    2015-01-01

    Spontaneous coronary artery dissection (SCAD) is an infrequent and often missed diagnosis among patients presenting with acute coronary syndrome (ACS). Unfortunately, SCAD can result in significant morbidities such as myocardial ischemia and infarction, ventricular arrhythmias and sudden cardiac death. Lack of angiographic recognition from clinicians is a major factor of under-diagnosis. With the advent of new imaging modalities, particularly with intracoronary imaging, there has been improved diagnosis of SCAD. The aim of this paper is to review the epidemiology, etiology, presentation, diagnosis and management of SCAD. PMID:25774346

  1. Left Atrial Wall Dissection after Mitral Valve Replacement

    PubMed Central

    Kim, Kyung Woo; Park, Se Hyeok; Lee, Sang-Il; Kim, Ji Yeon; Kim, Kyung-Tae; Choe, Won Joo; Park, Jang Su; Kim, Jung Won

    2013-01-01

    Left atrial dissection does occur, though rarely, after mitral valve surgery. A 68-year-old Korean female presented with moderate mitral stenosis, mild mitral regurgitation, moderate tricuspid regurgitation and mild aortic regurgitation. She was scheduled for mitral valve replacement and tricuspid annuloplasty. We experienced a left atrial dissection after weaning from cardiopulmonary bypass and decided not to repair it. The patient recovered uneventfully. We suggest that a specific type of left atrial dissection can be treated conservatively. PMID:24198922

  2. Isolated brachiocephalic artery dissection presenting as acute stroke.

    PubMed

    Mani, Hariharasudan; Ahluwalia, Sharat

    2015-01-01

    Isolated brachiocephalic artery dissection is an extremely rare condition. Its presentation as an acute stroke can pose a significant diagnostic challenge in patients because of its rarity. We present a case of isolated spontaneous brachiocephalic artery dissection presenting as acute cerebrovascular accident. This case also illustrates the treatment dilemma brachiocephalic artery dissection can present, whether to choose antithrombotic/anticoagulation therapy and/or surgery, and also the dilemma in blood pressure management. PMID:26315357

  3. Chronic pulmonary artery dissection associated with pulmonary arterial hypertension

    PubMed Central

    2013-01-01

    Abstract Pulmonary artery dissection is a complication associated with pulmonary arterial hypertension. This complication is described as acute in onset and is frequently fatal without intervention. We describe a patient with idiopathic pulmonary arterial hypertension and chest pain found to have an unsuspected chronic pulmonary artery dissection on postmortem examination. Chronic pulmonary artery dissection should be considered in patients with chest pain and worsening dyspnea, as the frequency this condition may be underestimated. PMID:24618553

  4. Spontaneous Coronary Dissection Masquerading as Benign Fascicular Ventricular Tachycardia.

    PubMed

    Ho, Sara Wei-Fen; Lin, Weiqin; Chan, Koo Hui; Seow, Swee-Chong

    2016-01-01

    Spontaneous coronary artery dissection is an uncommon cause of acute coronary syndrome. Diagnosis of coronary artery dissection is made on coronary angiogram and prompt revascularisation is the key in management. We present a case of coronary artery dissection with an atypical presentation of cardiac arrhythmia mimicking benign fascicular ventricular tachycardia. A high index of suspicion and early coronary angiogram allowed us to diagnose and treat this potentially life-threatening disease. PMID:26475648

  5. Iatrogenic left main artery dissection: A catastrophic complication

    PubMed Central

    Namazi, Mohammad Hassan; Rostami, Reza Tajik; Mohammadi, Afsaneh; Amini, Abdol Latifi; Safi, Morteza; Saadat, Habibollah; Vakili, Hosein; Motamedi, Mohammad Reza; Movahed, Mohammad Reza

    2012-01-01

    Iatrogenic left main artery (LM) dissection is a catastrophic complication of coronary angiography and angioplasty that requires prompt management using stenting. Although LM dissection can be prevented, it cannot always be avoided and has a reported incidence rate of 0.02%. In the present report, a case of iatrogenic LM dissection that was successfully treated with multiple stents is presented and followed by a brief review of the literature. PMID:23592948

  6. Aortic dissection after superior mesenteric artery percutaneous stenting. Case report.

    PubMed

    Socrate, A M; Locati, P; Marchetti, G

    2000-03-01

    We report an unusual case of aortic dissection after superior mesenteric artery percutaneous stenting. A 44-year-old patient, who suffered from back pain and fever, was diagnosed as having an aortic dissection. Aortic dissection, extending from the aortic arch (just after left subclavian artery origin) to the aortic carrefour, was successfully diagnosed by means of Duplex scan and CT scan examination. Two pathogenetic hypotheses, malformative and iatrogenic, were discussed. PMID:10838838

  7. Doing Dissections Differently: A Structured, Peer-Assisted Learning Approach to Maximizing Learning in Dissections

    ERIC Educational Resources Information Center

    Hall, Emma R.; Davis, Rachel C.; Weller, Renate; Powney, Sonya; Williams, Sarah B.

    2013-01-01

    Areas of difficulty faced by our veterinary medicine students, with respect to their learning in dissection classes, were identified. These challenges were both general adult-learning related and specific to the discipline of anatomy. Our aim was to design, implement, and evaluate a modified reciprocal peer-assisted/team-based learning…

  8. Background Music in the Dissection Laboratory: Impact on Stress Associated with the Dissection Experience

    ERIC Educational Resources Information Center

    Anyanwu, Emeka G.

    2015-01-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on

  9. Background Music in the Dissection Laboratory: Impact on Stress Associated with the Dissection Experience

    ERIC Educational Resources Information Center

    Anyanwu, Emeka G.

    2015-01-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on…

  10. Doing Dissections Differently: A Structured, Peer-Assisted Learning Approach to Maximizing Learning in Dissections

    ERIC Educational Resources Information Center

    Hall, Emma R.; Davis, Rachel C.; Weller, Renate; Powney, Sonya; Williams, Sarah B.

    2013-01-01

    Areas of difficulty faced by our veterinary medicine students, with respect to their learning in dissection classes, were identified. These challenges were both general adult-learning related and specific to the discipline of anatomy. Our aim was to design, implement, and evaluate a modified reciprocal peer-assisted/team-based learning

  11. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog

    NASA Astrophysics Data System (ADS)

    Akpan, Joseph Paul; Andre, Thomas

    1999-06-01

    Science teachers, school administrators, educators, and the scientific community are faced with ethical controversies over animal dissection in classrooms. Simulation has been proposed as a way of dealing with this issue. One intriguing previous finding was that use of an interactive videodisc dissection facilitated performance on a subsequent actual dissection. This study examined the prior use of simulation of frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. There were three experimental conditions: simulation before dissection (SBD); dissection before simulation (DBS); or dissection-only (DO). Results of the study indicated that students receiving SBD performed significantly better than students receiving DBS or DO on both actual dissection and knowledge of the anatomy and morphology. Students' attitudes toward the use of animals for dissection did not change significantly from pretest to posttest and did not interact with treatment. The genders did not differ in achievement, but males were more favorable towards dissection and computers than were females.

  12. Molecular Mechanisms of Thoracic Aortic Dissection

    PubMed Central

    Wu, Darrell; Shen, Ying H.; Russell, Ludivine; Coselli, Joseph S.; LeMaire, Scott A.

    2013-01-01

    Thoracic aortic dissection (TAD) is a highly lethal vascular disease. In many patients with TAD, the aorta progressively dilates and ultimately ruptures. Dissection formation, progression, and rupture cannot be reliably prevented pharmacologically because the molecular mechanisms of aortic wall degeneration are poorly understood. The key histopathologic feature of TAD is medial degeneration, a process characterized by smooth muscle cell depletion and extracellular matrix degradation. These structural changes have a profound impact on the functional properties of the aortic wall and can result from excessive protease-mediated destruction of the extracellular matrix, altered signaling pathways, and altered gene expression. Review of the literature reveals differences in the processes that lead to ascending versus descending and sporadic versus hereditary TAD. These differences add to the complexity of this disease. Although tremendous progress has been made in diagnosing and treating TAD, a better understanding of the molecular, cellular, and genetic mechanisms that cause this disease is necessary to developing more effective preventative and therapeutic treatment strategies. PMID:23856125

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

    PubMed

    Li, Yunfeng; Ye, Zhijian; Nie, Yanfang; Zhang, Jian; Wang, Guo-Liang; Wang, Zhenzhong

    2015-06-01

    Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most destructive disease of rice and causes tremendous losses of rice yield worldwide. To explore the molecular mechanisms involved in the rice-M. oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae. This data article contains additional results and analysis of M. oryzae-regulated phosphoproteins in rice leaves [1]. We report the analysis of M. oryzae-regulated phosphoproteins at all time points, including Venn diagram analysis, close-up views, relative intensities, and functional category, and the MS spectra of representative phosphoprotein and representative phosphorylated peptides. PMID:26217708

  14. A phosphoproteomics approach to identify candidate kinase inhibitor pathway targets in lymphoma-like primary cell lines.

    PubMed

    Vojvodic, Miliana; Hansford, Loen M; Morozova, Olena; Blakely, Kim M; Taylor, Paul; Fathers, Kelly E; Moffat, Jason; Marra, Marco; Smith, Kristen M; Moran, Michael F; Kaplan, David R

    2013-12-01

    Mass spectrometry-based technologies are increasingly utilized in drug discovery. Phosphoproteomics in particular has allowed for the efficient surveying of phosphotyrosine signaling pathways involved in various diseases states, most prominently in cancer. We describe a phosphotyrosine-based proteomics screening approach to identify signaling pathways and tyrosine kinase inhibitor targets in highly tumorigenic human lymphoma-like primary cells. We identified several receptor tyrosine kinase pathways and validated SRC family kinases (SFKs) as potential drug targets for targeted selection of small molecule inhibitors. BMS-354825 (dasatinib) and SKI-606 (bosutinib), second and third generation clinical SFK/ABL inhibitors, were found to be potent cytotoxic agents against tumorigenic cells with low toxicity to normal pediatric stem cells. Both SFK inhibitors reduced ERK1/2 and AKT phosphorylation and induced apoptosis. This study supports the adaptation of high-end mass spectrometry techniques for the efficient identification of candidate tyrosine kinases as novel therapeutic targets in primary cancer cell lines. PMID:23701117

  15. Diagnosis of Aortic Dissection in Emergency Department Patients is Rare

    PubMed Central

    Alter, Scott M.; Eskin, Barnet; Allegra, John R.

    2015-01-01

    Introduction Aortic dissection is a rare event. While the most frequent symptom is chest pain, that is a common emergency department (ED) chief complaint and other diseases causing chest pain occur much more often. Furthermore, 20% of dissections are without chest pain and 6% are painless. For these reasons, diagnosing dissections may be challenging. Our goal was to determine the number of total ED and atraumatic chest pain patients for every aortic dissection diagnosed by emergency physicians. Methods Design: Retrospective cohort. Setting: 33 suburban and urban New York and New Jersey EDs with annual visits between 8,000 and 80,000. Participants: Consecutive patients seen by emergency physicians from 1-1-1996 through 12-31-2010. Observations: We identified aortic dissection and atraumatic chest pain patients using the International Classification of Diseases 9th Revision and Clinical Modification codes. We then calculated the number of total ED and atraumatic chest pain patients for every aortic dissection, along with 95% confidence intervals (CIs). Results From a database of 9.5 million ED visits, we identified 782 aortic dissections or one for every 12,200 (95% CI [11,40013,100]) visits. The mean age of dissection patients was 6616 years and 38% were female. There were 763,000 (8%) with atraumatic chest pain diagnoses. Thus, there is one dissection for every 980 (95% CI [9101,050]) atraumatic chest pain patients. Conclusion The diagnosis of aortic dissections by emergency physicians is rare and challenging. An emergency physician seeing 3,000 to 4,000 patients a year would diagnose an aortic dissection approximately every three to four years. PMID:26587083

  16. Functional dissection of Odorant binding protein genes in Drosophila melanogaster.

    PubMed

    Swarup, S; Williams, T I; Anholt, R R H

    2011-08-01

    Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial. PMID:21605338

  17. Genetic dissection of agronomic traits in Capsicum baccatum var. pendulum.

    PubMed

    Moulin, M M; Rodrigues, R; Bento, C S; Gonalves, L S A; Santos, J O; Sudr, C P; Viana, A P

    2015-01-01

    Genetic mapping is very useful for dissecting complex agronomic traits. Genetic mapping allows for identification of quantitative trait loci (QTL), provide knowledge on a gene position and its adjacent region, and enable prediction of evolutionary mechanisms, in addition to contributing to synteny studies. The aim of this study was to predict genetic values associated with different agronomic traits evaluated in an F2 population of Capsicum baccatum var. pendulum. Previously, a reference genetic map for C. baccatum was constructed, which included 183 markers (42 microsatellite, 85 inter-simple sequence repeat, and 56 random amplification of polymorphic DNA) arranged in 16 linkage groups. The map was used to identify QTL associated with 11 agronomic traits, including plant height, crown diameter, number of days to flowering, days to fruiting, number of fruits per plant, average fruit weight, fruit length, fruit diameter, fruit pulp thickness, soluble solids, and fruit dry weight. QTL mapping was performed by standard interval mapping. The number of small QTL effects ranged from 3-11, with a total of 61 QTL detected in 9 linkage groups. This is the first report involving QTL analysis for C. baccatum species. PMID:25867359

  18. Phosphoproteomic Profiling of In Vivo Signaling in Liver by the Mammalian Target of Rapamycin Complex 1 (mTORC1)

    PubMed Central

    Demirkan, Gokhan; Yu, Kebing; Boylan, Joan M.; Salomon, Arthur R.; Gruppuso, Philip A.

    2011-01-01

    Background Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood. Methodology/Principal Findings We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates. Conclusions/Significance In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo. PMID:21738781

  19. Proteome and Phosphoproteome Characterization Reveals New Response and Defense Mechanisms of Brachypodium distachyon Leaves under Salt Stress*

    PubMed Central

    Lv, Dong-Wen; Subburaj, Saminathan; Cao, Min; Yan, Xing; Li, Xiaohui; Appels, Rudi; Sun, Dong-Fa; Ma, Wujun; Yan, Yue-Ming

    2014-01-01

    Salinity is a major abiotic stress affecting plant growth and development. Understanding the molecular mechanisms of salt response and defense in plants will help in efforts to improve the salt tolerance of crops. Brachypodium distachyon is a new model plant for wheat, barley, and several potential biofuel grasses. In the current study, proteome and phosphoproteome changes induced by salt stress were the focus. The Bd21 leaves were initially treated with salt in concentrations ranging from 80 to 320 mm and then underwent a recovery process prior to proteome analysis. A total of 80 differentially expressed protein spots corresponding to 60 unique proteins were identified. The sample treated with a median salt level of 240 mm and the control were selected for phosphopeptide purification using TiO2 microcolumns and LC-MS/MS for phosphoproteome analysis to identify the phosphorylation sites and phosphoproteins. A total of 1509 phosphoproteins and 2839 phosphorylation sites were identified. Among them, 468 phosphoproteins containing 496 phosphorylation sites demonstrated significant changes at the phosphorylation level. Nine phosphorylation motifs were extracted from the 496 phosphorylation sites. Of the 60 unique differentially expressed proteins, 14 were also identified as phosphoproteins. Many proteins and phosphoproteins, as well as potential signal pathways associated with salt response and defense, were found, including three 14-3-3s (GF14A, GF14B, and 14-3-3A) for signal transduction and several ABA signal-associated proteins such as ABF2, TRAB1, and SAPK8. Finally, a schematic salt response and defense mechanism in B. distachyon was proposed. PMID:24335353

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

    PubMed Central

    2010-01-01

    Background Sea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field. Results The present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919]. Conclusions The 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental phosphoproteins as phosphoryn or dentin matrix protein-1, phosphodontin may perform similar functions in the sea urchin tooth. More than half of the detected proteins were not previously identified at the protein level, thus confirming the existence of proteins only known as genomic sequences previously. PMID:20181113

  1. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog.

    ERIC Educational Resources Information Center

    Akpan, Joseph Paul; Andre, Thomas

    1999-01-01

    Reports on a study that examined the prior use of simulation on frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. Finds that students who performed the simulation before dissection performed significantly better than students who performed dissection before using the simulation or…

  2. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog.

    ERIC Educational Resources Information Center

    Akpan, Joseph Paul; Andre, Thomas

    1999-01-01

    Reports on a study that examined the prior use of simulation on frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. Finds that students who performed the simulation before dissection performed significantly better than students who performed dissection before using the simulation or

  3. Student Attitudes toward Cadaveric Dissection at a UK Medical School

    ERIC Educational Resources Information Center

    Quince, Thelma A.; Barclay, Stephen I. G.; Spear, Michelle; Parker, Richard A.; Wood, Diana F.

    2011-01-01

    A more humanistic approach toward dissection has emerged. However, student attitudes toward this approach are unknown and the influences on such attitudes are little understood. One hundred and fifty-six first-year medical students participated in a study examining firstly, attitudes toward the process of dissection and the personhood of the…

  4. Outcomes of a Rotational Dissection System in Gross Anatomy

    ERIC Educational Resources Information Center

    Marshak, David W.; Oakes, Joanne; Hsieh, Pei-Hsuan; Chuang, Alice Z.; Cleary, Leonard J.

    2015-01-01

    At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to…

  5. Student Attitudes toward Cadaveric Dissection at a UK Medical School

    ERIC Educational Resources Information Center

    Quince, Thelma A.; Barclay, Stephen I. G.; Spear, Michelle; Parker, Richard A.; Wood, Diana F.

    2011-01-01

    A more humanistic approach toward dissection has emerged. However, student attitudes toward this approach are unknown and the influences on such attitudes are little understood. One hundred and fifty-six first-year medical students participated in a study examining firstly, attitudes toward the process of dissection and the personhood of the

  6. Dissection Videos Do Not Improve Anatomy Examination Scores

    ERIC Educational Resources Information Center

    Mahmud, Waqas; Hyder, Omar; Butt, Jamaal; Aftab, Arsalan

    2011-01-01

    In this quasi-experimental study, we describe the effect of showing dissection videos on first-year medical students' performance in terms of test scores during a gross anatomy course. We also surveyed students' perception regarding the showing of dissection videos. Two hundred eighty-seven first-year medical students at Rawalpindi Medical College

  7. Outcomes of a Rotational Dissection System in Gross Anatomy

    ERIC Educational Resources Information Center

    Marshak, David W.; Oakes, Joanne; Hsieh, Pei-Hsuan; Chuang, Alice Z.; Cleary, Leonard J.

    2015-01-01

    At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to

  8. Traumatic Axillary Artery Dissection with Radial Artery Embolism

    SciTech Connect

    Chung, Hwan-Hoon; Cha, Sang Hoon Cho, Sung Bum; Kim, Jung Hyuck; Lee, Seung Hwa; Shin, Jae Seung; Park, Sang Woo

    2006-04-15

    This report describes a case of pathologically proven traumatic arterial dissection, presenting as complete occlusion of the axillary artery with radial artery embolism. Occlusion of the axillary artery by traumatic dissection mimicked transection and radial artery embolism mimicked congenital absence of the radial artery on the initial angiogram, but these were correctly diagnosed with the following sonogram.

  9. Multidetector CT of Aortic Dissection: A Pictorial Review.

    PubMed

    McMahon, Michelle A; Squirrell, Christopher A

    2010-03-01

    Aortic dissection is the most common acute emergency condition of the aorta and often has a fatal outcome. Outcome is determined by the type and extent of dissection and the presence of associated complications (eg, cerebral sequelae, aortic branch involvement, pericardial involvement, and visceral involvement), with early diagnosis and treatment being essential for improved prognosis. Aortic dissections are classified on the basis of the site of the intimal tear according to the Stanford classification system. Type A aortic dissection involves the ascending thoracic aorta and may extend into the descending aorta, whereas in a type B dissection the intimal tear is located distal to the left subclavian artery. Type A dissection typically requires urgent surgical intervention, whereas type B dissection can often be treated medically. Modern multidetector computed tomography (CT) is a fast, widely available imaging modality with high sensitivity and specificity. Multidetector CT allows the early recognition and characterization of aortic dissection as well as determination of the presence of any associated complications, findings that are essential for optimizing treatment and improving clinical outcomes. PMID:20228328

  10. Alternatives To Animal Dissection in School Science Classes. ERIC Digest.

    ERIC Educational Resources Information Center

    Haury, David L.

    Until recently, one of the most expected and accepted experiences among students in biology classrooms of the United States has been the dissection of vertebrate animals, from frogs and mice to cats and fetal pigs. However, resistance to animal dissection has grown during the past decade with concerns ranging from inhumane treatment of animals by…

  11. A Modified Dissection Method to Preserve Neck Structures

    ERIC Educational Resources Information Center

    Hankin, Mark H.; Stoller, Jeremy L.

    2009-01-01

    The neck is not only one of the more challenging anatomical regions to dissect but also has important application to clinical conditions, diseases, and procedures. In this study, we describe two simple modifications for dissection of the neck that (1) aid in the identification and preservation of the cutaneous branches of the cervical plexus and

  12. Dissection Videos Do Not Improve Anatomy Examination Scores

    ERIC Educational Resources Information Center

    Mahmud, Waqas; Hyder, Omar; Butt, Jamaal; Aftab, Arsalan

    2011-01-01

    In this quasi-experimental study, we describe the effect of showing dissection videos on first-year medical students' performance in terms of test scores during a gross anatomy course. We also surveyed students' perception regarding the showing of dissection videos. Two hundred eighty-seven first-year medical students at Rawalpindi Medical College…

  13. Justifying the Dissection of Animals in Biology Teaching.

    ERIC Educational Resources Information Center

    Wheeler, Anthony G.

    1993-01-01

    Presents the idea that several points should be considered in animal dissection: (1) ethics; (2) the animal's position; (3) the law; and (4) the actual benefits to society. Recommends that students be carefully prepared for animal dissection. Contains 21 references. (DDR)

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

    PubMed Central

    Nakedi, Kehilwe C.; Nel, Andrew J. M.; Garnett, Shaun; Blackburn, Jonathan M.; Soares, Nelson C.

    2015-01-01

    Ser/Thr/Tyr protein phosphorylation plays a critical role in regulating mycobacterial growth and development. Understanding the mechanistic link between protein phosphorylation signaling network and mycobacterial growth rate requires a global view of the phosphorylation events taking place at a given time under defined conditions. In the present study we employed a phosphopeptide enrichment and high throughput mass spectrometry-based strategy to investigate and qualitatively compare the phosphoproteome of two mycobacterial model organisms: the fast growing Mycobacterium smegmatis and the slow growing Mycobacterium bovis BCG. Cells were harvested during exponential phase and our analysis detected a total of 185 phospho-sites in M. smegmatis, of which 106 were confidently localized [localization probability (LP) = 0.75; PEP = 0.01]. By contrast, in M. bovis BCG the phosphoproteome comprised 442 phospho-sites, of which 289 were confidently localized. The percentage distribution of Ser/Thr/Tyr phosphorylation was 39.47, 57.02, and 3.51% for M. smegmatis and 35, 61.6, and 3.1% for M. bovis BCG. Moreover, our study identified a number of conserved Ser/Thr phosphorylated sites and conserved Tyr phosphorylated sites across different mycobacterial species. Overall a qualitative comparison of the fast and slow growing mycobacteria suggests that the phosphoproteome of M. smegmatis is a simpler version of that of M. bovis BCG. In particular, M. bovis BCG exponential cells exhibited a much more complex and sophisticated protein phosphorylation network regulating important cellular cycle events such as cell wall biosynthesis, elongation, cell division including immediately response to stress. The differences in the two phosphoproteomes are discussed in light of different mycobacterial growth rates. PMID:25904896

  15. Dissecting aortic aneurysms: accuracy of computed tomographic diagnosis

    SciTech Connect

    Thorsen, M.K.; San Dretto, M.A.; Lawson, T.L.; Foley, W.D.; Smith, D.F.; Berland, L.L.

    1983-09-01

    During a three-year period, fifty patients were evaluated for the possibility of dissecting aortic aneurysm using high-resolution computed tomography (CT). The diagnosis of dissection was made if two contrast-medium-filled channels were identified within the aortic lumen. Eighteen patients were diagnosed with CT as having dissecting aortic aneurysms. Eight patients were evaluated postoperatively and five of these patients had persistence of the double channel. Twenty-four patients had no evidence on CT of aortic dissection. Follow-up was obtained in all patients. There were no known false-negative diagnoses and one false-positive diagnosis. High-resolution CT offers an accurate, noninvasive means to evaluate patients for suspected dissecting aortic aneurysms.

  16. CrossFit-related cervical internal carotid artery dissection.

    PubMed

    Lu, Albert; Shen, Peter; Lee, Paul; Dahlin, Brian; Waldau, Ben; Nidecker, Anna E; Nundkumar, Anoop; Bobinski, Matthew

    2015-08-01

    CrossFit is a high-intensity strength and conditioning program that has gained popularity over the past decade. Potential injuries associated with CrossFit training have been suggested in past reports. We report three cases of cervical carotid dissection that are associated with CrossFit workouts. Patient 1 suffered a distal cervical internal carotid artery (ICA) dissection near the skull base and a small infarct in Wernicke's area. He was placed on anticoagulation and on follow-up has near complete recovery. Patient 2 suffered a proximal cervical ICA dissection that led to arterial occlusion and recurrent middle cerebral artery territory infarcts and significant neurological sequelae. Patient 3 had a skull base ICA dissection that led to a partial Horner's syndrome but no cerebral infarct. While direct causality cannot be proven, intense CrossFit workouts may have led to the ICA dissections in these patients. PMID:25917634

  17. Lymph node dissection for gastric cancer: a critical review

    PubMed Central

    Batista, Thales Paulo; Martins, Mário Rino

    2012-01-01

    Gastric cancer is one of the most common neoplasms and an important cause of cancer-related death worldwide. Efforts to reduce its high mortality rates are currently focused on multidisciplinary management. However, surgery remains a cornerstone in the management of patients with resectable disease. There is still some controversy as to the extent of lymph node dissection for potentially curable stomach cancer. Surgeons in eastern countries favor more extensive lymph node dissection, whereas those in the West favor less extensive dissection. Thus, extent of lymph node dissection remains one of the most hotly discussed aspects of gastric surgery, particularly because most stomach cancers are now often comprehensively treated by adding some perioperative chemotherapy or chemo-radiation. We provide a critical review of lymph nodes dissection for gastric cancer with a particular focus on its benefits in a multimodal approach. PMID:25992202

  18. Traditional versus Computer-Based Dissections in Enhancing Learning in a Tertiary Setting: A Student Perspective.

    ERIC Educational Resources Information Center

    Franklin, Sue; Peat, Mary; Lewis, Alison

    2002-01-01

    Describes a study that investigates both the use and usefulness of laboratory dissections and computer-based dissections in a tertiary, first-year human biology course. Explores attitudes toward dissection. (DDR)

  19. Dissecting Japan's Dengue Outbreak in 2014.

    PubMed

    Quam, Mikkel B; Sessions, October; Kamaraj, Uma Sangumathi; Rocklöv, Joacim; Wilder-Smith, Annelies

    2016-02-01

    Despite Japan's temperate climate, a dengue outbreak occurred in Tokyo for the first time in over 70 years in 2014. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam. With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come. PMID:26711518

  20. Cervical Artery Dissection: Emerging Risk Factors

    PubMed Central

    Micheli, S; Paciaroni, M; Corea, F; Agnelli, G; Zampolini, M; Caso, V

    2010-01-01

    Cervical artery dissection (CAD) represents an increasingly recognized cause of stroke and the most common cause of ischemic stroke in young adults. Many factors have been identified in association with CAD such as primary disease of arterial wall (fibrodysplasia) and other non-specific diseases related to CAD like Ehlers Danlos-syndrome IV, Marfans syndrome, vessel tortuosity. Moreover, an underlying arteriopathy which could be in part genetically determined, has been suspected. The rule of emerging risk factors for CAD such as recent respiratory tract infection, migraine and hyperhomocysteinemia are still a matter of research. Other known risks factors for CAD are major head/neck trauma like chiropractic maneuver, coughing or hyperextension injury associated to car. We examined emerging risks factors for CAD detected in the last years, as CAD pathogenesis is still not completely understood and needs further investigations. PMID:21270941

  1. Virtual dissection of aortic lesions from MRI

    NASA Astrophysics Data System (ADS)

    McColl, Roderick W.; Lo, Hao; Peshock, Ronald M.

    2004-04-01

    In this article we demonstrate the use of an automated technique to visualize lesions in the abdominal aorta, gathered from MRI imagery, and displayed as a stained pathology specimen. We have developed this technique in response to the suggestion from clinical colleagues that such a representation of the data is more understandable to the pathologist than presentation of axial MRI slices, even if the atheroma is of high contrast and very thick. This virtual dissection relies on an initial manual segmentation of the inner and outer walls of the vessel, which I achieved using a commercial cardiac analysis package. The algorithm consists of (1) decoding the file that describes the contours; (2) generating a center for each slice; (3) determining the appropriate posterior position on all slices; (4) interpolating along the (approximately circular) lumen and through all slices; (5) false color presentation of the wall thickness as a pathological stain.

  2. Proto-professionalism and the dissecting laboratory.

    PubMed

    Slotnick, Henry B; Hilton, Sean R

    2006-07-01

    This essay argues for medical students' dissection of cadavers because this activity offers medical students opportunities to have certain experiences and reflect on them in ways facilitating their development of mature medical professionalism at the time they enter clinical practice. Issues central to professionalism as we envision it are (1) cognitive abilities identified as reflective judgment and principled ethical reasoning as they are exercised in four practice domains and (2) learning to learn in medical settings. We argue further that a key feature of such setting is practitioners' having to deal with ill-structured problems, and so we identify their features and relate their management to the sophisticated cognitive and learning abilities required of medical professionals. PMID:16583422

  3. Dissecting Japan's Dengue Outbreak in 2014

    PubMed Central

    Quam, Mikkel B.; Sessions, October; Kamaraj, Uma Sangumathi; Rocklöv, Joacim; Wilder-Smith, Annelies

    2016-01-01

    Despite Japan's temperate climate, a dengue outbreak occurred in Tokyo for the first time in over 70 years in 2014. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam. With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come. PMID:26711518

  4. Spontaneous renal artery dissection with renal infarction

    PubMed Central

    Leray-Moragus, Hlne; Chenine, Leila; Vernhet-Kovacsik, Hlne

    2012-01-01

    Spontaneous renal artery dissection (SRAD) is a rare entity, which often presents diagnostic difficulties because of its non-specific clinical presentation. We report six cases complicated with renal infarction, occurring in middle-aged male patients without risk factors, illustrating the difficulty and delay for diagnosing SRAD. Ultrasound and Doppler imaging were not sensitive enough to confirm the diagnosis, and contrast-enhanced abdominal computed tomography was used to correct the diagnosis and allow the clinicians to propose appropriate treatment. We conclude that considering the urgency in diagnosing and treating SRAD, contrast enhanced abdominal tomography and/or abdominal magnetic resonance imaging should be proposed as soon as a suspicion of SRAD is evoked by the clinical presentation. PMID:26069781

  5. Immunostaining of dissected zebrafish embryonic heart.

    PubMed

    Yang, Jingchun; Xu, Xiaolei

    2012-01-01

    Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects. The expression of any gene can be manipulated via morpholino technology or RNA injection. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals. PMID:22258109

  6. Diamondiferous Peridotite Tomography: Precursor to Xenolith Dissections.

    NASA Astrophysics Data System (ADS)

    Taylor, L. A.; Taylor, D. S.; Anand, M.; Ketcham, R.; Carlson, W.; Sobolev, N. V.; Pokhilenko, N.

    2003-12-01

    Three-dimensional, high-resolution computed X-ray tomography (HRCXT) of diamondiferous peridotite xenoliths from Siberia has successfully imaged diamonds and their textural relationships with co-existing minerals. This is an extension to the tomography and xenolith dissections performed previously on diamondiferous eclogites (e.g., Taylor et al., 2000, Int'l Geol Rev 42; Anand et al., 2003, 8th Int'l Kimb Conf). Spatial relationships between diamonds and their surroundings provide clues to the processes that control diamond crystallization. These relationships can be determined by rotating and viewing the 3-D model at different perspectives and orientations to look for specific associations/alignments. It is possible to render some of the phases transparent and display only one or two minerals at a time. Then by rotating the model, it is possible to look for spatial relationships between different crystals of the same mineral or different minerals. The maps obtained from the tomography form the basis for the delicate dissections of the xenoliths, revealing the diamonds as they formed in their mantle host rocks. Diamondiferous peridotites appear to be sparse carriers of diamonds compared to eclogites, where one unusual eclogite of 65 g contained 74 macro-diamonds. Although kimberlites always contain considerably more peridotite xenoliths than eclogites, the diamond population may actually be more indicative of eclogitic origin. The diamonds appear to be preferentially located in zones with a prominent sub-planar fabric of secondary mineralization - i.e., zones of increased permeability. Diamond was never observed in direct contact with any fresh, primary minerals. Also, there is insufficient sulfide mineralization to call upon an immiscible-sulfide melt as the diamond-forming medium. The association of the diamonds with secondary minerals strongly suggests that the diamonds formed after the initial host peridotite or eclogite, perhaps in conjunction with introduction of metasomatic fluids.

  7. A comprehensive proteomic and phosphoproteomic analysis of yeast deletion mutants of 14-3-3 orthologs and associated effects of rapamycin

    PubMed Central

    Paulo, Joao A.; Gygi, Steven P.

    2015-01-01

    We applied a multiplexed, mass spectrometry-based strategy to interrogate the proteome and phosphoproteome of three yeast strains under two growth conditions. The yeast proteins Bmh1 and Bmh2, analogs to the 14-3-3 protein family, have a wide-array of cellular functions including the regulation of phosphorylation events. Similarly, rapamycin is a drug that can regulate phosphorylation events. By performing a series of TMT10-plex experiments, we investigated the alterations in the proteome and phosphoproteome of wildtype and two deletion strains (bmh1Δ and bmh2Δ) of S. cerevisiae treated with rapamycin and DMSO as a control. Our 3×3+1 strategy allowed for triplicate analysis of each of the three strains, plus an additional sample consisting of an equal mix of all samples. We quantified over 4000 proteins and 20,000 phosphorylation events. Of these, we quantified over 3700 proteins across all 20 samples and over 14,300 phosphorylation events within each drug treatment. In total, data collected from four TMT10-plex experiments required approximately one week of data collection on the mass spectrometer. This study underscores the complex cellular roles of Bmh1 and Bmh2 coupled with response to rapamycin treatment and emphasizes the utility of multiplexed proteomic techniques to elucidate comprehensive proteomes and phosphoproteomes. PMID:25315811

  8. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    ERIC Educational Resources Information Center

    Randler, Christoph; Demirhan, Eda; Wst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection.

  9. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    ERIC Educational Resources Information Center

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection.…

  10. The Effects of Computer Animated Dissection versus Preserved Animal Dissection on the Student Achievement in a High School Biology Class.

    ERIC Educational Resources Information Center

    Kariuki, Patrick; Paulson, Ronda

    The purpose of this study was to examine the effectiveness of computer-animated dissection techniques versus the effectiveness of traditional dissection techniques as related to student achievement. The sample used was 104 general biology students from a small, rural high school in Northeast Tennessee. Random selection was used to separate the

  11. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    PubMed Central

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection. We compared this treatment with another group that watched a life history video about the fish. The participants were 135 students studying to become biology teachers. Seventy received the treatment with the dissection video, and 65 viewed the life history video. We applied a pre/posttest treatment-comparison design and used the Positive and Negative Affect Schedule (PANAS), the State–Trait–Anxiety Inventory for State (STAI-S), and a self-efficacy measure three times: before the lesson (pretest), after the film treatment (posttest 1), and after the dissection (posttest 2). The dissection film group scored higher in PA, NA, and state anxiety (STAI-S) after the dissection video treatment and higher in self-efficacy after the dissection. The life history group showed no differences between the pretest and posttest 1. The dissection film has clear benefits—increasing PA and self-efficacy—that come at the cost of higher NA and higher STAI-S.

  12. A variant of nested dissection for solving n by n grid problems

    NASA Technical Reports Server (NTRS)

    George, A.; Poole, W. G., Jr.; Voigt, R. G.

    1976-01-01

    Nested dissection orderings are known to be very effective for solving the sparse positive definite linear systems which arise from n by n grid problems. In this paper nested dissection is shown to be the final step of incomplete nested dissection, an ordering which corresponds to the premature termination of dissection. Analyses of the arithmetic and storage requirements for incomplete nested dissection are given, and the ordering is shown to be competitive with nested dissection under certain conditions.

  13. Dissecting the iTRAQ Data Analysis.

    PubMed

    Aggarwal, Suruchi; Yadav, Amit Kumar

    2016-01-01

    In the era of large-scale quantitative biology, mass spectrometry-based quantitative proteomics is progressively becoming indispensable for gaining insights into the biological systems at molecular level. Various quantitative study designs rely on chemical tagging approaches to study disease, stress, or drug response and temporal studies aiming at disease/developmental progression in a biological system. Isobaric tags for relative and absolute quantitation (iTRAQ) is one of the most popular chemical labeling techniques which allows four, six, or eight samples to be multiplexed in a single run. As the iTRAQ tag has a balancer group to equalize all states of a labeled peptide to same mass, the differentially labeled iTRAQ peptides are mixed before chromatography and elute as a single combined peak in MS. This enhances the peptide signal and quantitation is performed during MS/MS along with sequencing, where reporter ions of different masses are released to give relative quantitation. Known amount of a spiked-in protein can also help in absolute quantitation of the proteins in a sample. PMID:26519184

  14. Painless Type B Aortic Dissection: Insights From the International Registry of Acute Aortic Dissection

    PubMed Central

    Tolenaar, Jip L.; Hutchison, Stuart J.; Montgomery, Dan; O'Gara, Patrick; Fattori, Rosella; Pyeritz, Reed E.; Pape, Linda; Suzuki, Toru; Evangelista, Arturo; Moll, Frans L.; Rampoldi, Vincenzo; Isselbacher, Eric M.; Nienaber, Cristoph A.; Eagle, Kim A.; Trimarchi, Santi

    2013-01-01

    Introduction: The classical presentation of a patient with Type B acute aortic dissection (TBAAD) is characterized by severe chest, back, or abdominal pain, ripping or tearing in nature. However, some patients present with painless acute aortic dissection, which can lead to a delay in diagnosis and treatment. We utilized the International Registry on Acute Aortic Dissections (IRAD) database to study these patients. Methods: We analyzed 43 painless TBAAD patients enrolled in the database between January 1996 and July 2012. The differences in presentation, diagnostics, management, and outcome were compared with patients presenting with painful TBAAD. Results: Among the 1162 TBAAD patients enrolled in IRAD, 43 patients presented with painless TBAAD (3.7%). The mean age of patients with painless TBAAD was significantly higher than normal TBAAD patients (69.2 versus 63.3 years, P = 0.020). The presence of atherosclerosis (46.4% versus 30.1%, P = 0.022), diabetes (17.9% versus 7.5%; P = 0.018), and other aortic diseases (8.6% versus 2.3%, P= 0.051), such as prior aortic aneurysm (31% versus 18.8% P = 0.049) was more common in these patients. Median delay time between presentation and diagnosis was longer in painless patients (median 34.0 versus 19.0 hours; P = 0.006). Dissection of iatrogenic origin (19.5% versus 1.3%; P < 0.001) was significantly more frequent in the painless group. The in-hospital mortality was 18.6% in the painless group, compared with an in-hospital mortality of 9.9% in the control group (P = 0.063). Conclusion: Painless TBAAD is a relatively rare presentation (3.7%) of aortic dissection, and is often associated with a history of atherosclerosis, diabetes, prior aortic disease including aortic aneurysm, and an iatrogenic origin. We observed a trend for increased in-hospital mortality in painless TBAAD patients, which may be the result of a delay in diagnosis and management. Therefore, physicians should be aware of this relative rare presentation of TBAAD.

  15. Quantitative analysis

    PubMed Central

    Nevin, John A.

    1984-01-01

    Quantitative analysis permits the isolation of invariant relations in the study of behavior. The parameters of these relations can serve as higher-order dependent variables in more extensive analyses. These points are illustrated by reference to quantitative descriptions of performance maintained by concurrent schedules, multiple schedules, and signal-detection procedures. Such quantitative descriptions of empirical data may be derived from mathematical theories, which in turn can lead to novel empirical analyses so long as their terms refer to behavioral and environmental events. Thus, quantitative analysis is an integral aspect of the experimental analysis of behavior. PMID:16812400

  16. Hydro-dissection - A simple Solution in Difficult Laparoscopic Cholecystectomy.

    PubMed

    Lubna, H; Masoom, M R

    2015-07-01

    This Quasi-experimental study was done to assess the effectiveness of hydro-dissection in difficult laparoscopic cholecystectomies in Hamdard University Hospital, Karachi, Pakistan, from April 2012 to March 2014. All consecutive patients who presented with cholelithiasis and planned for laparoscopic cholecystectomy were enrolled in this study. Per-operatively the degree of difficulty of the operation was assessed by Cuschieri's scale after grading; Grade II, III and IV cholecystectomies were included in this study. Hydro dissection with saline jet through 5mm simple irrigation and suction probe was used, Operative findings and the total number of patients, in whom anatomy of calot's triangle was clearly displayed with hydro-dissection, was recorded. A total 55 patients were included in the study after assessing the degree of difficulty per operatively by Cuschieri Scale. Thirty one (31) patients were in Group II, 22 in Group III and 02 were included in group IV of Cuschieri scale in which hydro-dissection was used. This method cleared the obscure anatomy in all patients in Group II but in 3 patients of Group III, dense adhesions required sharp dissection to clear the operative field. Two patients, in whom conversion was required, were grouped in Cuschieri's scale IV. Methods of dissection in difficult cholecystectomies are of paramount importance to avoid iatrogenic injuries. Hydro-dissection using suction irrigation probe is a safe and effective technique to clear the difficult anatomy. PMID:26329960

  17. Endovascular management of chronic post-dissection aneurysms

    PubMed Central

    Oikonomou, Kyriakos; Katsargyris, Athanasios; Ritter, Wolfgang; Spinelli, Domenico; Seto, Yuki

    2014-01-01

    Open repair is still the gold standard in acute type A dissection. Endovascular repair is advocated for complicated acute type B dissections. Recent evidence also supports the role of endovascular repair in a larger proportion of uncomplicated acute type B dissections. The role of endovascular repair in chronic post-dissection aneurysms, however, is still unclear. Most commonly, post-dissection aneurysms involve the thoracoabdominal aorta, making the use of fenestrated/branched stent-grafts to achieve complete aneurysm exclusion mandatory. These fenestrated/branched stent-grafts have been used with success in atherosclerotic thoracoabdominal aortic aneurysms (TAAAs). In chronic post-dissection aneurysms, however, additional technical challenges arise. The usually narrow true lumen makes the use of branches more tedious and overall planning difficult. A second technical challenge relates to the fact that visceral branches can also originate from the false lumen. In such cases, perforation of the stiff chronic dissection flap is required to obtain access to the vessel. During the period January 2010 to November 2013, 17 patients (13 males, mean age 65±7.8 years) with chronic thoracoabdominal aneurismal degeneration following acute dissection were treated in our department with the use of fenestrated/branched stent-grafts. Technical success was achieved in all cases (100%). Perioperative mortality was two (11.8%) patients. One patient died due to multiple organ failure and one due to cardiac failure. No case of paraplegia was observed. During a 12-month median follow-up (range, 4-28 months) no aneurysm-related deaths were observed. Reintervention was required in three cases to repair a type Ib endoleak from a side branch. Endovascular treatment with fenestrated/branched stent-grafts is feasible for chronic post-dissection aneurysms. Standard thoracic stent-grafting is an option in a minority of patients, when the aneurysm is limited to the thoracic segment. Fenestrated and branched devices can successfully be used for aneurysms extending to the thoracoabdominal aorta. PMID:24967171

  18. Thoraco-abdominal Aorta Dissection: Look Again Before You Leap

    PubMed Central

    Zeina, Abdel-Rauf; Trachtengerts, Victoria; Abadi, Sobhi; Jarchowsky, Jacob; Soimu, Uri; Nachtigal, Alicia

    2009-01-01

    Aortic dissection is a life-threatening condition that might require immediate assessment and therapy. We present the case of a 71-year-old woman with essential hypertension complaining about low back pain; unenhanced thoracic-lumbar spine computed tomography examination (CT) revealed a hyperdense thin line across the aorta with an appearance of "double aortic lumen". Enhanced CT scan confirmed the diagnosis of type B aortic dissection. Radiologists should be familiar with this finding that could be considered a new radiological sign of aortic dissection on unenhanced CT examination. PMID:22470686

  19. Chronic type B aortic dissection: indications and strategies for treatment.

    PubMed

    Rohlffs, F; Tsilimparis, N; Diener, H; Larena-Avellaneda, A; Von Kodolitsch, Y; Wipper, S; Debus, E S; Kölbel, T

    2015-04-01

    Chronic type B aortic dissection is a distinctive condition that needs individual treatment strategies and different considerations than in therapy of acute or subacute type B aortic dissection. The most common indication for treatment of this complex disease is aneurysmal dilatation of the dissected aortic segment. While open repair of the enlarged dissected aorta remains the best option for good-risk patients and patients with connective tissue disorders in high-volume centers with respective expertise, endovascular management of chronic type B aortic dissection with postdissection aneurysms has significantly gained ground in the past years. But the concept of TEVAR with implantation of a tubular stent-graft into the thoracic aorta to seal the proximal entry tear and reroute the blood flow into the true lumen alone, is not associated with satisfactory results. This is mainly due to the sparse remodeling capacity of the aortic tissue compared to earlier stages of the disease as the aortic wall and the dissection membrane are thickened and more rigid. On the other hand, it is restricted by the most limiting factor for endovascular success in chronic type B aortic dissection: persistent false lumen perfusion. This problem also affects patients with residual dissection after surgical repair of a DeBakey type I aortic dissection or dissection after ascending aortic repair for other pathologies. Hence, it is evident that strategies to achieve endovascular false lumen occlusion are of increasing importance and novel techniques have been introduced to solve the problem of persisting false lumen flow. Thus, the evolution of a large variety of techniques to address the false lumen perfusion issue indicates that complicated chronic type B dissection involves a high diversity in clinical presentation and morphology. A large armamentarium of catheter skills as well as critical individualized treatment strategies are required to address the heterogenous morphological disease pattern for each individual patient. The rapid development in endovascular techniques gives new directions for treatment indications and strategies in chronic aortic dissection and enables new insights into this old disease. PMID:25604323

  20. Dissecting innate immunity by germline mutagenesis.

    PubMed

    Rutschmann, Sophie; Hoebe, Kasper

    2008-04-01

    The innate arm of our immune system is the first line of defence against infections. In addition, it is believed to drive adaptive immune responses, which help fight pathogens and provide long-term memory. As such, the innate immune system is instrumental for protection against pathogens that would otherwise destroy their host. Although our understanding of the innate immune components involved in pathogen sensing and fighting is improving, it is still limited. This is particularly exemplified by increased documentation of innate immune deficiencies in humans that often result in high and recurrent susceptibility to infections or even death, without the genetic cause being evident. To provide further insight into the mechanisms by which pathogen sensing and eradication occur, several strategies can be used. The current review focuses on the forward genetic approaches that have been used to dissect innate immunity in the fruit fly and the mouse. For both animal models, forward genetics has been instrumental in the deciphering of innate immunity and has greatly improved our understanding of how we respond to invading pathogens. PMID:18205789

  1. Dissecting innate immunity by germline mutagenesis

    PubMed Central

    Rutschmann, Sophie; Hoebe, Kasper

    2008-01-01

    The innate arm of our immune system is the first line of defence against infections. In addition, it is believed to drive adaptive immune responses, which help fight pathogens and provide long-term memory. As such, the innate immune system is instrumental for protection against pathogens that would otherwise destroy their host. Although our understanding of the innate immune components involved in pathogen sensing and fighting is improving, it is still limited. This is particularly exemplified by increased documentation of innate immune deficiencies in humans that often result in high and recurrent susceptibility to infections or even death, without the genetic cause being evident. To provide further insight into the mechanisms by which pathogen sensing and eradication occur, several strategies can be used. The current review focuses on the forward genetic approaches that have been used to dissect innate immunity in the fruit fly and the mouse. For both animal models, forward genetics has been instrumental in the deciphering of innate immunity and has greatly improved our understanding of how we respond to invading pathogens. PMID:18205789

  2. [Esophagectomy after endoscopic submucosal dissection (ESD)].

    PubMed

    Nako, Yoshito; Shiozaki, Atushi; Fujiwara, Hitoshi; Konishi, Hirotaka; Kosuga, Toshiyuki; Morimura, Ryo; Murayama, Yasutoshi; Komatsu, Shuhei; Ikoma, Hisashi; Kuriu, Yoshiaki; Nakanishi, Masayoshi; Ichikawa, Daisuke; Okamoto, Kazuma; Sakakura, Chouhei; Otsuji, Eigo

    2014-11-01

    Herein, we report 9 patients who underwent esophagectomy after endoscopic submucosal dissection (ESD) between April 2003 and December 2013. All patients were men, with a mean age of 65 years. En bloc ESD was performed, and no complications arose in any patient. The mean surgical time of esophagectomy was 323 minutes, and mean blood loss was 295 mL. Postoperative complications were present in 5 patients(anastomotic leakage in 3, pulmonary complications in 2, and recurrent laryngeal nerve palsy in 1). In a patient diagnosed with pT1b-SM1 disease after ESD, a residual tumor(pT1a-MM, N0) was detected after esophagectomy. In another patient diagnosed with pT1b-SM2 disease, lymph node metastasis was detected after esophagectomy. In all patients, curative resection was performed, and no recurrences have been observed to date. This highlights the importance of additional esophagectomy after ESD for patients with pT1b disease. Esophagectomy after ESD can be considered a valid treatment because it provides high curative rates with acceptable safety. PMID:25731401

  3. Optogenetic dissection of medial prefrontal cortex circuitry

    PubMed Central

    Riga, Danai; Matos, Mariana R.; Glas, Annet; Smit, August B.; Spijker, Sabine; Van den Oever, Michel C.

    2014-01-01

    The medial prefrontal cortex (mPFC) is critically involved in numerous cognitive functions, including attention, inhibitory control, habit formation, working memory and long-term memory. Moreover, through its dense interconnectivity with subcortical regions (e.g., thalamus, striatum, amygdala and hippocampus), the mPFC is thought to exert top-down executive control over the processing of aversive and appetitive stimuli. Because the mPFC has been implicated in the processing of a wide range of cognitive and emotional stimuli, it is thought to function as a central hub in the brain circuitry mediating symptoms of psychiatric disorders. New optogenetics technology enables anatomical and functional dissection of mPFC circuitry with unprecedented spatial and temporal resolution. This provides important novel insights in the contribution of specific neuronal subpopulations and their connectivity to mPFC function in health and disease states. In this review, we present the current knowledge obtained with optogenetic methods concerning mPFC function and dysfunction and integrate this with findings from traditional intervention approaches used to investigate the mPFC circuitry in animal models of cognitive processing and psychiatric disorders. PMID:25538574

  4. Spontaneous coronary artery dissection presenting as acute myocardial infarction.

    PubMed Central

    Mahenthiran, J.; Revankar, R.; Koka, V.; Hoo, J.; Shenoy, M.

    2000-01-01

    Spontaneous coronary artery dissection is a rare entity being increasingly diagnosed as a cause of acute myocardial infarction, especially in cases of low cardiac risk female patients. This is one such case report of a black female patient, who suffered an acute anterior wall myocardial infarction due to an idiopathic spontaneous coronary artery dissection of the left anterior descending artery. She was treated with a thrombolytic agent in the acute phase, uneventfully. An urgent coronary angiogram demonstrated an intimal tear with a dissection of the left anterior descending artery. She survived the acute event and her subsequent hospital course was uncomplicated. Hence she was treated medically for her ischemic event and left ventricular systolic dysfunction with a favorable outcome. This case is yet another report of a survivor treated with a thrombolytic agent for the acute myocardial infarction due to spontaneous coronary artery dissection. Images Figure 2 Figure 3 PMID:10800297

  5. Transventricular aortic cannulation for repair of aortic dissection.

    PubMed

    Flege, J B; Aberg, T

    2001-09-01

    We have used transventricular aortic cannulation as arterial inflow from the heart-lung machine in seven consecutive operations done in 1 year for acute aortic dissection. Satisfactory cardiopulmonary bypass was achieved in all patients. PMID:11565703

  6. Non-invasive diagnosis of internal carotid artery dissections.

    PubMed Central

    Mllges, W; Ringelstein, E B; Leibold, M

    1992-01-01

    Arteriography is thought to be mandatory for the diagnosis of internal carotid artery (ICA) dissection. With the introduction of transcranial Doppler sonography (TCD) and magnetic resonance imaging (MRI), however, this is no longer the case. In 13 consecutive patients with ICA dissections the diagnosis was made by means of non-invasive tests including extracranial and transcranial Doppler sonography, contrast enhanced computed tomography (ceCT), and, in five patients, MRI. Intra-arterial digital subtraction angiography used as the gold standard in all cases was confirmative. Extracranial and transcranial ultrasound findings indicative of the diagnosis could be identified. MRI directly demonstrated the intramural haematoma and the false lumen of the dissected artery. These non-invasive techniques also allowed for repetitive follow up examinations. They were, however, unable to demonstrate false aneurysms in the chronic state. Results show that the diagnosis of carotid dissection can be made by means of cerebrovascular ultrasound and MRI. Images PMID:1538235

  7. Complicated Postpartum Type B Aortic Dissection and Endovascular Repair

    PubMed Central

    Rosenberger, Laura H.; Adams, Joshua D.; Kern, John A.; Tracci, Margaret C.; Angle, J. Fritz; Cherry, Kenneth J.

    2012-01-01

    BACKGROUND Fifty percent of aortic dissections in women younger than 40 years occur in association with pregnancy. Of these, half of type B dissections occur in the postpartum period. CASE A 30-year-old woman was status post spontaneous vaginal delivery at 30 weeks of gestation for fetal death, complicated by an eclamptic seizure. On post-partum day 4, she suffered an acute, complicated type B aortic dissection treated with endovascular stent graft placement. CONCLUSION Endovascular repair may be an attractive option for the treatment of complicated type B aortic dissections in pregnancy and the peripartum period, with reduced maternal and fetal mortality. This may allow the fetus to remain in situ and avoid the risks of surgery and possible cardiopulmonary bypass, with little radiation risk to the fetus. PMID:22270446

  8. Blindness after bilateral neck dissection: case report and review.

    PubMed

    Pazos, G A; Leonard, D W; Blice, J; Thompson, D H

    1999-01-01

    The primary objective of this review of the literature is to identify the probable causes of blindness after bilateral radical neck dissections. This case report and literature review also discusses possible preventive measures that may avert this catastrophic outcome. Cases of blindness after bilateral radical neck dissection were identified by an electronic literature search, as well as cross-checking all references of the above-identified papers. Eleven previous cases of blindness after bilateral neck dissection were identified. The most common cause was posterior ischemic optic neuropathy (PION), which was permanent. We present the only case in the literature in which blindness occurred after radical neck dissections separated by a span of 9 years. The cause of blindness in our patient was posterior ischemic optic neuropathy. Contributing factors included anemia, hypotension, and disruption of collateral venous return from the neck. PMID:10512147

  9. SMAD2 Mutations Are Associated with Arterial Aneurysms and Dissections.

    PubMed

    Micha, Dimitra; Guo, Dong-Chuan; Hilhorst-Hofstee, Yvonne; van Kooten, Fop; Atmaja, Dian; Overwater, Eline; Cayami, Ferdy K; Regalado, Ellen S; van Uffelen, Ren; Venselaar, Hanka; Faradz, Sultana M H; Vriend, Gerrit; Weiss, Marjan M; Sistermans, Erik A; Maugeri, Alessandra; Milewicz, Dianna M; Pals, Gerard; van Dijk, Fleur S

    2015-12-01

    We report three families with arterial aneurysms and dissections in which variants predicted to be pathogenic were identified in SMAD2. Moreover, one variant occurred de novo in a proband with unaffected parents. SMAD2 is a strong candidate gene for arterial aneurysms and dissections given its role in the TGF-? signaling pathway. Furthermore, although SMAD2 and SMAD3 probably have functionally distinct roles in cell signaling, they are structurally very similar. Our findings indicate that SMAD2 mutations are associated with arterial aneurysms and dissections and are in accordance with the observation that patients with pathogenic variants in genes encoding proteins involved in the TGF-? signaling pathway exhibit arterial aneurysms and dissections as key features. PMID:26247899

  10. The risk for type B aortic dissection in Marfan syndrome.

    PubMed

    Setacci, C; Galzerano, G; Setacci, F; Mazzitelli, G; de Donato, G; Ricci, C

    2015-12-01

    Marfan syndrome is the most prevalent connective tissue disorder, with an autosomal dominant inheritance with variable penetrance. This paper aims to summarize epidemiology and treatment for type B dissection in Marfan patients. PMID:26350976

  11. [Spontaneous dissection and stenosis of the vertebral artery].

    PubMed

    Frauchiger, B; Bernays, D R

    1991-08-31

    The case of a 21-year-old female patient with neck pain and brain stem symptomatology after appendectomy is reported. Duplex sonographic findings were compatible with right vertebral artery dissection and occlusion of the vessel. Angiography confirmed the diagnosis. The patient recovered within weeks and was free of symptoms 6 months after the acute episode. Dissection of the vertebral artery can occur with or without minor trauma. Associations with fibromuscular dysplasia, arterial hypertension and the use of oral contraceptives have been reported. As in our own patient, dissection can appear without any vessel pathology or risk situation. The most dangerous complication of the disease is rupture of the adventitia with subarachnoid hemorrhage. Without this complication, prognosis of vertebral artery dissection is favourable with complete recovery within weeks. Clinical findings, diagnostic procedure and therapy are discussed. PMID:1925454

  12. Dissecting intramyocardial hematoma after robotic mitral valve repair.

    PubMed

    McGrath, Tory; Ushukumari, Deepu; Canale, Leonardo; Gillinov, Marc

    2015-03-01

    We report the first case of a dissecting intramyocardial hematoma discovered intraoperatively after robotic mitral valve repair, potential etiologies relevant to robotic surgery, and its successful management. PMID:25742825

  13. [Thoracic aortic dissection revealed by systemic cholesterol embolism].

    PubMed

    Braem, L; Paule, P; Héno, P; Morand, J J; Mafart, B; La Folie, T; Varlet, P; Mioulet, D; Fourcade, L

    2006-10-01

    Systemic cholesterol embolism is a rare complication of atherosclerosis, and has various presentations. Arterial catheterisms are a common cause. However, the association with an aortic dissection has been exceptionally reported. We report the observation of a 70 year-old man, with coronary artery disease, hypertension, diabetes and dyslipidemia. Six months before hospitalization, a coronary angioplasty was performed due to recurrent angina. The association of purpuric lesions on the feet, with acute renal failure confirmed cholesterol embolism syndrome. Transoesophageal echocardiography showed a dissection of the descending thoracic aorta associated with complex atheroma. The evolution was marked by the pulpar necrosis of a toe and by a worsening of the renal failure, requiring definitive hemodialysis. Further echographic control highlighted the rupture of the intimal veil of the dissection. Cholesterol embolism syndrome may reveal an aortic dissection in patients without thoracic symptoms. In such cases, transoesophageal echocardiography is a useful and non-invasive examination. PMID:17078270

  14. Aortic dissection and bicuspid aortic valve: an autopsy study.

    PubMed

    Sawaimoon, Satyakam Krishna; Jadhav, Meenal Vitthal; Rane, Sharda Raju; Sagale, Mangesh; Khedkar, Bhushan

    2006-07-01

    Medico-legal post-mortems referred to the Department of Pathology, for the histopathological examination, revealed six cases of acute aortic dissection--two in isolation, three in combination with congenital bicuspid aortic valve; and one isolated case of congenital bicuspid aortic valve. One case of isolated aortic dissection was associated with Marfan's syndrome; and one case of aortic dissection with bicuspid aortic valve was associated with polycystic kidneys. History of hypertension could be elicited in two cases. Cystic medial degeneration of aorta was seen in three cases; one of which was associated with Marfan's syndrome. All five cases of aortic dissection belonged to type II of DeBakey classification. PMID:17001877

  15. Isolated Spontaneous Renal Artery Dissection Presented with Flank Pain

    PubMed Central

    Gandhi, Shruti P.; Patel, Kajal; Pal, Bipin C.

    2015-01-01

    Spontaneous renal artery dissection is a rare but important cause of flank pain. We report a case of isolated spontaneous renal artery dissection in 56-year-old man complicated by renal infarction presented with flank pain. Doppler study pointed towards vascular pathology. Computed tomography (CT) angiography was used to make final diagnosis which demonstrated intimal flap in main renal artery with renal infarction. PMID:26090259

  16. [Diagnosis of thoracic aorta dissection using transesophageal echocardiography].

    PubMed

    Hernndez Herrera, C; Morn-Malek, A; Romero-Crdenas, A; Rijlaarsdam, M; Vargas-Barrn, J

    1994-01-01

    During a 36 month period there were 20 patients in our hospital with aortic dissection suspected clinically. All of them were examined with transesophageal echocardiography (TEE); 17 were examined with transthoracic echocardiography (TTE); six with computed tomography (CT) and seven with aortography. Twelve patients required surgery: eight with proximal aortic dissection (Type-A), two with distal dissection (Type-B) and two with aortic aneurysm without dissection. With the goal of investigating the utility of TEE for the diagnosis of aortic dissection in our hospital, we compared this and other available methods to the surgery findings. The sensitivity to TEE was 100% and the specificity 92%, with test accuracy at 92%. The sensitivity of the other tests was low: 66% with TTE; 50% with TAC; 57%, with aortography. The specificity was 90% with TTE, and higher with CT and aortography (100%). The ultrasound tests reveal additional information about complications like aortic regurgitation. Transesophageal echocardiography is the best test to examine patients with aortic dissection in our hospital. Computed tomography, aortography and magnetic resonance imaging have indication only to answer specific doubts. PMID:8074589

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

    PubMed Central

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

    2015-01-01

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

  18. Ultrasonic Dissection versus Conventional Dissection for Pancreatic Surgery: A Meta-Analysis

    PubMed Central

    2016-01-01

    Background. The role of ultrasonic dissection (UD) in pancreatic surgery remains controversial. The aim of this meta-analysis was to evaluate the clinical effect of UD in pancreatic surgery when compared with conventional dissection (CD). Materials and Methods. A comprehensive literature search was performed to identify eligible studies that compared UD with CD for pancreatic surgery in PubMed, EMBASE, Web of Science, and the Cochrane Library. Risk ratio (RR) or mean difference with 95% confidence interval (CI) was calculated. Results. Six studies were included with a total of 215 patients undergoing UD and 210 undergoing CD. In comparison with CD in distal pancreatectomy, UD was associated with lower rates of pancreatic fistula (RR = 0.46, 95% CI: 0.270.76) and abdominal abscess and shorter operation time and hospital stay (P < 0.05). In pancreaticoduodenectomy, there was no significant difference in pancreatic fistula rate between two groups (RR = 0.79, 95% CI: 0.481.29). However, the significantly less intraoperative blood loss and the transfused blood unit were found in patients receiving UD (P < 0.05). Conclusions. The results of this meta-analysis show that, in comparison with CD, UD is associated with better perioperative outcomes in pancreatic surgery. PMID:26880891

  19. Implantation of esophageal cancer onto post-dissection ulcer after gastric endoscopic submucosal dissection.

    PubMed

    Asai, Satoshi; Takeshita, Koutarou; Kano, Yuki; Nakao, Eisuke; Ichinona, Takumi; Fujimoto, Naoki; Akamine, Eisuke; Mori, Takuji; Ogawa, Atsuhiro

    2016-03-01

    A case in which implantation of esophageal squamous cell carcinoma onto a post-dissection gastric ulcer was strongly suspected is presented. A 72-year-old man with alcoholic liver cirrhosis underwent esophagogastroduodenoscopy (EGD). Esophageal cancer (EC) (Mt, 20 mm, 0-Is) and gastric cancer (GC) (antrum, 15 mm, 0-IIc) were identified. Biopsy specimens revealed moderately differentiated squamous cell carcinoma (SCC) and differentiated adenocarcinoma, respectively. The GC was resected by endoscopic submucosal dissection (ESD) [14 mm × 9 mm, type 0-IIc, tub1, pT1a(M), ly0, v0, HM(-), VM(-)]. Two months after ESD, radiation therapy was started for the EC, and an almost complete response was obtained. Nine months after the ESD, a follow-up EGD showed a submucosal tumor-like lesion with ulceration, located immediately under the post-ESD scar, and biopsy specimens showed moderately differentiated SCC. There were no similar lesions suggesting hematogenous or lymphatic metastasis in the stomach. PMID:26973424

  20. Implantation of esophageal cancer onto post-dissection ulcer after gastric endoscopic submucosal dissection

    PubMed Central

    Asai, Satoshi; Takeshita, Koutarou; Kano, Yuki; Nakao, Eisuke; Ichinona, Takumi; Fujimoto, Naoki; Akamine, Eisuke; Mori, Takuji; Ogawa, Atsuhiro

    2016-01-01

    A case in which implantation of esophageal squamous cell carcinoma onto a post-dissection gastric ulcer was strongly suspected is presented. A 72-year-old man with alcoholic liver cirrhosis underwent esophagogastroduodenoscopy (EGD). Esophageal cancer (EC) (Mt, 20 mm, 0-Is) and gastric cancer (GC) (antrum, 15 mm, 0-IIc) were identified. Biopsy specimens revealed moderately differentiated squamous cell carcinoma (SCC) and differentiated adenocarcinoma, respectively. The GC was resected by endoscopic submucosal dissection (ESD) [14 mm × 9 mm, type 0-IIc, tub1, pT1a(M), ly0, v0, HM(-), VM(-)]. Two months after ESD, radiation therapy was started for the EC, and an almost complete response was obtained. Nine months after the ESD, a follow-up EGD showed a submucosal tumor-like lesion with ulceration, located immediately under the post-ESD scar, and biopsy specimens showed moderately differentiated SCC. There were no similar lesions suggesting hematogenous or lymphatic metastasis in the stomach. PMID:26973424

  1. Ultrasonic Dissection versus Conventional Dissection for Pancreatic Surgery: A Meta-Analysis.

    PubMed

    Lei, Haiming; Xu, Dong; Shi, Xinghua; Han, Koulan

    2016-01-01

    Background. The role of ultrasonic dissection (UD) in pancreatic surgery remains controversial. The aim of this meta-analysis was to evaluate the clinical effect of UD in pancreatic surgery when compared with conventional dissection (CD). Materials and Methods. A comprehensive literature search was performed to identify eligible studies that compared UD with CD for pancreatic surgery in PubMed, EMBASE, Web of Science, and the Cochrane Library. Risk ratio (RR) or mean difference with 95% confidence interval (CI) was calculated. Results. Six studies were included with a total of 215 patients undergoing UD and 210 undergoing CD. In comparison with CD in distal pancreatectomy, UD was associated with lower rates of pancreatic fistula (RR = 0.46, 95% CI: 0.27-0.76) and abdominal abscess and shorter operation time and hospital stay (P < 0.05). In pancreaticoduodenectomy, there was no significant difference in pancreatic fistula rate between two groups (RR = 0.79, 95% CI: 0.48-1.29). However, the significantly less intraoperative blood loss and the transfused blood unit were found in patients receiving UD (P < 0.05). Conclusions. The results of this meta-analysis show that, in comparison with CD, UD is associated with better perioperative outcomes in pancreatic surgery. PMID:26880891

  2. Integrated analysis of global proteome, phosphoproteome, and glycoproteome enables complementary interpretation of disease-related protein networks.

    PubMed

    Park, Jong-Moon; Park, Ji-Hwan; Mun, Dong-Gi; Bae, Jingi; Jung, Jae Hun; Back, Seunghoon; Lee, Hangyeore; Kim, Hokeun; Jung, Hee-Jung; Kim, Hark Kyun; Lee, Hookeun; Kim, Kwang Pyo; Hwang, Daehee; Lee, Sang-Won

    2015-01-01

    Multi-dimensional proteomic analyses provide different layers of protein information, including protein abundance and post-translational modifications. Here, we report an integrated analysis of protein expression, phosphorylation, and N-glycosylation by serial enrichments of phosphorylation and N-glycosylation (SEPG) from the same tissue samples. On average, the SEPG identified 142,106 unmodified peptides of 8,625 protein groups, 18,846 phosphopeptides (15,647 phosphosites), and 4,019 N-glycopeptides (2,634 N-glycosites) in tumor and adjacent normal tissues from three gastric cancer patients. The combined analysis of these data showed that the integrated analysis additively improved the coverages of gastric cancer-related protein networks; phosphoproteome and N-glycoproteome captured predominantly low abundant signal proteins, and membranous or secreted proteins, respectively, while global proteome provided abundances for general population of the proteome. Therefore, our results demonstrate that the SEPG can serve as an effective approach for multi-dimensional proteome analyses, and the holistic profiles of protein expression and PTMs enabled improved interpretation of disease-related networks by providing complementary information. PMID:26657352

  3. Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

    PubMed

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima; Fisher-Wellman, Kelsey H; Kleinert, Maximilian; Humphrey, Sean J; Yang, Pengyi; Holliday, Mira; Trefely, Sophie; Fazakerley, Daniel J; Stckli, Jacqueline; Burchfield, James G; Jensen, Thomas E; Jothi, Raja; Kiens, Bente; Wojtaszewski, Jrgen F P; Richter, Erik A; James, David E

    2015-11-01

    Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted invitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry. PMID:26437602

  4. The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

    PubMed

    Tampella, Giacomo; Kerns, Hannah M; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A; Garrett, Meghan E; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A; Rawlings, David J; James, Richard G

    2015-07-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K. PMID:26026062

  5. 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

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

    PubMed

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

    2016-02-01

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

  7. Combined Integrin Phosphoproteomic Analyses and siRNA-based Functional Screening Identified Key Regulators for Cancer Cell Adhesion and Migration

    PubMed Central

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

    2009-01-01

    Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using SILAC-MS, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to siRNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2 and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration. PMID:19351860

  8. Phosphoproteomic analysis of basal and therapy-induced adaptive signaling networks in BRAF and NRAS mutant melanoma

    PubMed Central

    Fedorenko, Inna V.; Fang, Bin; Munko, A. Cecelia; Gibney, Geoffrey T.; Koomen, John M.; Smalley, Keiran S.M.

    2015-01-01

    Basal and kinase inhibitor-driven adaptive signaling has been examined in a panel of melanoma cell lines using phosphoproteomics in conjunction with pathway analysis. A considerable divergence in the spectrum of tyrosine-phosphorylated peptides was noted at the cell line level. The unification of genotype-specific cell line data revealed the enrichment for the tyrosine-phosphorylated cytoskeletal proteins to be associated with the presence of a BRAF mutation and oncogenic NRAS to be associated with increased receptor tyrosine kinase phosphorylation. A number of proteins including cell cycle regulators (CDK1, CDK2 and CDK3), MAPK pathway components (ERK1 and ERK2), interferon regulators (TYK2), GTPase regulators (RIN1) and controllers of protein tyrosine phosphorylation (DYR1A and PTPRA) were common to all genotypes. Treatment of a BRAF-mutant/PTEN-null melanoma cell line with vemurafenib led to decreased phosphorylation of ERK, phospholipase C1 and β-catenin with increases in RTK phosphorylation, STAT3 and GSK3α noted. In NRAS-mutant melanoma, MEK inhibition led to increased phosphorylation of EGFR signaling pathway components, Src family kinases and PKCδ with decreased phosphorylation seen in STAT3 and ERK1/2. Together these data present the first systems level view of adaptive and basal phosphotyrosine signaling in BRAF- and NRAS-mutant melanoma. PMID:25339196

  9. 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

  10. 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. PMID:26633130

  11. Differential Phosphoproteome Regulation of Nucleus Accumbens in Environmentally Enriched and Isolated Rats in Response to Acute Stress

    PubMed Central

    Fan, Xiuzhen; Li, Dingge; Zhang, Yafang; Green, Thomas A.

    2013-01-01

    Increasing evidence shows that stress contributes to the pathogenesis of major depressive disorder which is a severe neuropsychiatric disorder and influences over 10% of the world's population. Our previous studies revealed that rats reared in an enriched environment display less depression-related behavior compared to rats raised in an isolated environment, which implies that environmental enrichment produces an antidepressant-like behavioral phenotype. However, the molecular mechanisms are not fully understood. Protein phosphorylation rapidly changes signaling pathway function and alters the function of proteins associated with the stress-induced depressive disorder. Thus, in this study, a phosphoproteomic approach was used to uncover differential phosphoprotein regulation in rat nucleus accumbens between isolated (IC) and enriched environmental conditions (EC) under basal conditions, and in response to acute stress. We found 23 phosphoproteins were regulated in EC vs. IC rats under basal conditions; 10 phosphoproteins regulated by stress in IC rats; and 15 regulated by stress in EC rats. Among all significantly regulated phosphoproteins, 11 of them were represented in at least two conditions. The regulated phosphoproteins represent signaling pathway proteins (including ERK2), enzymes, transcriptional regulators, protein translation regulators, transporters, chaperones and cytoskeletal proteins. These findings provide a global view for further understanding the contribution of protein phosphorylation in depression pathogenesis and antidepressant action. PMID:24278208

  12. Integrated analysis of global proteome, phosphoproteome, and glycoproteome enables complementary interpretation of disease-related protein networks

    PubMed Central

    Park, Jong-Moon; Park, Ji-Hwan; Mun, Dong-Gi; Bae, Jingi; Jung, Jae Hun; Back, Seunghoon; Lee, Hangyeore; Kim, Hokeun; Jung, Hee-Jung; Kim, Hark Kyun; Lee, Hookeun; Kim, Kwang Pyo; Hwang, Daehee; Lee, Sang-Won

    2015-01-01

    Multi-dimensional proteomic analyses provide different layers of protein information, including protein abundance and post-translational modifications. Here, we report an integrated analysis of protein expression, phosphorylation, and N-glycosylation by serial enrichments of phosphorylation and N-glycosylation (SEPG) from the same tissue samples. On average, the SEPG identified 142,106 unmodified peptides of 8,625 protein groups, 18,846 phosphopeptides (15,647 phosphosites), and 4,019 N-glycopeptides (2,634 N-glycosites) in tumor and adjacent normal tissues from three gastric cancer patients. The combined analysis of these data showed that the integrated analysis additively improved the coverages of gastric cancer-related protein networks; phosphoproteome and N-glycoproteome captured predominantly low abundant signal proteins, and membranous or secreted proteins, respectively, while global proteome provided abundances for general population of the proteome. Therefore, our results demonstrate that the SEPG can serve as an effective approach for multi-dimensional proteome analyses, and the holistic profiles of protein expression and PTMs enabled improved interpretation of disease-related networks by providing complementary information. PMID:26657352

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

    PubMed Central

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

    2011-01-01

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

  14. Iatrogenic Acute Aortic Dissection during Percutaneous Coronary Intervention for Acute Myocardial Infarction

    PubMed Central

    Noguchi, Kenichiro; Hori, Daijiro; Nomura, Yohei; Tanaka, Hiroyuki

    2012-01-01

    Iatrogenic acute aortic dissection during percutaneous coronary intervention is an extremely rare, but critical complication. Localized aortic dissections have been treated by sealing the entry with a coronary stent. Extensive dissections may require a surgical intervention. We present a case of type A extensive aortic dissection occurring during angioplasty of the left circumflex artery for acute myocardial infarction. This iatrogenic aortic dissection required emergent surgical repair with supracoronary replacement of the ascending aorta. PMID:23555491

  15. On the necessity of dissecting sequence similarity scores into segment-specific contributions for inferring protein homology, function prediction and annotation

    PubMed Central

    2014-01-01

    Background Protein sequence similarities to any types of non-globular segments (coiled coils, low complexity regions, transmembrane regions, long loops, etc. where either positional sequence conservation is the result of a very simple, physically induced pattern or rather integral sequence properties are critical) are pertinent sources for mistaken homologies. Regretfully, these considerations regularly escape attention in large-scale annotation studies since, often, there is no substitute to manual handling of these cases. Quantitative criteria are required to suppress events of function annotation transfer as a result of false homology assignments. Results The sequence homology concept is based on the similarity comparison between the structural elements, the basic building blocks for conferring the overall fold of a protein. We propose to dissect the total similarity score into fold-critical and other, remaining contributions and suggest that, for a valid homology statement, the fold-relevant score contribution should at least be significant on its own. As part of the article, we provide the DissectHMMER software program for dissecting HMMER2/3 scores into segment-specific contributions. We show that DissectHMMER reproduces HMMER2/3 scores with sufficient accuracy and that it is useful in automated decisions about homology for instructive sequence examples. To generalize the dissection concept for cases without 3D structural information, we find that a dissection based on alignment quality is an appropriate surrogate. The approach was applied to a large-scale study of SMART and PFAM domains in the space of seed sequences and in the space of UniProt/SwissProt. Conclusions Sequence similarity core dissection with regard to fold-critical and other contributions systematically suppresses false hits and, additionally, recovers previously obscured homology relationships such as the one between aquaporins and formate/nitrite transporters that, so far, was only supported by structure comparison. PMID:24890864

  16. Case Misclassification in Studies of Spinal Manipulation and Arterial Dissection

    PubMed Central

    Cai, Xuemei; Razmara, Ali; Paulus, Jessica K; Switkowski, Karen; Fariborz, Pari J; Goryachev, Sergey D; DAvolio, Leonard; Feldmann, Edward; Thaler, David E

    2014-01-01

    Goals Spinal manipulation has been associated with cervical arterial dissection and stroke but a causal relationship has been questioned by population-based studies. Earlier studies identified cases using International Classification of Diseases-9 codes specific to anatomical stroke location rather than stroke etiology. We hypothesize that case misclassification occurred in these previous studies and an underestimation of the strength of the association. We also predicted that case misclassification would differ by patient age. Materials and methods We identified cases in the Veterans Health Administration database using the same strategy as the prior studies. The electronic medical record was then screened for the word dissection. The presence of atraumatic dissection was determined by medical record review by a neurologist. Findings Of 3690 patients found by International Classification of Diseases-9 codes over a 30-month period, 414 (11.2%) had confirmed cervical artery dissection with a positive predictive value of 10.5% (95% CI 9.611.5%). The positive predictive value was higher in patients <45 versus ?45 years (41% vs. 9%, p<0.001). We reanalyzed a previous study which reported no association between spinal manipulation and cervical artery dissection (OR=1.12, 95% CI 0.771.63), and re-calculated an odds ratio of 2.15 (95% CI 0.984.69). For patients under age 45, the OR was 6.91 (95% CI 2.5913.74). Conclusions Prior studies grossly misclassified cases of cervical dissection and mistakenly dismissed a causal association with manipulation. Our study indicates that the odds ratio for spinal manipulation exposure in cervical artery dissection is higher than previously reported. PMID:25085345

  17. "And afterwards your body to be given for public dissection": a history of the murderers dissected in Glasgow and the west of Scotland.

    PubMed

    Kennedy, S S; McLeod, K J; McDonald, S W

    2001-02-01

    Between 1752 and 1832, the bodies of hanged murderers were dissected or gibbeted. During this period, 38 murderers were executed in the West of Scotland. The bodies of at least 23 were dissected in Glasgow. The stories of these murders are recounted. Insight is also given into the attitudes of the public and the anatomists to dissection of executed murderers. PMID:11310358

  18. A comparison of retention of anatomical knowledge in an introductory college biology course: Traditional dissection vs. virtual dissection

    NASA Astrophysics Data System (ADS)

    Taeger, Kelli Rae

    Dissection has always played a crucial role in biology and anatomy courses at all levels of education. However, in recent years, ethical concerns, as well as improved technology, have brought to the forefront the issue of whether virtual dissection is as effective or whether it is more effective than traditional dissection. Most prior research indicated the two methods produced equal results. However, none of those studies examined retention of information past the initial test of knowledge. Two groups of college students currently enrolled in an introductory level college biology course were given one hour to complete a frog dissection. One group performed a traditional frog dissection, making cuts in an actual preserved frog specimen with scalpels and scissors. The other group performed a virtual frog dissection, using "The Digital Frog 2" software. Immediately after the dissections were completed, each group was given an examination consisting of questions on actual specimens, pictures generated from the computer software, and illustrations that neither group had seen. Two weeks later, unannounced, the groups took the same exam in order to test retention. The traditional dissection group scored significantly higher on two of the three sections, as well as the total score on the initial exam. However, with the exception of specimen questions (on which the traditional group retained significantly more information), there was no significant difference in the retention from exam 1 to exam 2 between the two groups. These results, along with the majority of prior studies, show that the two methods produce, for the most part, the same end results. Therefore, the decision of which method to employ should be based on the goals and preferences of the instructor(s) and the department. If that department's goals include: Being at the forefront of new technology, increasing time management, increasing student: teacher ratio for economic reasons, and/or ethical issues, then the choice should be the use of computer software. If the goals include: Students gaining a 3-dimensional feel for the location and relationship of parts to one another, students being able to see various naturally occurring anomalies, and increased experience with manipulation of dissection tools, then the choice should be dissection of actual specimens. It is important to note, however, that regardless of which method is chosen, the effectiveness of that method is very much dependent on the skill and enthusiasm of the instructor.

  19. Applications of quantitative whole body autoradiographic technique in radiopharmaceutical research

    SciTech Connect

    Som, P.; Oster, Z.H.; Yonekura, Y.; Meyer, M.A.; Fand, I.; Brill, A.B.

    1982-01-01

    The routine evaluation of radiopharmaceuticals involves dissecting tissue distribution studies (DTDS) and gamma or positron imaging. DTDS have the following disadvantages: since not all tissues can always be sampled, sites of radiopharmaceutical uptake may be missed and because the procedure involves weighing of dissected tissue samples, the spatial resolution of this method is low and determined by the smallest amount that can be weighed accurately. Gamma camera imaging and positron emission tomography though more comprehensive in evaluating the global distribution of a compound, have relative low spatial resolution. Whole body autoradiography of small animals has a much higher spatial resolution as compared to the above and depicts the global distribution of radiopharmaceuticals. A computer-assisted quantification method of WBARG applied to positron, beta, and gamma emitters will complement the method by producing quantitative values comparable to those obtained by dissection and direct tissue counting, with the advantages of depicting the global distribution at high spatial resolution.

  20. Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics

    PubMed Central

    Tsai, Chia-Feng; Wang, Yi-Ting; Yen, Hsin-Yung; Tsou, Chih-Chiang; Ku, Wei-Chi; Lin, Pei-Yi; Chen, Hsuan-Yu; Nesvizhskii, Alexey I.; Ishihama, Yasushi; Chen, Yu-Ju

    2015-01-01

    Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase–substrate network associated with acquired drug resistance. PMID:25814448

  1. Computed Tomography of Aortic Wall Calcifications in Aortic Dissection Patients

    PubMed Central

    de Jong, Pim A.; Hellings, Willem E.; Takx, Richard A. P.; Igum, Ivana; van Herwaarden, Joost A.; Mali, Willem P. Th. M.

    2014-01-01

    Objectives To investigate the frequency of aortic calcifications at the outer edge of the false lumen and the frequency of fully circular aortic calcifications in a consecutive series of patients with aortic dissection who underwent contrast-enhanced CT. Methods The study population compromised of 69 consecutive subjects aged 60 years and older with a contrast-enhanced CT scan demonstrating an aortic dissection. All CT scans were evaluated for the frequency of aortic calcifications at the outer edge of the false lumen and the frequency of fully circular aortic calcifications by two experienced observers. Between observer reliability was evaluated by using Cohens Kappa. Differences between groups were tested using unpaired T test and Chi-square test. Results Presumed media calcifications were observed in 22 (32%) patients of 60 years and older and were found more frequently in chronic aortic dissection (N?=?12/23, 52%) than in acute aortic dissection (N?=?10/46, 22%). Conclusion As the intima has been torn away by the aortic dissection it is highly likely that CT scans can visualize the calcifications in the tunica media of the aorta. PMID:25003993

  2. Congenital Incomplete Fusion of Superior Mesenteric Artery Mimicking Dissection

    PubMed Central

    Sharma, Vasu Keshav; Hng, Martin Weng Chin

    2015-01-01

    Patient: Male, 62 Final Diagnosis: Superior mesenteric artery anatomic variant Symptoms: Abdominal pain diarrhea transcient ischemic attacks Medication: Clinical Procedure: CT of abdomen and pelvis Specialty: Surgery Objective: Congenital defects/diseases Background: Both spontaneous SMA dissection and anatomical variants of GIT vasculature are well known entities. We present a case initially diagnosed as an SMA dissection on CT, but upon detailed review of the imaging findings was considered to be incompletely fused ventral segmental arteries a rare anatomic variant not well described before. This finding is clinically significant, as it can mimic a vascular dissection and such a wrong diagnosis will lead to unnecessary investigation and intervention. Case Report: A 62-year-old male patient presented with abdominal pain of uncertain etiology. The initial CT revealed an abnormal appearance of the superior mesenteric artery (SMA) which was diagnosed as SMA dissection. However, the appearance of this dissection was unusual and there was a mismatch between the clinical presentation and radiological findings. The scan was reviewed and a 3D reconstruction of the abdominal aortal and visceral arteries was performed. The abnormal appearance of the SMA was deemed to be from a congenital anatomical variant. A review of the embryological origin of gut vasculature provides a likely explanation for this appearance. Conclusions: Ours is an unusual case of a developmental variant that has not been well described hitherto. Attention to the ancillary radiological signs and understanding the embryological origin of the abdominal vasculature is important to distinguish such variants from pathology. PMID:25623118

  3. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

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

  4. Quantitative Thinking.

    ERIC Educational Resources Information Center

    DuBridge, Lee A.

    An appeal for more research to determine how to educate children as effectively as possible is made. Mathematics teachers can readily examine the educational problems of today in their classrooms since learning progress in mathematics can easily be measured and evaluated. Since mathematics teachers have learned to think in quantitative terms and…

  5. On Quantitizing

    ERIC Educational Resources Information Center

    Sandelowski, Margarete; Voils, Corrine I.; Knafl, George

    2009-01-01

    "Quantitizing", commonly understood to refer to the numerical translation, transformation, or conversion of qualitative data, has become a staple of mixed methods research. Typically glossed are the foundational assumptions, judgments, and compromises involved in converting disparate data sets into each other and whether such conversions advance

  6. Digital dissection system for medical school anatomy training

    NASA Astrophysics Data System (ADS)

    Augustine, Kurt E.; Pawlina, Wojciech; Carmichael, Stephen W.; Korinek, Mark J.; Schroeder, Kathryn K.; Segovis, Colin M.; Robb, Richard A.

    2003-05-01

    As technology advances, new and innovative ways of viewing and visualizing the human body are developed. Medicine has benefited greatly from imaging modalities that provide ways for us to visualize anatomy that cannot be seen without invasive procedures. As long as medical procedures include invasive operations, students of anatomy will benefit from the cadaveric dissection experience. Teaching proper technique for dissection of human cadavers is a challenging task for anatomy educators. Traditional methods, which have not changed significantly for centuries, include the use of textbooks and pictures to show students what a particular dissection specimen should look like. The ability to properly carry out such highly visual and interactive procedures is significantly constrained by these methods. The student receives a single view and has no idea how the procedure was carried out. The Department of Anatomy at Mayo Medical School recently built a new, state-of-the-art teaching laboratory, including data ports and power sources above each dissection table. This feature allows students to access the Mayo intranet from a computer mounted on each table. The vision of the Department of Anatomy is to replace all paper-based resources in the laboratory (dissection manuals, anatomic atlases, etc.) with a more dynamic medium that will direct students in dissection and in learning human anatomy. Part of that vision includes the use of interactive 3-D visualization technology. The Biomedical Imaging Resource (BIR) at Mayo Clinic has developed, in collaboration with the Department of Anatomy, a system for the control and capture of high resolution digital photographic sequences which can be used to create 3-D interactive visualizations of specimen dissections. The primary components of the system include a Kodak DC290 digital camera, a motorized controller rig from Kaidan, a PC, and custom software to synchronize and control the components. For each dissection procedure, the images are captured automatically, and then processed to generate a Quicktime VR sequence, which permits users to view an object from multiple angles by rotating it on the screen. This provides 3-D visualizations of anatomy for students without the need for special '3-D glasses' that would be impractical to use in a laboratory setting. In addition, a digital video camera may be mounted on the rig for capturing video recordings of selected dissection procedures being carried out by expert anatomists for playback by the students. Anatomists from the Department of Anatomy at Mayo have captured several sets of dissection sequences and processed them into Quicktime VR sequences. The students are able to look at these specimens from multiple angles using this VR technology. In addition, the student may zoom in to obtain high-resolution close-up views of the specimen. They may interactively view the specimen at varying stages of dissection, providing a way to quickly and intuitively navigate through the layers of tissue. Electronic media has begun to impact all areas of education, but a 3-D interactive visualization of specimen dissections in the laboratory environment is a unique and powerful means of teaching anatomy. When fully implemented, anatomy education will be enhanced significantly by comparison to traditional methods.

  7. Spontaneous coronary artery dissection: the management dilemma continues.

    PubMed

    Ahmed, Zaheer; Bajwa, Ata; Bhardwaj, Bhaskar; Laster, Steven B; Magalski, Anthony

    2015-01-01

    Spontaneous coronary artery dissection (SCAD) is an increasingly recognised cause of acute coronary syndrome, particularly in women. A 36-year-old Caucasian woman presented to our hospital with sudden onset chest pain and was diagnosed with a non-ST elevation myocardial infarction. Coronary angiography revealed mid and distal left anterior descending artery (LAD) dissection with distal LAD occlusion. A short segment of apical LAD filled late with incomplete opacification (Thrombolysis In Myocardial Infarction (TIMI) 1 flow). A decision was made to treat the patient conservatively, with subsequent resolution of dissection over the next 3 months. Our patient made a good clinical recovery with healing of her affected coronary vasculature on subsequent angiogram. The case illustrates that SCAD can be managed conservatively with antiplatelet agents, ?-blockers, heparin and statins, if the patient is haemodynamically stable and coronary flow is adequate. PMID:26272965

  8. The birth and evolution of neuroscience through cadaveric dissection.

    PubMed

    Moon, Karam; Filis, Andreas K; Cohen, Alan R

    2010-09-01

    Although interest in the art of dissection and vivisection has waxed and waned throughout the ages, the past century has seen it accepted as commonplace in medical schools across the country. No other practice in medicine has contributed more to the understanding of neuroanatomy and the neurosciences as dissection of the human cadaver, the origins of which are widely documented to have been in Alexandrian Greece. This article chronicles the fascinating and often controversial use of dissection and vivisection in these fields through the ages, beginning with Herophilus of Alexandria, among the first systematic dissectors in the history of Western medicine. The authors comment on its role in the development of modern neurosurgery and conclude with remarks about use of this educational tool today in the United States. PMID:20657312

  9. Expression microdissection adapted to commercial laser dissection instruments

    PubMed Central

    Hanson, Jeffrey C; Tangrea, Michael A; Kim, Skye; Armani, Michael D; Pohida, Thomas J; Bonner, Robert F; Rodriguez-Canales, Jaime; Emmert-Buck, Michael R

    2016-01-01

    Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis. PMID:21412274

  10. Dissection of the atrial wall after mitral valve replacement.

    PubMed Central

    Lukcs, L; Kassai, I; Lengyel, M

    1996-01-01

    We describe an unusual sequela of mitral valve replacement in a 50-year-old woman who had undergone a closed mitral commissurotomy in 1975. She was admitted to our hospital because of mitral restenosis in November 1993, at which time her mitral valve was replaced with a mechanical prosthesis. On the 8th postoperative day, the patient developed symptoms of heart failure; transesophageal echocardiography revealed dissection and rupture of the left atrial wall. At prompt reoperation, we found an interlayer dissection and rupture of the atrial wall into the left atrium. We repaired the ruptured atrial wall with a prosthetic patch. The postoperative course was uneventful, and postoperative transesophageal echocardiography showed normal prosthetic valve function and no dissection. Images PMID:8680278

  11. Isolated middle cerebral artery dissection: a systematic review.

    PubMed

    Asaithambi, Ganesh; Saravanapavan, Pradeepan; Rastogi, Vaibhav; Khan, Sheema; Bidari, Sharatchandra; Khanna, Anna Y; Ganti, Latha; Qureshi, Adnan I; Hedna, Vishnumurthy Shushrutha

    2014-01-01

    Acute stroke can be missed in the emergency department, particularly in younger patients and in those with more vague symptoms such as headache or dizziness. Cervicocephalic dissections are one group of etiologies for acute stroke in the young. While cervicocephalic dissections are not uncommon in clinical practice, isolated middle cerebral artery dissection (MCAD) has been rarely reported as a cause for stroke. We sought to review the clinical implications and pathophysiology of an isolated MCAD. We searched the medical literature for isolated MCAD in clinical stroke patients using MEDLINE, HighWire, and Google Scholar databases from 1966 to 2013 using the keywords 'middle cerebral artery dissection,' 'intracerebral artery dissection,' and 'middle cerebral artery dissection stroke.' We reviewed cases to learn various characteristics of isolated MCAD. A total of 61 cases (62.3% male, mean age 44.16??19.17 years) were reviewed from 54 publications. Most cases were reported from Asian countries (78.7%). Ischemic strokes were more common than hemorrhagic strokes (68.9%). Digital subtraction angiography was the most common imaging modality used to diagnose isolated MCAD (75.4%). Surgery was the preferred form of therapeutic intervention (39.3%). Males (n?=?27/48, p?=?0.0008) and those who presented with only ischemic syndromes (n?=?22/48, p?=?0.0009) had significantly higher rates of favorable outcome. Isolated MCAD is a rare disease that can contribute to the stroke burden of young patients. Further studies are needed to better characterize optimal treatment strategies and define outcomes for this rare condition. PMID:25593617

  12. Lymphedema After Complete Axillary Node Dissection for Melanoma

    PubMed Central

    Starritt, Emma C.; Joseph, David; McKinnon, J Gregory; Lo, Sing Kai; de Wilt, Johannes H. W.; Thompson, John F.

    2004-01-01

    Objectives: The objectives of this study were to define appropriate criteria for assessing the presence of lymphedema, and to report the prevalence and risk factors for development of upper limb lymphedema after level IIII axillary dissection for melanoma. Summary Background Data: The lack of a consistent and reliable objective definition for lymphedema remains a significant barrier to appreciating its prevalence after axillary dissection for melanoma (or breast carcinoma). Methods: Lymphedema was assessed in 107 patients (82 male, 25 female) who had previously undergone complete level IIII axillary dissection. Of the 107 patients, 17 had also received postoperative axillary radiotherapy. Arm volume was measured using a water displacement technique. Change in volume of the arm on the side of the dissection was referenced to the volume of the other (control) arm. Volume measurements were corrected for the effect of handedness using corrections derived from a control group. Classification and regression tree (CART) analysis was used to determine a threshold fractional arm volume increase above which volume changes were considered to indicate lymphedema. Results: Based on the CART analysis results, lymphedema was defined as an increase in arm volume greater than 16% of the volume of the control arm. Using this definition, lymphedema prevalence for patients in the present study was 10% after complete level IIII axillary dissection for melanoma and 53% after additional axillary radiotherapy. Radiotherapy and wound complications were independent risk factors for the development of lymphedema. Conclusions: A suggested objective definition for arm lymphedema after axillary dissection is an arm volume increase of greater than 16% of the volume of the control arm. PMID:15492570

  13. Phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide: TBTO affects proliferation and energy sensing pathways.

    PubMed

    Osman, Ahmed M; van Loveren, Henk

    2012-03-01

    We report the results of phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide (TBTO), an immunotoxic compound. After cell lysis, phosphoproteins were isolated using Phosphoprotein Purification Kit, separated by SDS-PAGE and subsequently digested with trypsin. Phosphopeptides were enriched employing titanium dioxide, and the obtained fractions were analyzed by nano-LC-MS/MS. A total of 160 phosphoproteins and 328 phosphorylation sites were identified in thymoma cells. Among the differentially phosphorylated proteins identified in TBTO-treated cells were key enzymes, which catalyze rate-limiting steps in pathways that are sensitive to cellular energy status. These proteins included acetyl-CoA carboxylase isoform 1, which catalyzes the rate-limiting step of fatty acid synthesis. Another enzyme was glutamine: fructose-6-phosphate amidotransferase, GFAT1, the first and rate-limiting enzyme for the hexoamine synthesis pathway. Pyruvate dehydrogenase (PDH), a multicomplex enzyme that catalyzes the rate-limiting step of aerobic oxidation of fuel carbohydrates, was identified in both TBTO-treated and control cells; however, phosphorylation at residue S293, known to inhibit PDH activity, was identified only in control cells. A lower expression level of ribosomal protein S6 kinase 1, a downstream kinase of the mammalian target of rapamycin signaling pathway implicated in protein synthesis through phosphorylation of 40 ribosomal S6, was observed in the treated cells. Giant kinases like AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKAR1A), which are known to mediate the phosphorylation of these enzymes, were identified in TBTO-treated cells. Downregulation of proteins, such as MAPK, matrin-3 and ribonucleotide reductase, subunit RRM2, which are implicated in cell proliferation, was also observed in TBTO-treated cells. Together, the results show that TBTO affects proliferation and energy sensor pathways. PMID:22174045

  14. Proteome and phosphoproteome analysis of honeybee (Apis mellifera) venom collected from electrical stimulation and manual extraction of the venom gland

    PubMed Central

    2013-01-01

    Background Honeybee venom is a complicated defensive toxin that has a wide range of pharmacologically active compounds. Some of these compounds are useful for human therapeutics. There are two major forms of honeybee venom used in pharmacological applications: manually (or reservoir disrupting) extracted glandular venom (GV), and venom extracted through the use of electrical stimulation (ESV). A proteome comparison of these two venom forms and an understanding of the phosphorylation status of ESV, are still very limited. Here, the proteomes of GV and ESV were compared using both gel-based and gel-free proteomics approaches and the phosphoproteome of ESV was determined through the use of TiO2 enrichment. Results Of the 43 proteins identified in GV, < 40% were venom toxins, and > 60% of the proteins were non-toxic proteins resulting from contamination by gland tissue damage during extraction and bee death. Of the 17 proteins identified in ESV, 14 proteins (>80%) were venom toxic proteins and most of them were found in higher abundance than in GV. Moreover, two novel proteins (dehydrogenase/reductase SDR family member 11-like and histone H2B.3-like) and three novel phosphorylation sites (icarapin (S43), phospholipase A-2 (T145), and apamin (T23)) were identified. Conclusions Our data demonstrate that venom extracted manually is different from venom extracted using ESV, and these differences may be important in their use as pharmacological agents. ESV may be more efficient than GV as a potential pharmacological source because of its higher venom protein content, production efficiency, and without the need to kill honeybee. The three newly identified phosphorylated venom proteins in ESV may elicit a different immune response through the specific recognition of antigenic determinants. The two novel venom proteins extend our proteome coverage of honeybee venom. PMID:24199871

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

    PubMed Central

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

    2008-01-01

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

  16. 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 components of the host cell machinery via modulation of RSK activity. Thus, this study has important implications for the pathobiology of KSHV and other diseases in which RSK activity is dysregulated. PMID:26133373

  17. Two-dimensional gel electrophoresis maps of the proteome and phosphoproteome of primitively cultured rat mesangial cells.

    PubMed

    Jiang, Xiao-Sheng; Tang, Liu-Ya; Cao, Xing-Jun; Zhou, Hu; Xia, Qi-Chang; Wu, Jia-Rui; Zeng, Rong

    2005-12-01

    Mesangial cells (MC) play an important role in maintaining the structure and function of the glomerulus. The proliferation of MC is a prominent feature of many kinds of glomerular disease. The first reference 2-DE maps of rat mesangial cells (RMC), stained with silver staining or Pro-Q Diamond dye, have been established here to describe the proteome and phosphoproteome of RMC, respectively. A total of 157 selected protein spots, corresponding to 118 unique proteins, have been identified by MALDI-TOF-MS or LC-ESI-IT-MS/MS, in which 37 protein spots representing 28 unique proteins have also been stained with Pro-Q Diamond, indicating that they are in phosphorylated forms. All the identified proteins were bioinformatically annotated in detail according to their physiochemical characteristics, subcellular location, and function. Most of the separated or identified protein spots are distributed in the area of mass 10-70 kDa and pI 5.0-8.0. The identified proteins include mainly cytoplasmic and nuclear proteins and some mitochondrial, endoplasmic reticulum, and membrane proteins. These proteins are classified into different functional groups such as structure and mobility proteins (21.2%), metabolic enzymes (16.9%), protein folding and metabolism proteins (13.6%), signaling proteins (14.4%), heat-shock proteins (7.6%), and other functional proteins (12.7%). While structure and mobility proteins are mostly represented by protein spots with high abundance, signaling proteins are mostly represented by protein spots with relatively low abundance. Such a 2-DE database for RMC, especially with many signaling proteins and phosphoproteins characterized, will provide a valuable resource for comparative proteomics analysis of normal and pathologic conditions affecting MC function or pathologic progress. PMID:16315178

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

    SciTech Connect

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

    2010-11-30

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

  19. Quantitative radiography

    SciTech Connect

    Logan, C.M.; Hernandez, J.M.; Devine, G.J.

    1991-02-01

    We have developed a system of quantitative radiography in order to produce quantitative images displaying homogeneity of parts. The materials that we characterize are synthetic composites and may contain important subtle density variations not discernable by examining a raw film x-radiograph. In order to quantitatively interpret film radiographs, it is necessary to digitize, interpret, and display the images. Our integrated system of quantitative radiography displays accurate, high-resolution pseudocolor images in units of density. We characterize approximately 10,000 parts per year in hundreds of different configurations and compositions with this system. Images are captured using DuPont NDT55 industrial x-ray film in Daypack{trademark} packages. X-ray cabinets are of custom design, with helium flight path and a filter wheel for positioning filters if desired. The cabinets contain baffles to reduce scattered radiation and are equipped with drawer for rapid load/unload of parts. Separate units with tungsten-anode or copper-anode tubes are available. The usual operating voltage is 15 to 35 kVp. Fixturing provides for rough part positioning and precise alignment with respect to the x-ray source. Areal density standards are placed at several locations on each film. In interpreting the image, we use the standards nearest the image of the part being quantified. Because of this, small variations in x-ray flux uniformity (heel effects) are unimportant. The usual standard is a step wedge of aluminum containing 13 steps. Films are permanently labeled by imaging a perforated metal numbering strip. Data such as part number, step wedge identification, etc. are read from barcode labels and transferred to a data base for later retrieval and use in quantifying the image.

  20. Superior Mesenteric Artery Dissection after Extracorporeal Shockwave Lithotripsy

    PubMed Central

    Bakoyiannis, Christos; Anastasiou, Ioannis; Koutsoumpelis, Andreas; Fragiadis, Evangelos; Felesaki, Eleni; Kafeza, Marina; Georgopoulos, Sotirios; Tsigris, Christos

    2012-01-01

    The use of shockwave lithotripsy is currently the mainstay of treatment in renal calculosis. Several complications including vessel injuries have been implied to extracorporeal shockwave lithotripsy. We report an isolated dissection of the superior mesenteric artery in a 60-year-old male presenting with abdominal pain which occurred three days after extracorporeal shockwave lithotripsy. The patient was treated conservatively and the abdominal pain subsided 24 hours later. The patient's history, the course of his disease, and the timing may suggest a correlation between the dissection and the ESWL. PMID:23304627