Sample records for quantitative phosphoproteomics reveals

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

    PubMed

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

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

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

    PubMed Central

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

    2017-01-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chan, Chi Yuet X’avia; Gritsenko, Marina A.; Smith, Richard D.

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

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

    PubMed

    Chen, Kai; Lv, Fan; Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-11-15

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

  5. In vivo SILAC-based proteomics reveals phosphoproteome changes during mouse skin carcinogenesis.

    PubMed

    Zanivan, Sara; Meves, Alexander; Behrendt, Kristina; Schoof, Erwin M; Neilson, Lisa J; Cox, Jürgen; Tang, Hao R; Kalna, Gabriela; van Ree, Janine H; van Deursen, Jan M; Trempus, Carol S; Machesky, Laura M; Linding, Rune; Wickström, Sara A; Fässler, Reinhard; Mann, Matthias

    2013-02-21

    Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Comparison of three quantitative phosphoproteomic strategies to study receptor tyrosine kinase signaling.

    PubMed

    Zhang, Guoan; Neubert, Thomas A

    2011-12-02

    There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.

  7. Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation

    PubMed Central

    Ji, Qinqin; Salomon, Arthur R.

    2015-01-01

    The activation of T-lymphocytes through antigen-mediated T-cell receptor (TCR) clustering is vital in regulating the adaptive-immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperature initiated some of the hallmarks of TCR signaling such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum as well as coalesce T-cell membrane microdomains. The precise mechanism of TCR signaling initiation due to temperature change remains obscure. One critical question is whether signaling initiated by cold treatment of T cells differs from signaling initiated by crosslinking of the T cell receptor. To address this uncertainty, a wide-scale, quantitative mass spectrometry-based phosphoproteomic analysis was performed on T cells stimulated either by temperature shift or through crosslinking of the TCR. Careful statistical comparison between the two stimulations revealed a striking level of identity between the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, at unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns nearly identical to soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells. PMID:25839225

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

    PubMed

    Schmidt, Andreas; Trentini, Débora 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.

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

    PubMed

    Huang, Honggang; Larsen, Martin R; Palmisano, Giuseppe; Dai, Jie; Lametsch, René

    2014-06-25

    Protein phosphorylation can regulate most of the important processes in muscle, such as metabolism and contraction. The postmortem (PM) metabolism and rigor mortis have essential effects on meat quality. In order to identify and characterize the protein phosphorylation events involved in meat quality development, a quantitative mass spectrometry-based phosphoproteomic study was performed to analyze the porcine muscle within 24h PM using dimethyl labeling combined with the TiSH phosphopeptide enrichment strategy. In total 305 unique proteins were identified, including 160 phosphoproteins with 784 phosphorylation sites. Among these, 184 phosphorylation sites on 93 proteins had their phosphorylation levels significantly changed. The proteins involved in glucose metabolism and muscle contraction were the two largest clusters of phosphoproteins with significantly changed phosphorylation levels in muscle within 24 h PM. The high phosphorylation level of heat shock proteins (HSPs) in early PM may be an adaptive response to slaughter stress and protect muscle cell from apoptosis, as observed in the serine 84 of HSP27. This work indicated that PM muscle proteins underwent significant changes at the phosphorylation level but were relatively stable at the total protein level, suggesting that protein phosphorylation may have important roles in meat quality development through the regulation of proteins involved in glucose metabolism and muscle contraction, thereby affecting glycolysis and rigor mortis development in PM muscle. The manuscript describes the characterization of postmortem (PM) porcine muscle within 24 h postmortem from the perspective of protein phosphorylation using advanced phosphoproteomic techniques. In the study, the authors employed the dimethyl labeling combined with the TiSH phosphopeptide enrichment and LC-MS/MS strategy. This was the first high-throughput quantitative phosphoproteomic study in PM muscle of farm animals. In the work, both the proteome

  10. 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

    2015-12-04

    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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-05-01

    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

  13. SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

    PubMed

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

    2015-01-30

    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. Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-06-01

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

  15. Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate

    PubMed Central

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

    2015-01-01

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

  16. Quantitative phosphoproteomics analysis reveals a key role of insulin growth factor 1 receptor (IGF1R) tyrosine kinase in human sperm capacitation.

    PubMed

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

    2015-04-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Identification of MAPK Substrates Using Quantitative Phosphoproteomics.

    PubMed

    Zhang, Tong; Schneider, Jacqueline D; Zhu, Ning; Chen, Sixue

    2017-01-01

    Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

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

    PubMed

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

    2016-02-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nguyen, Tran H.; Brechenmacher, Laurent; Aldrich, Joshua T.

    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 insightmore » 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.« less

  20. 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; Jørgensen, 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

  1. Quantitative phosphoproteomic analysis of RIP3-dependent protein phosphorylation in the course of TNF-induced necroptosis.

    PubMed

    Zhong, Chuan-Qi; Li, Yuanyue; Yang, Daowei; Zhang, Na; Xu, Xiaozheng; Wu, Yaying; Chen, Jinan; Han, Jiahuai

    2014-03-01

    Tumor necrosis factor (TNF) induced cell death in murine fibrosarcoma L929 cells is a model system in studying programed necrosis (also known as necroptosis). Receptor interacting protein 3 (RIP3), a serine-threonine kinase, is known to play an essential role in TNF-induced necroptosis; however, the phosphorylation events initiated by RIP3 activation in necroptotic process is still largely unknown. Here, we performed a quantitative MS based analysis to compare TNF-induced changes in the global phosphoproteome of wild-type (RIP3(+/+) ) and RIP3-knockdown L929 cells at different time points after TNF treatment. A total of 8058 phosphopeptides spanning 6892 phosphorylation sites in 2762 proteins were identified in the three experiments, in which cells were treated with TNF for 0.5, 2, and 4 h. By comparing the phosphorylation sites in wild-type and RIP3-knockdown L929 cells, 174, 167, and 177 distinct phosphorylation sites were revealed to be dependent on RIP3 at the 0.5, 2, and 4 h time points after TNF treatment, respectively. Notably, most of them were not detected in a previous phosphoproteomic analysis of RIP3-dependent phosphorylation in lipopolysaccharide-stimulated peritoneal macrophages and TNF-treated murine embryonic fibroblasts (MEFs), suggesting that the data presented in this report are highly relevant to the study of TNF-induced necroptosis of L929 cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Quantitative Phosphoproteomics Dissection of Seven-transmembrane Receptor Signaling Using Full and Biased Agonists*

    PubMed Central

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

    2010-01-01

    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 (AT1R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. “Biased agonists” with intrinsic “functional selectivity” that simultaneously blocks Gαq protein activity and activates G protein-independent pathways of the AT1R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT1R 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 AT1R agonist angiotensin II and the biased agonist [Sar1,Ile4,Ile8]angiotensin II (SII angiotensin II), which only activates the Gαq 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 AT1R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Gαq protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Gαq-dependent and -independent AT1R 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 Gαq 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

  3. Quantitation of the phosphoproteome using the library-assisted extracted ion chromatogram (LAXIC) strategy.

    PubMed

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

    2014-01-01

    Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.

  4. Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome.

    PubMed

    Panizza, Elena; Branca, Rui M M; Oliviusson, Peter; Orre, Lukas M; Lehtiö, Janne

    2017-07-03

    Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5-3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.

  5. SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast.

    PubMed

    Baro, Barbara; Játiva, Soraya; Calabria, Inés; Vinaixa, Judith; Bech-Serra, Joan-Josep; de LaTorre, Carolina; Rodrigues, João; Hernáez, María Luisa; Gil, Concha; Barceló-Batllori, Silvia; Larsen, Martin R; Queralt, Ethel

    2018-05-01

    Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.

  6. Quantitative phosphoproteomics using acetone-based peptide labeling: Method evaluation and application to a cardiac ischemia/reperfusion model

    PubMed Central

    Wijeratne, Aruna B.; Manning, Janet R.; Schultz, Jo El J.; Greis, Kenneth D.

    2013-01-01

    Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. In this report, an MS-based strategy utilizing an inexpensive acetone-based peptide labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Since the chemistry for RABA-labeling for phosphorylation profiling had not been previously reported, it was first validated using a standard phosphoprotein and identical phosphoproteomes from cardiac tissue extracts. A workflow was then utilized to compare cardiac tissue phosphoproteomes from mouse hearts not expressing FGF2 vs. hearts expressing low molecular weight fibroblast growth factor-2 (LMW FGF2) to relate low molecular weight fibroblast growth factor-2 (LMW FGF2) mediated cardioprotective phenomena induced by ischemia/reperfusion (I/R) injury of hearts, with downstream phosphorylation changes in LMW FGF2 signaling cascades. Statistically significant phosphorylation changes were identified at 14 different sites on 10 distinct proteins including some with mechanisms already established for LMW FGF2-mediated cardioprotective signaling (e.g. connexin-43), some with new details linking LMW FGF2 to the cardioprotective mechanisms (e.g. cardiac myosin binding protein C or cMyBPC), and also several new downstream effectors not previously recognized for cardio-protective signaling by LMW FGF2. Additionally, one of the phosphopeptides, cMyBPC/pSer-282, identified was further verified with site-specific quantification using an SRM (selected reaction monitoring)-based approach that also relies on isotope labeling of a synthetic phosphopeptide with deuterated acetone as an internal standard. Overall, this study confirms that the inexpensive acetone-based peptide labeling can be used in both exploratory and targeted quantification

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

    PubMed Central

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

    2016-01-01

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

  8. 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 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

  9. Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium.

    PubMed

    Soares, Nelson C; Spät, Philipp; Krug, Karsten; Macek, Boris

    2013-06-07

    Recent phosphoproteomics studies have generated relatively large data sets of bacterial proteins phosphorylated on serine, threonine, and tyrosine, implicating this type of phosphorylation in the regulation of vital processes of a bacterial cell; however, most phosphoproteomics studies in bacteria were so far qualitative. Here we applied stable isotope labeling by amino acids in cell culture (SILAC) to perform a quantitative analysis of proteome and phosphoproteome dynamics of Escherichia coli during five distinct phases of growth in the minimal medium. Combining two triple-SILAC experiments, we detected a total of 2118 proteins and quantified relative dynamics of 1984 proteins in all measured phases of growth, including 570 proteins associated with cell wall and membrane. In the phosphoproteomic experiment, we detected 150 Ser/Thr/Tyr phosphorylation events, of which 108 were localized to a specific amino acid residue and 76 were quantified in all phases of growth. Clustering analysis of SILAC ratios revealed distinct sets of coregulated proteins for each analyzed phase of growth and overrepresentation of membrane proteins in transition between exponential and stationary phases. The proteomics data indicated that proteins related to stress response typically associated with the stationary phase, including RpoS-dependent proteins, had increasing levels already during earlier phases of growth. Application of SILAC enabled us to measure median occupancies of phosphorylation sites, which were generally low (<12%). Interestingly, the phosphoproteome analysis showed a global increase of protein phosphorylation levels in the late stationary phase, pointing to a likely role of this modification in later phases of growth.

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

    PubMed

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

    2015-01-01

    To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. The phosphoproteome of toll-like receptor-activated macrophages

    PubMed Central

    Weintz, Gabriele; Olsen, Jesper V; Frühauf, Katja; Niedzielska, Magdalena; Amit, Ido; Jantsch, Jonathan; Mages, Jörg; Frech, Cornelie; Dölken, Lars; Mann, Matthias; Lang, Roland

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression. PMID:20531401

  12. Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus.

    PubMed

    Shobahah, Jauharotus; Xue, Shengjie; Hu, Dongbing; Zhao, Cui; Wei, Ming; Quan, Yanping; Yu, Wei

    2017-06-19

    Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

  13. Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

    PubMed Central

    Joshi, Rubin N.; Binai, Nadine A.; Marabita, Francesco; Sui, Zhenhua; Altman, Amnon; Heck, Albert J. R.; Tegnér, Jesper; Schmidt, Angelika

    2017-01-01

    Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25− T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer. PMID:28993769

  14. Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

    PubMed

    Francavilla, Chiara; Lupia, Michela; Tsafou, Kalliopi; Villa, Alessandra; Kowalczyk, Katarzyna; Rakownikow Jersie-Christensen, Rosa; Bertalot, Giovanni; Confalonieri, Stefano; Brunak, Søren; Jensen, Lars J; Cavallaro, Ugo; Olsen, Jesper V

    2017-03-28

    Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Functional phosphoproteomic mass spectrometry-based approaches

    PubMed Central

    2012-01-01

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

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

    PubMed

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

    2013-07-02

    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.

  17. Quantitative measurement of phosphoproteome response to osmotic stress in arabidopsis based on Library-Assisted eXtracted Ion Chromatogram (LAXIC).

    PubMed

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

    2013-08-01

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

  18. An Integrated Phosphoproteomics Work Flow Reveals Extensive Network Regulation in Early Lysophosphatidic Acid Signaling*

    PubMed Central

    Schreiber, Thiemo B.; Mäusbacher, Nina; Kéri, György; Cox, Jürgen; Daub, Henrik

    2010-01-01

    Lysophosphatidic acid (LPA) induces a variety of cellular signaling pathways through the activation of its cognate G protein-coupled receptors. To investigate early LPA responses and assess the contribution of epidermal growth factor (EGF) receptor transactivation in LPA signaling, we performed phosphoproteomics analyses of both total cell lysate and protein kinase-enriched fractions as complementary strategies to monitor phosphorylation changes in A498 kidney carcinoma cells. Our integrated work flow enabled the identification and quantification of more than 5,300 phosphorylation sites of which 224 were consistently regulated by LPA. In addition to induced phosphorylation events, we also obtained evidence for early dephosphorylation reactions due to rapid phosphatase regulation upon LPA treatment. Phosphorylation changes induced by direct heparin-binding EGF-like growth factor-mediated EGF receptor activation were typically weaker and only detected on a subset of LPA-regulated sites, indicating signal integration among EGF receptor transactivation and other LPA-triggered pathways. Our results reveal rapid phosphoregulation of many proteins not yet implicated in G protein-coupled receptor signaling and point to various additional mechanisms by which LPA might regulate cell survival and migration as well as gene transcription on the molecular level. Moreover, our phosphoproteomics analysis of both total lysate and kinase-enriched fractions provided highly complementary parts of the LPA-regulated signaling network and thus represents a useful and generic strategy toward comprehensive signaling studies on a system-wide level. PMID:20071362

  19. Methods for and Insights from Phosphoproteome Analysis in Marine Microbes

    NASA Astrophysics Data System (ADS)

    Held, N. A.; Saito, M. A.; McIlvin, M.

    2016-02-01

    Phosphorylation, the dynamic addition of a phosphate group to specific amino acids, is a key regulator of protein activity in both prokaryotes and eukaryotes. Protein phosphorylation is known to modulate nutrient acquisition, metabolism, growth and reproduction in model organisms, yet little is known about the role of phosphorylation marine organisms. Recent developments in LC-MS/MS make it possible to identify phosphorylation events in the proteome. We tested various methods in marine bacteria and developed a simple approach to phosphoproteome analysis. We then applied this method to cultured isolates of Prochlorococcus and diatom-associated Alteromonas sp. BB2AT2. We began by comparing the phosphoproteomes of these organisms in exponential and stationary phase growth. We conducted iterative experiments to assess completeness of our analysis, similar to the rarefaction approach used to determine sequence depth in ecology. We also explored semi-quantitative changes in protein phosphorylation when cells were subject to phosphate deplete media and/or phosphatase inhibitors. These early studies demonstrate the promise of phosphoproteomics to advance our understanding of bacterial biochemistry and microbe-environment interactions.

  20. Phosphoproteomic network analysis in the sea urchin Strongylocentrotus purpuratus reveals new candidates in egg activation.

    PubMed

    Guo, Hongbo; Garcia-Vedrenne, Ana Elisa; Isserlin, Ruth; Lugowski, Andrew; Morada, Anthony; Sun, Alex; Miao, Yishen; Kuzmanov, Uros; Wan, Cuihong; Ma, Hongyue; Foltz, Kathy; Emili, Andrew

    2015-12-01

    Fertilization triggers a dynamic symphony of molecular transformations induced by a rapid rise in intracellular calcium. Most prominent are surface alterations, metabolic activation, cytoskeletal reorganization, and cell-cycle reentry. While the activation process appears to be broadly evolutionarily conserved, and protein phosphorylation is known to play a key role, the signaling networks mediating the response to fertilization are not well described. To address this gap, we performed a time course phosphoproteomic analysis of egg activation in the sea urchin Strongylocentrotus purpuratus, a system that offers biochemical tractability coupled with exquisite synchronicity. By coupling large-scale phosphopeptide enrichment with unbiased quantitative MS, we identified striking changes in global phosphoprotein patterns at 2- and 5-min postfertilization as compared to unfertilized eggs. Overall, we mapped 8796 distinct phosphosite modifications on 2833 phosphoproteins, of which 15% were differentially regulated in early egg activation. Activated kinases were identified by phosphosite mapping, while enrichment analyses revealed conserved signaling cascades not previously associated with egg activation. This work represents the most comprehensive study of signaling associated with egg activation to date, suggesting novel mechanisms that can be experimentally tested and providing a valuable resource for the broader research community. All MS data have been deposited in the ProteomeXchange with identifier PXD002239 (http://proteomecentral.proteomexchange.org/dataset/PXD002239). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

    PubMed

    Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

    2017-07-01

    TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed

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

    2016-09-13

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

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

    PubMed

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

    2016-01-04

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2015-01-01

    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 combined with bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5 min 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

  6. Quantitative phosphoproteomic analysis of caprine muscle with high and low meat quality.

    PubMed

    Liu, Manshun; Wei, Yanchao; Li, Xin; Quek, Siew Young; Zhao, Jing; Zhong, Huazhen; Zhang, Dequan; Liu, Yongfeng

    2018-07-01

    During the conversion of muscle to meat, protein phosphorylation can regulate various biological processes that have important effects on meat quality. To investigate the phosphorylation pattern of protein on rigor mortis, goat longissimus thoracis and external intercostals were classified into two groups (high quality and low quality), and meat quality was evaluated according to meat quality attributes (Warner-Bratzler shear force, Color, pH and drip loss). A quantitative mass spectrometry-based phosphoproteomic study was conducted to analyze the caprine muscle at 12h postmortem applying the TiO 2 -SIMAC-HILIC (TiSH) phosphopeptide enrichment strategy. A total of 2125 phosphopeptides were identified from 750 phosphoproteins. Among them, 96 proteins had differed in phosphorylation levels. The majority of these proteins are involved in glucose metabolism and muscle contraction. The differential phosphorylation level of proteins (PFK, MYL2 and HSP27) in two groups may be the crucial factors of regulating muscle rigor mortis. This study provides a comprehensive view for the phosphorylation status of caprine muscle at rigor mortis, it also gives a better understanding of the regulation of protein phosphorylation on various biological processes that affect the final meat quality attributes. Copyright © 2018. Published by Elsevier Ltd.

  7. Quantitative cardiac phosphoproteomics profiling during ischemia-reperfusion in an immature swine model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ledee, Dolena R.; Kang, Min A.; Kajimoto, Masaki

    Ischemia-reperfusion (I/R) results in altered metabolic and molecular responses, and phosphorylation is one of the most noted regulatory mechanisms mediating signaling mechanisms during physiological stresses. To expand our knowledge of the potential phosphoproteomic changes in the myocardium during I/R, we used Isobaric Tags for Relative and Absolute Quantitation-based analyses in left ventricular samples obtained from porcine hearts under control or I/R conditions. The data are available via ProteomeXchange with identifier PXD006066. We identified 1,896 phosphopeptides within left ventricular control and I/R porcine samples. Significant differential phosphorylation between control and I/R groups was discovered in 111 phosphopeptides from 86 proteins. Analysismore » of the phosphopeptides using Motif-x identified five motifs: (..R..S..), (..SP..), (..S.S..), (..S…S..), and (..S.T..). Semiquantitative immunoblots confirmed site location and directional changes in phosphorylation for phospholamban and pyruvate dehydrogenase E1, two proteins known to be altered by I/R and identified by this study. Novel phosphorylation sites associated with I/R were also identified. Functional characterization of the phosphopeptides identified by our methodology could expand our understanding of the signaling mechanisms involved during I/R damage in the heart as well as identify new areas to target therapeutic strategies.« less

  8. Proteomes and Phosphoproteomes of Anther and Pollen: Availability and Progress.

    PubMed

    Zhang, Zaibao; Hu, Menghui; Feng, Xiaobing; Gong, Andong; Cheng, Lin; Yuan, Hongyu

    2017-10-01

    In flowering plants, anther development plays crucial role in sexual reproduction. Within the anther, microspore mother cells meiosis produces microspores, which further develop into pollen grains that play decisive role in plant reproduction. Previous studies on anther biology mainly focused on single gene functions relying on genetic and molecular methods. Recently, anther development has been expanded from multiple OMICS approaches like transcriptomics, proteomics/phosphoproteomics, and metabolomics. The development of proteomics techniques allowing increased proteome coverage and quantitative measurements of proteins which can characterize proteomes and their modulation during normal development, biotic and abiotic stresses in anther development. In this review, we summarize the achievements of proteomics and phosphoproteomics with anther and pollen organs from model plant and crop species (i.e. Arabidopsis, rice, tobacco). The increased proteomic information facilitated translation of information from the models to crops and thus aid in agricultural improvement. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. In vivo phosphoproteome characterization reveals key starch granule-binding phosphoproteins involved in wheat water-deficit response.

    PubMed

    Chen, Guan-Xing; Zhen, Shou-Min; Liu, Yan-Lin; Yan, Xing; Zhang, Ming; Yan, Yue-Ming

    2017-10-23

    Drought stress during grain development causes significant yield loss in cereal production. The phosphorylated modification of starch granule-binding proteins (SGBPs) is an important mechanism regulating wheat starch biosynthesis. In this study, we performed the first proteomics and phosphoproteomics analyses of SGBPs in elite Chinese bread wheat (Triticum aestivum L.) cultivar Jingdong 17 under well-watered and water-stress conditions. Water stress treatment caused significant reductions in spike grain numbers and weight, total starch and amylopectin content, and grain yield. Two-dimensional gel electrophoresis revealed that the quantity of SGBPs was reduced significantly by water-deficit treatment. Phosphoproteome characterization of SGBPs under water-deficit treatment demonstrated a reduced level of phosphorylation of main starch synthesis enzymes, particularly for granule-bound starch synthase (GBSS I), starch synthase II-a (SS II-a), and starch synthase III (SS III). Specifically, the Ser34 site of the GBSSI protein, the Tyr358 site of SS II-a, and the Ser837 site of SS III-a exhibited significant less phosphorylation under water-deficit treatment than well-watered treatment. Furthermore, the expression levels of several key genes related with starch biosynthesis detected by qRT-PCR were decreased significantly at 15 days post-anthesis under water-deficit treatment. Immunolocalization showed a clear movement of GBSS I from the periphery to the interior of starch granules during grain development, under both water-deficit and well-watered conditions. Our results demonstrated that the reduction in gene expression or transcription level, protein expression and phosphorylation levels of starch biosynthesis related enzymes under water-deficit conditions is responsible for the significant decrease in total starch content and grain yield.

  10. Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks.

    PubMed

    Jers, Carsten; Soufi, Boumediene; Grangeasse, Christophe; Deutscher, Josef; Mijakovic, Ivan

    2008-08-01

    Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion.

    PubMed

    Ramsubramaniam, Nikhil; Harris, Steven D; Marten, Mark R

    2014-11-01

    We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra-long reverse phase gradient, and identified using a "high-high" strategy (high mass accuracy on the parent and fragment ions) with higher-energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: "sites of polar growth," "vesicle mediated transport," and "cytoskeleton organization." In these same GO categories, kinase-substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase-substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An-Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 (http://proteomecentral.proteomexchange.org/dataset/PXD000715). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Phosphoproteomics reveals that glycogen synthase kinase-3 phosphorylates multiple splicing factors and is associated with alternative splicing

    PubMed Central

    Shinde, Mansi Y.; Sidoli, Simone; Kulej, Katarzyna; Mallory, Michael J.; Radens, Caleb M.; Reicherter, Amanda L.; Myers, Rebecca L.; Barash, Yoseph; Lynch, Kristen W.; Garcia, Benjamin A.; Klein, Peter S.

    2017-01-01

    Glycogen synthase kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. In vitro kinase assays and genetic and pharmacological manipulations of GSK-3 have identified more than 100 putative GSK-3 substrates in diverse cell types. Many more have been predicted on the basis of a recurrent GSK-3 consensus motif ((pS/pT)XXX(S/T)), but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. Using stable isotope labeling of amino acids in culture (SILAC) and MS techniques to analyze the repertoire of GSK-3–dependent phosphorylation in mouse embryonic stem cells (ESCs), we found that ∼2.4% of (pS/pT)XXX(S/T) sites are phosphorylated in a GSK-3–dependent manner. A comparison of WT and Gsk3a;Gsk3b knock-out (Gsk3 DKO) ESCs revealed prominent GSK-3–dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis as well as proteins that regulate transcription, translation, and cell division. Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro. RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3–dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. The MS data also identified posttranscriptional regulation of protein abundance by GSK-3, with ∼47 proteins (1.4%) whose levels increased and ∼78 (2.4%) whose levels decreased in the absence of GSK-3. This study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence that GSK-3 broadly regulates alternative splicing. PMID:28916722

  15. Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection.

    PubMed

    Dapat, Clyde; Saito, Reiko; Suzuki, Hiroshi; Horigome, Tsuneyoshi

    2014-01-22

    The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited. In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC-MS/MS. We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host-virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway

    PubMed Central

    Yang, Shuping; Li, Xin; Liu, Xinfeng; Ding, Xiangbin; Xin, Xiangbo; Jin, Congfei; Zhang, Sheng; Li, Guangpeng; Guo, Hong

    2018-01-01

    MSTN-encoded myostatin is a negative regulator of skeletal muscle development. Here, we utilized the gluteus tissues from MSTN gene editing and wild type Luxi beef cattle which are native breed of cattle in China, performed tandem mass tag (TMT) -based comparative proteomics and phosphoproteomics analyses to investigate the regulatory mechanism of MSTN related to cellular metabolism and signaling pathway in muscle development. Out of 1,315 proteins, 69 differentially expressed proteins (DEPs) were found in global proteomics analysis. Meanwhile, 149 differentially changed phosphopeptides corresponding to 76 unique phosphorylated proteins (DEPPs) were detected from 2,600 identified phosphopeptides in 702 phosphorylated proteins. Bioinformatics analyses suggested that majority of DEPs and DEPPs were closely related to glycolysis, glycogenolysis, and muscle contractile fibre processes. The global discovery results were validated by Multiple Reaction Monitoring (MRM)-based targeted peptide quantitation analysis, western blotting, and muscle glycogen content measurement. Our data revealed that increase in abundance of key enzymes and phosphorylation on their regulatory sites appears responsible for the enhanced glycogenolysis and glycolysis in MSTN−/−. The elevated glycogenolysis was assocaited with an enhanced phosphorylation of Ser1018 in PHKA1, and Ser641/Ser645 in GYS1, which were regulated by upstream phosphorylated AKT-GSK3β pathway and highly consistent with the lower glycogen content in gluteus of MSTN−/−. Collectively, this study provides new insights into the regulatory mechanisms of MSTN involved in energy metabolism and muscle growth. PMID:29541418

  17. Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection.

    PubMed

    Labots, Mariette; van der Mijn, Johannes C; Beekhof, Robin; Piersma, Sander R; de Goeij-de Haas, Richard R; Pham, Thang V; Knol, Jaco C; Dekker, Henk; van Grieken, Nicole C T; Verheul, Henk M W; Jiménez, Connie R

    2017-06-06

    Mass spectrometry-based phosphoproteomics of cancer cell and tissue lysates provides insight in aberrantly activated signaling pathways and potential drug targets. For improved understanding of individual patient's tumor biology and to allow selection of tyrosine kinase inhibitors in individual patients, phosphoproteomics of small clinical samples should be feasible and reproducible. We aimed to scale down a pTyr-phosphopeptide enrichment protocol to biopsy-level protein input and assess reproducibility and applicability to tumor needle biopsies. To this end, phosphopeptide immunoprecipitation using anti-phosphotyrosine beads was performed using 10, 5 and 1mg protein input from lysates of colorectal cancer (CRC) cell line HCT116. Multiple needle biopsies from 7 human CRC resection specimens were analyzed at the 1mg-level. The total number of phosphopeptides captured and detected by LC-MS/MS ranged from 681 at 10mg input to 471 at 1mg HCT116 protein. ID-reproducibility ranged from 60.5% at 10mg to 43.9% at 1mg. Per 1mg-level biopsy sample, >200 phosphopeptides were identified with 57% ID-reproducibility between paired tumor biopsies. Unsupervised analysis clustered biopsies from individual patients together and revealed known and potential therapeutic targets. This study demonstrates the feasibility of label-free pTyr-phosphoproteomics at the tumor biopsy level based on reproducible analyses using 1mg of protein input. The considerable number of identified phosphopeptides at this level is attributed to an effective down-scaled immuno-affinity protocol as well as to the application of ID propagation in the data processing and analysis steps. Unsupervised cluster analysis reveals patient-specific profiles. Together, these findings pave the way for clinical trials in which pTyr-phosphoproteomics will be performed on pre- and on-treatment biopsies. Such studies will improve our understanding of individual tumor biology and may enable future pTyr-phosphoproteomics

  18. Analytical challenges translating mass spectrometry-based phosphoproteomics from discovery to clinical applications

    PubMed Central

    Iliuk, Anton B.; Arrington, Justine V.; Tao, Weiguo Andy

    2014-01-01

    Phosphoproteomics is the systematic study of one of the most common protein modifications in high throughput with the aim of providing detailed information of the control, response, and communication of biological systems in health and disease. Advances in analytical technologies and strategies, in particular the contributions of high-resolution mass spectrometers, efficient enrichments of phosphopeptides, and fast data acquisition and annotation, have catalyzed dramatic expansion of signaling landscapes in multiple systems during the past decade. While phosphoproteomics is an essential inquiry to map high-resolution signaling networks and to find relevant events among the apparently ubiquitous and widespread modifications of proteome, it presents tremendous challenges in separation sciences to translate it from discovery to clinical practice. In this mini-review, we summarize the analytical tools currently utilized for phosphoproteomic analysis (with focus on MS), progresses made on deciphering clinically relevant kinase-substrate networks, MS uses for biomarker discovery and validation, and the potential of phosphoproteomics for disease diagnostics and personalized medicine. PMID:24890697

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

    PubMed Central

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

    2013-01-01

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

  20. Quantitative Phosphoproteomics Reveals SLP-76 Dependent Regulation of PAG and Src Family Kinases in T Cells

    PubMed Central

    Cao, Lulu; Ding, Yiyuan; Hung, Norris; Yu, Kebing; Ritz, Anna; Raphael, Benjamin J.; Salomon, Arthur R.

    2012-01-01

    The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway. PMID:23071622

  1. Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

    PubMed

    Cao, Lulu; Ding, Yiyuan; Hung, Norris; Yu, Kebing; Ritz, Anna; Raphael, Benjamin J; Salomon, Arthur R

    2012-01-01

    The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2010-11-01

    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.

  4. Quantitative Phosphoproteomic Analysis Provides Insight into the Response to Short-Term Drought Stress in Ammopiptanthus mongolicus Roots.

    PubMed

    Sun, Huigai; Xia, Bolin; Wang, Xue; Gao, Fei; Zhou, Yijun

    2017-10-17

    Drought is one of the major abiotic stresses that negatively affects plant growth and development. Ammopiptanthus mongolicus is an ecologically important shrub in the mid-Asia desert region and used as a model for abiotic tolerance research in trees. Protein phosphorylation participates in the regulation of various biological processes, however, phosphorylation events associated with drought stress signaling and response in plants is still limited. Here, we conducted a quantitative phosphoproteomic analysis of the response of A. mongolicus roots to short-term drought stress. Data are available via the iProx database with project ID IPX0000971000. In total, 7841 phosphorylation sites were found from the 2019 identified phosphopeptides, corresponding to 1060 phosphoproteins. Drought stress results in significant changes in the abundance of 103 phosphopeptides, corresponding to 90 differentially-phosphorylated phosphoproteins (DPPs). Motif-x analysis identified two motifs, including [pSP] and [RXXpS], from these DPPs. Functional enrichment and protein-protein interaction analysis showed that the DPPs were mainly involved in signal transduction and transcriptional regulation, osmotic adjustment, stress response and defense, RNA splicing and transport, protein synthesis, folding and degradation, and epigenetic regulation. These drought-corresponsive phosphoproteins, and the related signaling and metabolic pathways probably play important roles in drought stress signaling and response in A. mongolicus roots. Our results provide new information for understanding the molecular mechanism of the abiotic stress response in plants at the posttranslational level.

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

    PubMed

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

    2016-01-01

    Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S(88)VS(90)K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. 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

  7. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry.

    PubMed

    Swaney, Danielle L; Wenger, Craig D; Thomson, James A; Coon, Joshua J

    2009-01-27

    Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology--collision-activated dissociation (CAD)--and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors--OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved.

  8. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry

    PubMed Central

    Swaney, Danielle L.; Wenger, Craig D.; Thomson, James A.; Coon, Joshua J.

    2009-01-01

    Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology—collision-activated dissociation (CAD)—and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors—OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD (8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved. PMID:19144917

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

    PubMed Central

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

    2016-01-01

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

  10. Phosphoproteomics analyses show subnetwork systems in T-cell receptor signaling.

    PubMed

    Hatano, Atsushi; Matsumoto, Masaki; Nakayama, Keiichi I

    2016-10-01

    A key issue in the study of signal transduction is how multiple signaling pathways are systematically integrated into the cell. We have now performed multiple phosphoproteomics analyses focused on the dynamics of the T-cell receptor (TCR) signaling network and its subsystem mediated by the Ca 2+ signaling pathway. Integration of these phosphoproteomics data sets and extraction of components of the TCR signaling network dependent on Ca 2+ signaling showed unexpected phosphorylation kinetics for candidate substrates of the Ca 2+ -dependent phosphatase calcineurin (CN) during TCR stimulation. Detailed characterization of the TCR-induced phosphorylation of a novel CN substrate, Itpkb, showed that phosphorylation of this protein is regulated by both CN and the mitogen-activated protein kinase Erk in a competitive manner. Phosphorylation of additional CN substrates was also found to be regulated by Erk and CN in a similar manner. The combination of multiple phosphoproteomics approaches thus showed two major subsystems mediated by Erk and CN in the TCR signaling network, with these subsystems regulating the phosphorylation of a group of proteins in a competitive manner. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  11. Chemical Visualization of Phosphoproteomes on Membrane*

    PubMed Central

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

    2012-01-01

    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

  12. Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study.

    PubMed

    Soman, Kizhake V; Stafford, Susan J; Pazdrak, Konrad; Wu, Zheng; Luo, Xuemei; White, Wendy I; Wiktorowicz, John E; Calhoun, William J; Kurosky, Alexander

    2017-08-04

    Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

  13. Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum.

    PubMed

    Bai, Xue; Ji, Zhihong

    2012-07-01

    In this study, we employed TiO₂ enrichment and high accuracy liquid chromatography-mass spectrometry-mass spectrometry to identify the phosphoproteome of Clostridium acetobutyicum ATCC824 in acidogenesis and solventogenesis. As many as 82 phosphopeptides in 61 proteins, with 107 phosphorylated sites on serine, threonine, or tyrosine, were identified with high confidence. We detected 52 phosphopeptides from 44 proteins in acidogenesis and 70 phosphopeptides from 51 proteins in solventogenesis, respectively. Bioinformatic analysis revealed most of the phosphoproteins located in cytoplasm and participated in carbon metabolism. Based on comparison between the two stages, we found 27 stage-specific phosphorylated proteins (10 in acidogenesis and 17 in solventogenesis), some of which were solvent production-related enzymes and metabolic regulators, showed significantly different phosphorylated status. Further analysis indicated that protein phosphorylation could be involved in the shift of stages or in solvent production pathway directly. Comparison against several other organisms revealed the evolutionary diversity among them on phosphorylation level in spite of their high homology on protein sequence level.

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

    PubMed Central

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

    2015-01-01

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

  15. Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence*

    PubMed Central

    Lin, Miao-Hsia; Hsu, Tung-Li; Lin, Shu-Yu; Pan, Yi-Jiun; Jan, Jia-Tsrong; Wang, Jin-Town; Khoo, Kay-Hooi; Wu, Shih-Hsiung

    2009-01-01

    Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD50 increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD50 increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. PMID:19696081

  16. A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells.

    PubMed

    Stuart, Scott A; Houel, Stephane; Lee, Thomas; Wang, Nan; Old, William M; Ahn, Natalie G

    2015-06-01

    Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to

  17. Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.

    PubMed

    Söderholm, Sandra; Kainov, Denis E; Öhman, Tiina; Denisova, Oxana V; Schepens, Bert; Kulesskiy, Evgeny; Imanishi, Susumu Y; Corthals, Garry; Hintsanen, Petteri; Aittokallio, Tero; Saelens, Xavier; Matikainen, Sampsa; Nyman, Tuula A

    2016-10-01

    Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we

  18. Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages*

    PubMed Central

    Söderholm, Sandra; Kainov, Denis E.; Öhman, Tiina; Denisova, Oxana V.; Schepens, Bert; Kulesskiy, Evgeny; Imanishi, Susumu Y.; Corthals, Garry; Hintsanen, Petteri; Aittokallio, Tero; Saelens, Xavier; Matikainen, Sampsa; Nyman, Tuula A.

    2016-01-01

    Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase–, mitogen-activated protein kinase–, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion

  19. 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.

  20. Construction of large signaling pathways using an adaptive perturbation approach with phosphoproteomic data.

    PubMed

    Melas, Ioannis N; Mitsos, Alexander; Messinis, Dimitris E; Weiss, Thomas S; Rodriguez, Julio-Saez; Alexopoulos, Leonidas G

    2012-04-01

    Construction of large and cell-specific signaling pathways is essential to understand information processing under normal and pathological conditions. On this front, gene-based approaches offer the advantage of large pathway exploration whereas phosphoproteomic approaches offer a more reliable view of pathway activities but are applicable to small pathway sizes. In this paper, we demonstrate an experimentally adaptive approach to construct large signaling pathways from phosphoproteomic data within a 3-day time frame. Our approach--taking advantage of the fast turnaround time of the xMAP technology--is carried out in four steps: (i) screen optimal pathway inducers, (ii) select the responsive ones, (iii) combine them in a combinatorial fashion to construct a phosphoproteomic dataset, and (iv) optimize a reduced generic pathway via an Integer Linear Programming formulation. As a case study, we uncover novel players and their corresponding pathways in primary human hepatocytes by interrogating the signal transduction downstream of 81 receptors of interest and constructing a detailed model for the responsive part of the network comprising 177 species (of which 14 are measured) and 365 interactions.

  1. 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

  2. The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress.

    PubMed

    Zhong, Min; Li, Sanfeng; Huang, Fenglin; Qiu, Jiehua; Zhang, Jian; Sheng, Zhonghua; Tang, Shaoqing; Wei, Xiangjin; Hu, Peisong

    2017-09-27

    The environmental damage caused by cadmium (Cd) pollution is of increasing concern in China. While the overall plant response to Cd has been investigated in some depth, the contribution (if any) of protein phosphorylation to the detoxification of Cd and the expression of tolerance is uncertain. Here, the molecular basis of the plant response has been explored in hydroponically raised rice seedlings exposed to 10 μΜ and 100 μΜ Cd 2+ stress. An analysis of the seedlings' quantitative phosphoproteome identified 2454 phosphosites, associated with 1244 proteins. A total of 482 of these proteins became differentially phosphorylated as a result of exposure to Cd stress; the number of proteins affected in this way was six times greater in the 100 μΜ Cd 2+ treatment than in the 10 μΜ treatment. A functional analysis of the differentially phosphorylated proteins implied that a significant number was involved in signaling, in stress tolerance and in the neutralization of reactive oxygen species, while there was also a marked representation of transcription factors.

  3. The Phosphoproteomic Response of Rice Seedlings to Cadmium Stress

    PubMed Central

    Zhong, Min; Li, Sanfeng; Huang, Fenglin; Qiu, Jiehua; Sheng, Zhonghua; Tang, Shaoqing; Wei, Xiangjin; Hu, Peisong

    2017-01-01

    The environmental damage caused by cadmium (Cd) pollution is of increasing concern in China. While the overall plant response to Cd has been investigated in some depth, the contribution (if any) of protein phosphorylation to the detoxification of Cd and the expression of tolerance is uncertain. Here, the molecular basis of the plant response has been explored in hydroponically raised rice seedlings exposed to 10 μΜ and 100 μΜ Cd2+ stress. An analysis of the seedlings’ quantitative phosphoproteome identified 2454 phosphosites, associated with 1244 proteins. A total of 482 of these proteins became differentially phosphorylated as a result of exposure to Cd stress; the number of proteins affected in this way was six times greater in the 100 μΜ Cd2+ treatment than in the 10 μΜ treatment. A functional analysis of the differentially phosphorylated proteins implied that a significant number was involved in signaling, in stress tolerance and in the neutralization of reactive oxygen species, while there was also a marked representation of transcription factors. PMID:28953215

  4. Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening.

    PubMed

    Zeng, Yunliu; Pan, Zhiyong; Wang, Lun; Ding, Yuduan; Xu, Qiang; Xiao, Shunyuan; Deng, Xiuxin

    2014-02-01

    Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post-translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome-wide mapping of in vivo phosphorylation sites in chromoplast-enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide-based affinity chromatography for phosphoprotein enrichment with LC-MS/MS. A total of 109 plastid-localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif-X analysis, two distinct types of phosphorylation sites, one as proline-directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P(3) DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high-level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening. © 2013 Scandinavian Plant Physiology Society.

  5. Phosphoproteome of Pristionchus pacificus provides insights into architecture of signaling networks in nematode models.

    PubMed

    Borchert, Nadine; Krug, Karsten; Gnad, Florian; Sinha, Amit; Sommer, Ralf J; Macek, Boris

    2012-12-01

    Pristionchus pacificus is a nematode that is increasingly used as a model organism in evolutionary biology. The genome of P. pacificus differs markedly from that of C. elegans, with a high number of orphan genes that are restricted to P. pacificus and have no homologs in other species. To gain insight into the architecture of signal transduction networks in model nematodes, we performed a large-scale qualitative phosphoproteome analysis of P. pacificus. Using two-stage enrichment of phosphopeptides from a digest of P. pacificus proteins and their subsequent analysis via high accuracy MS, we detected and localized 6,809 phosphorylation events on 2,508 proteins. We compared the detected P. pacificus phosphoproteome to the recently published phosphoproteome of C. elegans. The overall numbers and functional classes of phosphoproteins were similar between the two organisms. Interestingly, the products of orphan genes were significantly underrepresented among the detected P. pacificus phosphoproteins. We defined the theoretical kinome of P. pacificus and compared it to that of C. elegans. While tyrosine kinases were slightly underrepresented in the kinome of P. pacificus, all major classes of kinases were present in both organisms. Application of our kinome annotation to a recent transcriptomic study of dauer and mixed stage populations showed that Ser/Thr and Tyr kinases show similar expression levels in P. pacificus but not in C. elegans. This study presents the first systematic comparison of phosphoproteomes and kinomes of two model nematodes and, as such, will be a useful resource for comparative studies of their signal transduction networks.

  6. Chronic Cigarette Smoke Mediated Global Changes in Lung Mucoepidermoid Cells: A Phosphoproteomic Analysis.

    PubMed

    Solanki, Hitendra S; Advani, Jayshree; Khan, Aafaque Ahmad; Radhakrishnan, Aneesha; Sahasrabuddhe, Nandini A; Pinto, Sneha M; Chang, Xiaofei; Prasad, Thottethodi Subrahmanya Keshava; Mathur, Premendu Prakash; Sidransky, David; Gowda, Harsha; Chatterjee, Aditi

    2017-08-01

    Proteomics analysis of chronic cigarette smoke exposure is a rapidly emerging postgenomics research field. While smoking is a major cause of lung cancer, functional studies using proteomics approaches could enrich our mechanistic understanding of the elusive lung cancer global molecular signaling and cigarette smoke relationship. We report in this study on a stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analysis of a human lung mucoepidermoid carcinoma cell line, H292 cells, chronically exposed to cigarette smoke. Using high resolution Orbitrap Velos mass spectrometer, we identified the hyperphosphorylation of 493 sites, which corresponds to 341 proteins and 195 hypophosphorylated sites, mapping to 142 proteins upon smoke exposure (2.0-fold change). We report differential phosphorylation of multiple kinases, including PAK6, EPHA4, LYN, mitogen-activated protein kinase, and phosphatases, including TMEM55B, PTPN14, TIGAR, among others, in response to chronic cigarette smoke exposure. Bioinformatics analysis revealed that the molecules differentially phosphorylated upon chronic exposure of cigarette smoke are associated with PI3K/AKT/mTOR and CDC42-PAK signaling pathways. These signaling networks are involved in multiple cellular processes, including cell polarity, cytoskeletal remodeling, cellular migration, protein synthesis, autophagy, and apoptosis. The present study contributes to emerging proteomics insights on cigarette smoke mediated global signaling in lung cells, which in turn may aid in development of precision medicine therapeutics and postgenomics biomarkers.

  7. Dynamic Interaction- and Phospho-Proteomics Reveal Lck as a Major Signaling Hub of CD147 in T Cells.

    PubMed

    Supper, Verena; Hartl, Ingrid; Boulègue, Cyril; Ohradanova-Repic, Anna; Stockinger, Hannes

    2017-03-15

    Numerous publications have addressed CD147 as a tumor marker and regulator of cytoskeleton, cell growth, stress response, or immune cell function; however, the molecular functionality of CD147 remains incompletely understood. Using affinity purification, mass spectrometry, and phosphopeptide enrichment of isotope-labeled peptides, we examined the dynamic of the CD147 microenvironment and the CD147-dependent phosphoproteome in the Jurkat T cell line upon treatment with T cell stimulating agents. We identified novel dynamic interaction partners of CD147 such as CD45, CD47, GNAI2, Lck, RAP1B, and VAT1 and, furthermore, found 76 CD147-dependent phosphorylation sites on 57 proteins. Using the STRING protein network database, a network between the CD147 microenvironment and the CD147-dependent phosphoproteins was generated and led to the identification of key signaling hubs around the G proteins RAP1B and GNB1, the kinases PKCβ, PAK2, Lck, and CDK1, and the chaperone HSPA5. Gene ontology biological process term analysis revealed that wound healing-, cytoskeleton-, immune system-, stress response-, phosphorylation- and protein modification-, defense response to virus-, and TNF production-associated terms are enriched within the microenvironment and the phosphoproteins of CD147. With the generated signaling network and gene ontology biological process term grouping, we identify potential signaling routes of CD147 affecting T cell growth and function. Copyright © 2017 by The American Association of Immunologists, Inc.

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

    PubMed Central

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

    2016-01-01

    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

  9. Phosphoproteomic biomarkers predicting histologic nonalcoholic steatohepatitis and fibrosis.

    PubMed

    Younossi, Zobair M; Baranova, Ancha; Stepanova, Maria; Page, Sandra; Calvert, Valerie S; Afendy, Arian; Goodman, Zachary; Chandhoke, Vikas; Liotta, Lance; Petricoin, Emanuel

    2010-06-04

    The progression of nonalcoholic fatty liver disease (NAFLD) has been linked to deregulated exchange of the endocrine signaling between adipose and liver tissue. Proteomic assays for the phosphorylation events that characterize the activated or deactivated state of the kinase-driven signaling cascades in visceral adipose tissue (VAT) could shed light on the pathogenesis of nonalcoholic steatohepatitis (NASH) and related fibrosis. Reverse-phase protein microarrays (RPMA) were used to develop biomarkers for NASH and fibrosis using VAT collected from 167 NAFLD patients (training cohort, N = 117; testing cohort, N = 50). Three types of models were developed for NASH and advanced fibrosis: clinical models, proteomics models, and combination models. NASH was predicted by a model that included measurements of two components of the insulin signaling pathway: AKT kinase and insulin receptor substrate 1 (IRS1). The models for fibrosis were less reliable when predictions were based on phosphoproteomic, clinical, or the combination data. The best performing model relied on levels of the phosphorylation of GSK3 as well as on two subunits of cyclic AMP regulated protein kinase A (PKA). Phosphoproteomics technology could potentially be used to provide pathogenic information about NASH and NASH-related fibrosis. This information can lead to a clinically relevant diagnostic/prognostic biomarker for NASH.

  10. Evaluation of Quantitative Performance of Sequential Immobilized Metal Affinity Chromatographic Enrichment for Phosphopeptides

    PubMed Central

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

    2014-01-01

    We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium-dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. While our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semi-quantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. PMID:24096195

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis. © 2016 WILEY

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

    PubMed

    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

    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. 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. 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.

  14. 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

  15. 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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

    Adams, Nancy E.; Maurelli, Anthony T.

    2015-01-01

    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

  18. 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.

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

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-05

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

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

    PubMed Central

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

    2014-01-01

    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

  1. 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

  2. Quantitative- and Phospho-Proteomic Analysis of the Yeast Response to the Tyrosine Kinase Inhibitor Imatinib to Pharmacoproteomics-Guided Drug Line Extension

    PubMed Central

    dos Santos, Sandra C.; Mira, Nuno P.; Moreira, Ana S.

    2012-01-01

    Abstract Imatinib mesylate (IM) is a potent tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, a disease caused by the oncogenic kinase Bcr-Abl. Although the clinical success of IM set a new paradigm in molecular-targeted therapy, the emergence of IM resistance is a clinically significant problem. In an effort to obtain new insights into the mechanisms of adaptation and tolerance to IM, as well as the signaling pathways potentially affected by this drug, we performed a two-dimensional electrophoresis-based quantitative- and phospho-proteomic analysis in the eukaryotic model Saccharomyces cerevisiae. We singled out proteins that were either differentially expressed or differentially phosphorylated in response to IM, using the phosphoselective dye Pro-Q® Diamond, and identified 18 proteins in total. Ten were altered only at the content level (mostly decreased), while the remaining 8 possessed IM-repressed phosphorylation. These 18 proteins are mainly involved in cellular carbohydrate processes (glycolysis/gluconeogenesis), translation, protein folding, ion homeostasis, and nucleotide and amino acid metabolism. Remarkably, all 18 proteins have human functional homologs. A role for HSP70 proteins in the response to IM, as well as decreased glycolysis as a metabolic marker of IM action are suggested, consistent with findings from studies in human cell lines. The previously-proposed effect of IM as an inhibitor of vacuolar H+-ATPase function was supported by the identification of an underexpressed protein subunit of this complex. Taken together, these findings reinforce the role of yeast as a valuable eukaryotic model for pharmacological studies and identification of new drug targets, with potential clinical implications in drug reassignment or line extension under a personalized medicine perspective. PMID:22775238

  3. Phosphoproteomics links glycogen synthase kinase-3 to RNA splicing.

    PubMed

    Khoa, Le Tran Phuc; Dou, Yali

    2017-11-03

    Protein kinases play essential biological roles by phosphorylating a diverse range of signaling molecules, but deciphering their direct physiological targets remains a challenge. A new study by Shinde et al. uses phosphoproteomics to identify glycogen synthase kinase-3 (GSK-3) substrates in mouse embryonic stem cells (mESCs), providing a broad profile of GSK-3 activity and defining a new role for this central kinase in regulating RNA splicing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. 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

  5. Proteome and phosphoproteome analysis of commensally induced dendritic cell maturation states.

    PubMed

    Korkmaz, Ali Giray; Popov, Todor; Peisl, Loulou; Codrea, Marius Cosmin; Nahnsen, Sven; Steimle, Alexander; Velic, Ana; Macek, Boris; von Bergen, Martin; Bernhardt, Joerg; Frick, Julia-Stefanie

    2018-05-30

    Dendritic cells (DCs) can shape the immune system towards an inflammatory or tolerant state depending on the bacterial antigens and the environment they encounter. In this study we provide a proteomic catalogue of differentially expressed proteins between distinct DC maturation states, brought about by bacteria that differ in their endotoxicity. To achieve this, we have performed proteomics and phosphoproteomics on murine DC cultures. Symbiont and pathobiont bacteria were used to direct dendritic cells into a semi-mature and fully-mature state, respectively. The comparison of semi-mature and fully-mature DCs revealed differential expression in 103 proteins and differential phosphorylation in 118 phosphosites, including major regulatory factors of central immune processes. Our analyses predict that these differences are mediated by upstream elements such as SOCS1, IRF3, ABCA1, TLR4, and PTGER4. Our analyses indicate that the symbiont bacterial strain affects DC proteome in a distinct way, by downregulating inflammatory proteins and activating anti-inflammatory upstream regulators. Biological significance In this study we have investigated the responses of immune cells to distinct bacterial stimuli. We have used the symbiont bacterial strain B. vulgatus and the pathobiont E. coli strain to stimulate cultured primary dendritic cells and performed a shotgun proteome analysis to investigate the protein expression and phosphorylation level differences on a genome level. We have observed expression and phosphorylation level differences in key immune regulators, transcription factors and signal transducers. Moreover, our subsequent bioinformatics analysis indicated regulation at several signaling pathways such as PPAR signaling, LXR/RXR activation and glucocorticoid signaling pathways, which are not studied in detail in an inflammation and DC maturation context. Our phosphoproteome analysis showed differential phosphorylation in 118 phosphosites including those belonging

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

    PubMed Central

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

    2015-01-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

  7. Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach.

    PubMed

    Heerma van Voss, Marise R; Kammers, Kai; Vesuna, Farhad; Brilliant, Justin; Bergman, Yehudit; Tantravedi, Saritha; Wu, Xinyan; Cole, Robert N; Holland, Andrew; van Diest, Paul J; Raman, Venu

    2018-06-01

    DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-01-01

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

  9. Phosphoproteomic Analysis Identifies Signaling Pathways Regulated by Curcumin in Human Colon Cancer Cells.

    PubMed

    Sato, Tatsuhiro; Higuchi, Yutaka; Shibagaki, Yoshio; Hattori, Seisuke

    2017-09-01

    Curcumin, a major polyphenol of the spice turmeric, acts as a potent chemopreventive and chemotherapeutic agent in several cancer types, including colon cancer. Although various proteins have been shown to be affected by curcumin, how curcumin exerts its anticancer activity is not fully understood. Phosphoproteomic analyses were performed using SW480 and SW620 human colon cancer cells to identify curcumin-affected signaling pathways. Curcumin inhibited the growth of the two cell lines in a dose-dependent manner. Thirty-nine curcumin-regulated phosphoproteins were identified, five of which are involved in cancer signaling pathways. Detailed analyses revealed that the mTORC1 and p53 signaling pathways are main targets of curcumin. Our results provide insight into the molecular mechanisms of the anticancer activities of curcumin and future molecular targets for its clinical application. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  10. A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations.

    PubMed

    Litichevskiy, Lev; Peckner, Ryan; Abelin, Jennifer G; Asiedu, Jacob K; Creech, Amanda L; Davis, John F; Davison, Desiree; Dunning, Caitlin M; Egertson, Jarrett D; Egri, Shawn; Gould, Joshua; Ko, Tak; Johnson, Sarah A; Lahr, David L; Lam, Daniel; Liu, Zihan; Lyons, Nicholas J; Lu, Xiaodong; MacLean, Brendan X; Mungenast, Alison E; Officer, Adam; Natoli, Ted E; Papanastasiou, Malvina; Patel, Jinal; Sharma, Vagisha; Toder, Courtney; Tubelli, Andrew A; Young, Jennie Z; Carr, Steven A; Golub, Todd R; Subramanian, Aravind; MacCoss, Michael J; Tsai, Li-Huei; Jaffe, Jacob D

    2018-04-25

    Although the value of proteomics has been demonstrated, cost and scale are typically prohibitive, and gene expression profiling remains dominant for characterizing cellular responses to perturbations. However, high-throughput sentinel assays provide an opportunity for proteomics to contribute at a meaningful scale. We present a systematic library resource (90 drugs × 6 cell lines) of proteomic signatures that measure changes in the reduced-representation phosphoproteome (P100) and changes in epigenetic marks on histones (GCP). A majority of these drugs elicited reproducible signatures, but notable cell line- and assay-specific differences were observed. Using the "connectivity" framework, we compared signatures across cell types and integrated data across assays, including a transcriptional assay (L1000). Consistent connectivity among cell types revealed cellular responses that transcended lineage, and consistent connectivity among assays revealed unexpected associations between drugs. We further leveraged the resource against public data to formulate hypotheses for treatment of multiple myeloma and acute lymphocytic leukemia. This resource is publicly available at https://clue.io/proteomics. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Temporal dynamics of the Saccharopolyspora erythraea phosphoproteome.

    PubMed

    Licona-Cassani, Cuauhtemoc; Lim, Sooa; Marcellin, Esteban; Nielsen, Lars K

    2014-05-01

    Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest ("metabolic switch") preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO(2) enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE_6340/NC_009142_6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE_1406/NC_009142_1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE_7351/NC_009142_7233) and S32 (SACE_6101/NC_009142_5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of

  12. Temporal Dynamics of the Saccharopolyspora erythraea Phosphoproteome*

    PubMed Central

    Licona-Cassani, Cuauhtemoc; Lim, SooA; Marcellin, Esteban; Nielsen, Lars K.

    2014-01-01

    Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest (“metabolic switch”) preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO2 enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE_6340/NC_009142_6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE_1406/NC_009142_1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE_7351/NC_009142_7233) and S32 (SACE_6101/NC_009142_5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of

  13. Age-Dependent Effects of Acute Alcohol Administration in the Hippocampal Phosphoproteome.

    PubMed

    Contreras, Ana; Morales, Lidia; Tebourbi, Ali; Miguéns, Miguel; Olmo, Nuria Del; Pérez-García, Carmen

    2017-12-18

    Alcohol consumption during adolescence is deleterious to the developing brain and leads to persistent deficits in adulthood. Several results provide strong evidence for ethanol-associated alterations in glutamatergic signaling and impaired synaptic plasticity in the hippocampus. Protein phosphorylation is a well-known and well-documented mechanism in memory processes, but information on phosphoprotein alterations in hippocampus after ethanol exposure is limited. This study focuses on age-related changes in the hippocampal phosphoproteome after acute alcohol administration. We have compared the phosphoprotein expression in the hippocampus of adult and adolescent Wistar rats treated with a single dose of ethanol (5 g/kg i.p.), using a proteomic approach including phosphoprotein enrichment by immobilized metal affinity chromatography (IMAC). Our proteomic analysis revealed that 13 proteins were differentially affected by age, ethanol administration, or both. Most of these proteins are involved in neuroprotection and are expressed less in young rats treated with ethanol. We conclude that acute alcohol induces important changes in the expression of phosphoproteins in the hippocampus that could increase the risk of neurodegenerative disorders, especially when the alcohol exposure begins in adolescence.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.

    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 cellularmore » 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.« less

  15. Is Phosphoproteomics Ready for Clinical Research?

    PubMed Central

    Iliuk, Anton B.; Tao, W. Andy

    2012-01-01

    Background For many diseases such as cancer where phosphorylation-dependent signaling is the foundation of disease onset and progression, single-gene testing and genomic profiling alone are not sufficient in providing most critical information. The reason for this is that in these activated pathways the signaling changes and drug resistance are often not directly correlated with changes in protein expression levels. In order to obtain the essential information needed to evaluate pathway activation or the effects of certain drugs and therapies on the molecular level, the analysis of changes in protein phosphorylation is critical. Methods Existing approaches do not differentiate clinical disease subtypes on the protein and signaling pathway level, and therefore hamper the predictive management of the disease and the selection of therapeutic targets. Conclusions The mini-review examines the impact of emerging systems biology tools and the possibility of applying phosphoproteomics to clinical research. PMID:23159844

  16. Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells.

    PubMed

    Weber, Christoph; Schreiber, Thiemo B; Daub, Henrik

    2012-02-02

    Small molecule inhibitors of protein kinases have emerged as a major class of therapeutic agents for the treatment of hematological malignancies. Both in vitro studies and patient case reports suggest therapeutic potential of the clinical kinase inhibitors erlotinib and gefitinib in acute myeloid leukemia (AML). The drugs' cellular modes of action in AML warrant further investigation as their primary therapeutic target, the epidermal growth factor receptor, is not expressed. We therefore performed SILAC-based quantitative mass spectrometry analyses to a depth of 10,975 distinct phosphorylation sites to characterize the phosphoproteome of KG1 AML cells and its regulation upon erlotinib and gefitinib treatment. Less than 50 site-specific phosphorylations changed significantly, indicating rather specific interference with AML cell signaling. Many drug-induced changes occurred within a network of tyrosine phosphorylated proteins that included Src family kinases (SFKs) and the tyrosine kinases Btk and Syk. We further performed quantitative chemical proteomics in KG1 cell extracts and identified SFKs and Btk as direct cellular targets of both erlotinib and gefitinib. Taken together, our data suggest that cellular perturbation of SFKs and/or Btk translates into rather specific signal transduction inhibition, which in turn contributes to the antileukemic activity of erlotinib and gefitinib in AML. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Global Phosphoproteomics of Activated B Cells Using Complementary Metal Ion Functionalized Soluble Nanopolymers

    PubMed Central

    2015-01-01

    Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration. PMID:24905233

  18. Quantitative Analysis of Signaling Networks across Differentially Embedded Tumors Highlights Interpatient Heterogeneity in Human Glioblastoma

    PubMed Central

    2015-01-01

    Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor, with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a more physiologically relevant view of oncogenic signaling networks. However, clinical samples are not widely available and may be embedded for histopathologic analysis. With the goal of accurately identifying activated signaling networks in GBM tumor samples, we investigated the impact of embedding in optimal cutting temperature (OCT) compound followed by flash freezing in LN2 vs immediate flash freezing (iFF) in LN2 on protein expression and phosphorylation-mediated signaling networks. Quantitative proteomic and phosphoproteomic analysis of 8 pairs of tumor specimens revealed minimal impact of the different sample processing strategies and highlighted the large interpatient heterogeneity present in these tumors. Correlation analyses of the differentially processed tumor sections identified activated signaling networks present in selected tumors and revealed the differential expression of transcription, translation, and degradation associated proteins. This study demonstrates the capability of quantitative mass spectrometry for identification of in vivo oncogenic signaling networks from human tumor specimens that were either OCT-embedded or immediately flash-frozen. PMID:24927040

  19. Phosphoproteomic analysis of the posterior silk gland of Bombyx mori provides novel insight into phosphorylation regulating the silk production.

    PubMed

    Song, Jia; Che, Jiaqian; You, Zhengying; Ye, Lupeng; Li, Jisheng; Zhang, Yuyu; Qian, Qiujie; Zhong, Boxiong

    2016-10-04

    To understand phosphorylation event regulating silk synthesis in the posterior silk gland of Bombyx mori, phosphoproteome was profiled in a pair of near-isogenic lines, a normally cocooning strain (IC) and a nakedly pupated strain (IN) that the silk production is much lower than IC. In the posterior silk gland of the IC and IN, 714 and 658 phosphosites resided on 554 and 507 phosphopeptides from 431 and 383 phosphoproteins, were identified, respectively. Of all the phosphosites, the single phosphosite was the dominate phosphorylation form, comprising>60% of all the phosphosites in two phenotypic of silk production. All these phosphosites were classified as acidophilic and proline-directed kinase classes, and three motifs were uniquely identified in the IC. The motif S-P-P might be important for regulating phosphorylation network of silk protein synthesis. The dynamically phosphorylated proteins participated in ribosome, protein transport and energy metabolism suggest that phosphorylation may play key roles in regulating silk protein synthesis and secretion. Furthermore, fibroin heavy chain, an important component of silk protein, was specifically phosphorylated in the IC strain, suggesting its role to ensure the normal formation of silk structure and silk secretion. The data gain new understanding of the regulatory processes of silk protein synthesis and offer as starting point for further research on the silk production at phosphoproteome level. Despite the knowledge on regulation of silk protein synthesis in the posterior silk gland has gained at the gene or protein levels, how phosphorylation event influences the silk yield is largely unknown. To this end, we constructed a pair of silkworm near-isogenic lines that showed different cocooning phenotypes, and the phosphoproteome of the posterior silk gland of two isolines was compared. Here, we reported the first phosphoproteome data on the silkworm and found several key pathways related protein synthesis are

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang, Zhenlie; Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550; Ichihara, Sahoko

    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.more » Changes in selected proteins were further confirmed by Manganese II (Mn{sup 2+})-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p < 0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn{sup 2+}-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity. - Highlights: • 1-BP modified hippocampal phosphoproteome in rat and 23 altered proteins were identified. • 1-BP changed

  1. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

    PubMed Central

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

    2015-01-01

    Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. IKAP: A heuristic framework for inference of kinase activities from Phosphoproteomics data.

    PubMed

    Mischnik, Marcel; Sacco, Francesca; Cox, Jürgen; Schneider, Hans-Christoph; Schäfer, Matthias; Hendlich, Manfred; Crowther, Daniel; Mann, Matthias; Klabunde, Thomas

    2016-02-01

    Phosphoproteomics measurements are widely applied in cellular biology to detect changes in signalling dynamics. However, due to the inherent complexity of phosphorylation patterns and the lack of knowledge on how phosphorylations are related to functions, it is often not possible to directly deduce protein activities from those measurements. Here, we present a heuristic machine learning algorithm that infers the activities of kinases from Phosphoproteomics data using kinase-target information from the PhosphoSitePlus database. By comparing the estimated kinase activity profiles to the measured phosphosite profiles, it is furthermore possible to derive the kinases that are most likely to phosphorylate the respective phosphosite. We apply our approach to published datasets of the human cell cycle generated from HeLaS3 cells, and insulin signalling dynamics in mouse hepatocytes. In the first case, we estimate the activities of 118 at six cell cycle stages and derive 94 new kinase-phosphosite links that can be validated through either database or motif information. In the second case, the activities of 143 kinases at eight time points are estimated and 49 new kinase-target links are derived. The algorithm is implemented in Matlab and be downloaded from github. It makes use of the Optimization and Statistics toolboxes. https://github.com/marcel-mischnik/IKAP.git. marcel.mischnik@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions*

    PubMed Central

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

    2017-01-01

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

  6. Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions.

    PubMed

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

    2017-05-01

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

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

    PubMed

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

    2014-12-05

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

  8. Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.

    PubMed

    Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

    2016-04-12

    Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-07-01

    This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Feng; Waters, Katrina M.; Miller, John H.

    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 peakmore » 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-20

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

  13. Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes.

    PubMed

    Zhang, Yuanyuan; Zhang, Yajie; Yu, Yonghao

    2017-08-04

    Insulin resistance is a hallmark of type 2 diabetes. Although multiple genetic and physiological factors interact to cause insulin resistance, deregulated signaling by phosphorylation is a common underlying mechanism. In particular, the specific phosphorylation-dependent regulatory mechanisms and signaling outputs of insulin are poorly understood in hepatocytes, which represents one of the most important insulin-responsive cell types. Using primary rat hepatocytes as a model system, we performed reductive dimethylation (ReDi)-based quantitative mass spectrometric analysis and characterized the phosphoproteome that is regulated by insulin as well as its key downstream kinases including Akt, mTORC1, and S6K. We identified a total of 12 294 unique, confidently localized phosphorylation sites and 3805 phosphorylated proteins in this single cell type. Detailed bioinformatic analysis on each individual data set identified both known and previously unrecognized targets of this key insulin downstream effector pathway. Furthermore, integrated analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the delineation of the substrate specificity of several close-related kinases within the insulin signaling pathway. We expect that the data sets will serve as an invaluable resource, providing the foundation for future hypothesis-driven research that helps delineate the molecular mechanisms that underlie the pathogenesis of type 2 diabetes and related metabolic syndrome.

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

    PubMed

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

    2015-01-01

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

  15. 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

  16. Phosphoproteomics and Bioinformatics Analyses of Spinal Cord Proteins in Rats with Morphine Tolerance

    PubMed Central

    Liaw, Wen-Jinn; Tsao, Cheng-Ming; Huang, Go-Shine; Wu, Chin-Chen; Ho, Shung-Tai; Wang, Jhi-Joung; Tao, Yuan-Xiang; Shui, Hao-Ai

    2014-01-01

    Introduction Morphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance. Methods To better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL) for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins. Results Our proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism. Conclusions Repeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance. PMID:24392096

  17. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

    PubMed

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  18. Trespassing cancer cells: ‘fingerprinting’ invasive protrusions reveals metastatic culprits

    PubMed Central

    Klemke, Richard L.

    2012-01-01

    Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial “omics” approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. PMID:22980730

  19. Trespassing cancer cells: 'fingerprinting' invasive protrusions reveals metastatic culprits.

    PubMed

    Klemke, Richard L

    2012-10-01

    Metastatic cancer cells produce invasive membrane protrusions called invadopodia and pseudopodia, which play a central role in driving cancer cell dissemination in the body. Malignant cells use these structures to attach to and degrade extracellular matrix proteins, generate force for cell locomotion, and to penetrate the vasculature. Recent work using unique subcellular fractionation methodologies combined with spatial genomic, proteomic, and phosphoproteomic profiling has provided insight into the invadopodiome and pseudopodiome signaling networks that control the protrusion of invasive membranes. Here I highlight how these powerful spatial 'omics' approaches reveal important signatures of metastatic cancer cells and possible new therapeutic targets aimed at treating metastatic disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. 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

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

    PubMed Central

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

    2013-01-01

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

  2. In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling*

    PubMed Central

    Boersema, Paul J.; Foong, Leong Yan; Ding, Vanessa M. Y.; Lemeer, Simone; van Breukelen, Bas; Philp, Robin; Boekhorst, Jos; Snel, Berend; den Hertog, Jeroen; Choo, Andre B. H.; Heck, Albert J. R.

    2010-01-01

    Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative

  3. Quantitative Proteomics Reveals Fundamental Regulatory Differences in Oncogenic HRAS and Isocitrate Dehydrogenase (IDH1) Driven Astrocytoma.

    PubMed

    Doll, Sophia; Urisman, Anatoly; Oses-Prieto, Juan A; Arnott, David; Burlingame, Alma L

    2017-01-01

    Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our

  4. Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions†

    PubMed Central

    Naegle, Kristen M.; White, Forest M.; Lauffenburger, Douglas A.; Yaffe, Michael B.

    2012-01-01

    Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein–protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein–protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein–protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein–protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein–protein interactions in a dynamic context- and phosphorylation site-specific manner. PMID:22851037

  5. Proteomics/phosphoproteomics of left ventricular biopsies from patients with surgical coronary revascularization and pigs with coronary occlusion/reperfusion: remote ischemic preconditioning.

    PubMed

    Gedik, Nilgün; Krüger, Marcus; Thielmann, Matthias; Kottenberg, Eva; Skyschally, Andreas; Frey, Ulrich H; Cario, Elke; Peters, Jürgen; Jakob, Heinz; Heusch, Gerd; Kleinbongard, Petra

    2017-08-09

    Remote ischemic preconditioning (RIPC) by repeated brief cycles of limb ischemia/reperfusion reduces myocardial ischemia/reperfusion injury. In left ventricular (LV) biopsies from patients undergoing coronary artery bypass grafting (CABG), only the activation of signal transducer and activator of transcription 5 was associated with RIPC's cardioprotection. We have now used an unbiased, non-hypothesis-driven proteomics and phosphoproteomics approach to analyze LV biopsies from patients undergoing CABG and from pigs undergoing coronary occlusion/reperfusion without (sham) and with RIPC. False discovery rate-based statistics identified a higher prostaglandin reductase 2 expression at early reperfusion with RIPC than with sham in patients. In pigs, the phosphorylation of 116 proteins was different between baseline and early reperfusion with RIPC and/or with sham. The identified proteins were not identical for patients and pigs, but in-silico pathway analysis of proteins with ≥2-fold higher expression/phosphorylation at early reperfusion with RIPC in comparison to sham revealed a relation to mitochondria and cytoskeleton in both species. Apart from limitations of the proteomics analysis per se, the small cohorts, the sampling/sample processing and the number of uncharacterized/unverifiable porcine proteins may have contributed to this largely unsatisfactory result.

  6. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics

    PubMed Central

    Bennett, Eric J.; Rush, John; Gygi, Steven P.; Harper, J. Wade

    2010-01-01

    Dynamic reorganization of signaling systems frequently accompany pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex Absolute Quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules while only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. PMID:21145461

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

    PubMed

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

    2016-02-01

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

  8. Integration of Proteomic, Transcriptional, and Interactome Data Reveals Hidden Signaling Components

    PubMed Central

    Huang, Shao-shan Carol; Fraenkel, Ernest

    2009-01-01

    Cellular signaling and regulatory networks underlie fundamental biological processes such as growth, differentiation, and response to the environment. Although there are now various high-throughput methods for studying these processes, knowledge of them remains fragmentary. Typically, the vast majority of hits identified by transcriptional, proteomic, and genetic assays lie outside of the expected pathways. These unexpected components of the cellular response are often the most interesting, because they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree problem, that uses previously reported protein-protein and protein-DNA interactions to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. Applied simultaneously to phosphoproteomic and transcriptional data for the yeast pheromone response, it identifies changes in diverse cellular processes that extend far beyond the expected pathways. PMID:19638617

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

    PubMed Central

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

    2016-01-01

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

  10. A systematic understanding of signaling by ErbB2 in cancer using phosphoproteomics.

    PubMed

    Sidhanth, C; Manasa, P; Krishnapriya, S; Sneha, S; Bindhya, S; Nagare, R P; Garg, M; Ganesan, T S

    2018-06-01

    ErbB2 is an important receptor tyrosine kinase and a member of the ErbB family. Although it does not have a specific ligand, it transmits signals downstream by heterodimerization with other receptors in the family. It plays a major role in a variety of cellular responses like proliferation, differentiation, and adhesion. ErbB2 is amplified at the DNA level in breast cancer (20%-30%) and gastric cancer (10%-20%), and trastuzumab is effective as a therapeutic antibody. This review is a critical analysis of the currently published data on the signaling pathways of ErbB2 and the interacting proteins. It also focuses on the techniques that are currently available to evaluate the entire phosphoproteome following activation of ErbB2. Identification of new and relevant phosphoproteins can not only serve as new therapeutic targets but also as a surrogate marker in patients to assess the activity of compounds that inhibit ErbB2. Overall, such analysis will improve understanding of signaling by ErbB2.

  11. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.

    PubMed

    Bennett, Eric J; Rush, John; Gygi, Steven P; Harper, J Wade

    2010-12-10

    Dynamic reorganization of signaling systems frequently accompanies pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex absolute quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules, whereas only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2010-02-05

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

  13. Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

    PubMed

    Chu, Trang T T; Sinha, Ameya; Malleret, Benoit; Suwanarusk, Rossarin; Park, Jung E; Naidu, Renugah; Das, Rupambika; Dutta, Bamaprasad; Ong, Seow Theng; Verma, Navin K; Chan, Jerry K; Nosten, François; Rénia, Laurent; Sze, Siu K; Russell, Bruce; Chandramohanadas, Rajesh

    2018-01-01

    Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes. © 2017 John Wiley & Sons Ltd.

  14. Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy

    PubMed Central

    Kuzmanov, Uros; Guo, Hongbo; Buchsbaum, Diana; Cosme, Jake; Abbasi, Cynthia; Isserlin, Ruth; Sharma, Parveen; Gramolini, Anthony O.; Emili, Andrew

    2016-01-01

    Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function. PMID:27742792

  15. Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58

    PubMed Central

    Hareza, Agnieszka; Bakun, Magda; Świderska, Bianka; Dudkiewicz, Małgorzata; Koscielny, Alicja; Bajur, Anna; Jaworski, Jacek

    2018-01-01

    Many kinases are still ‘orphans,’ which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography–tandem mass spectrometry proteomic approach with phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK293T cells, and phosphosites dependent on the presence of DIA1 are elucidated. Most of these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, the proteomics of proteins immunoprecipitated with DIA1 reported its probable interactors. This pilot study provides the basis for deeper studies of DIA1 signalling. PMID:29666759

  16. Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58.

    PubMed

    Hareza, Agnieszka; Bakun, Magda; Świderska, Bianka; Dudkiewicz, Małgorzata; Koscielny, Alicja; Bajur, Anna; Jaworski, Jacek; Dadlez, Michał; Pawłowski, Krzysztof

    2018-01-01

    Many kinases are still 'orphans,' which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography-tandem mass spectrometry proteomic approach with phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK293T cells, and phosphosites dependent on the presence of DIA1 are elucidated. Most of these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, the proteomics of proteins immunoprecipitated with DIA1 reported its probable interactors. This pilot study provides the basis for deeper studies of DIA1 signalling.

  17. Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways*

    PubMed Central

    Rispal, Delphine; Eltschinger, Sandra; Stahl, Michael; Vaga, Stefania; Bodenmiller, Bernd; Abraham, Yann; Filipuzzi, Ireos; Movva, N. Rao; Aebersold, Ruedi; Helliwell, Stephen B.; Loewith, Robbie

    2015-01-01

    Target of rapamycin is a Ser/Thr kinase that operates in two conserved multiprotein complexes, TORC1 and TORC2. Unlike TORC1, TORC2 is insensitive to rapamycin, and its functional characterization is less advanced. Previous genetic studies demonstrated that TORC2 depletion leads to loss of actin polarization and loss of endocytosis. To determine how TORC2 regulates these readouts, we engineered a yeast strain in which TORC2 can be specifically and acutely inhibited by the imidazoquinoline NVP-BHS345. Kinetic analyses following inhibition of TORC2, supported with quantitative phosphoproteomics, revealed that TORC2 regulates these readouts via distinct pathways as follows: rapidly through direct protein phosphorylation cascades and slowly through indirect changes in the tensile properties of the plasma membrane. The rapid signaling events are mediated in large part through the phospholipid flippase kinases Fpk1 and Fpk2, whereas the slow signaling pathway involves increased plasma membrane tension resulting from a gradual depletion of sphingolipids. Additional hits in our phosphoproteomic screens highlight the intricate control TORC2 exerts over diverse aspects of eukaryote cell physiology. PMID:25882841

  18. Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

    PubMed Central

    Degryse, S; de Bock, C E; Demeyer, S; Govaerts, I; Bornschein, S; Verbeke, D; Jacobs, K; Binos, S; Skerrett-Byrne, D A; Murray, H C; Verrills, N M; Van Vlierberghe, P; Cools, J; Dun, M D

    2018-01-01

    Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens. PMID:28852199

  19. Phosphoproteome of the Oleaginous Green Alga, Chlorella vulgaris UTEX 395, under Nitrogen-Replete and -Deplete Conditions

    DOE PAGES

    Guarnieri, Michael T.; Gerritsen, Alida T.; Henard, Calvin A.; ...

    2018-03-06

    The unicellular green alga, Chlorella vulgaris UTEX 395, represents a promising biocatalyst for renewable biofuel production due to its relatively rapid growth rate and high lipid accumulation capacity (Guarnieri et al., 2011, 2012; Gerken et al., 2013; Griffiths et al., 2014; Zuniga et al., 2016). Prior analyses have unveiled the global proteome dynamics of C. vulgaris following nitrogen depletion, which induces a high lipid accumulation phenotype (Guarnieri et al., 2011, 2013). More recently, we have reported a draft genome, genome-scale model, and nitrosoproteome for this alga (Zuniga et al., 2016; Henard et al., 2017)1 providing further insight into lipid biosynthetic-,more » nutrient response-, and post-transcriptional-regulatory mechanisms. To further our understanding of these regulatory mechanisms and expand the knowledge base surrounding this organism, comparative phosphoproteomic analyses were conducted under nitrogen-replete and -deplete conditions to identify differentially phosphorylated proteins that will aid in the evaluation of the potential role of phosphoregulation in lipogenesis.« less

  20. Phosphoproteome of the Oleaginous Green Alga, Chlorella vulgaris UTEX 395, under Nitrogen-Replete and -Deplete Conditions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guarnieri, Michael T.; Gerritsen, Alida T.; Henard, Calvin A.

    The unicellular green alga, Chlorella vulgaris UTEX 395, represents a promising biocatalyst for renewable biofuel production due to its relatively rapid growth rate and high lipid accumulation capacity (Guarnieri et al., 2011, 2012; Gerken et al., 2013; Griffiths et al., 2014; Zuniga et al., 2016). Prior analyses have unveiled the global proteome dynamics of C. vulgaris following nitrogen depletion, which induces a high lipid accumulation phenotype (Guarnieri et al., 2011, 2013). More recently, we have reported a draft genome, genome-scale model, and nitrosoproteome for this alga (Zuniga et al., 2016; Henard et al., 2017)1 providing further insight into lipid biosynthetic-,more » nutrient response-, and post-transcriptional-regulatory mechanisms. To further our understanding of these regulatory mechanisms and expand the knowledge base surrounding this organism, comparative phosphoproteomic analyses were conducted under nitrogen-replete and -deplete conditions to identify differentially phosphorylated proteins that will aid in the evaluation of the potential role of phosphoregulation in lipogenesis.« less

  1. Fibrotic-like changes in degenerate human intervertebral discs revealed by quantitative proteomic analysis.

    PubMed

    Yee, A; Lam, M P Y; Tam, V; Chan, W C W; Chu, I K; Cheah, K S E; Cheung, K M C; Chan, D

    2016-03-01

    Intervertebral disc degeneration (IDD) can lead to symptomatic conditions including sciatica and back pain. The purpose of this study is to understand the extracellular matrix (ECM) changes in disc biology through comparative proteomic analysis of degenerated and non-degenerated human intervertebral disc (IVD) tissues of different ages. Seven non-degenerated (11-46 years of age) and seven degenerated (16-53 years of age) annulus fibrosus (AF) and nucleus pulposus (NP) samples were used. Proteins were extracted using guanidine hydrochloride, separated from large proteoglycans (PGs) by caesium chloride (CsCl) density gradient ultracentrifugation, and identified using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). For quantitative comparison, proteins were labeled with iTRAQ reagents. Collagen fibrils in the NP were assessed using scanning electron microscopy (SEM). In the AF, quantitative analysis revealed increased levels of HTRA1, COMP and CILP in degeneration when compared with samples from older individuals. Fibronectin showed increment with age and degeneration. In the NP, more CILP and CILP2 were present in degenerated samples of younger individuals. Reduced protein solubility was observed in degenerated and older non-degenerated samples correlated with an accumulation of type I collagen in the insoluble fibers. Characterization of collagen fibrils in the NP revealed smaller mean fibril diameters and decreased porosity in the degenerated samples. Our study identified distinct matrix changes associated with aging and degeneration in the intervertebral discs (IVDs). The nature of the ECM changes, together with observed decreased in solubility and changes in fibril diameter is consistent with a fibrotic-like environment. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  2. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yingchun; Yang, Feng; Fu, Yi

    Abstract - Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signalingmore » pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.« less

  3. Quantitative proteomics reveal a feedforward mechanism for mitochondrial PARKIN translocation and ubiquitin chain synthesis.

    PubMed

    Ordureau, Alban; Sarraf, Shireen A; Duda, David M; Heo, Jin-Mi; Jedrychowski, Mark P; Sviderskiy, Vladislav O; Olszewski, Jennifer L; Koerber, James T; Xie, Tiao; Beausoleil, Sean A; Wells, James A; Gygi, Steven P; Schulman, Brenda A; Harper, J Wade

    2014-11-06

    Phosphorylation is often used to promote protein ubiquitylation, yet we rarely understand quantitatively how ligase activation and ubiquitin (UB) chain assembly are integrated with phosphoregulation. Here we employ quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway disrupted in Parkinson's disease. PINK1 plays a dual role by phosphorylating PARKIN on its UB-like domain and poly-UB chains on mitochondria. PARKIN activation by PINK1 produces canonical and noncanonical UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB (poly-p-UB) on mitochondria. In vitro, PINK1 directly activates PARKIN's ability to assemble canonical and noncanonical UB chains and promotes association of PARKIN with both p-UB and poly-p-UB. Our data reveal a feedforward mechanism that explains how PINK1 phosphorylation of both PARKIN and poly-UB chains synthesized by PARKIN drives a program of PARKIN recruitment and mitochondrial ubiquitylation in response to mitochondrial damage. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

    PubMed

    Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

    2016-10-07

    Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

  5. Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth.

    PubMed

    Sankar, Martial; Nieminen, Kaisa; Ragni, Laura; Xenarios, Ioannis; Hardtke, Christian S

    2014-02-11

    Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001.

  6. Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth

    PubMed Central

    Sankar, Martial; Nieminen, Kaisa; Ragni, Laura; Xenarios, Ioannis; Hardtke, Christian S

    2014-01-01

    Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001 PMID:24520159

  7. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  8. Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

    PubMed Central

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-01-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  9. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

    PubMed

    Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

    2018-06-01

    Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Network inference reveals novel connections in pathways regulating growth and defense in the yeast salt response.

    PubMed

    MacGilvray, Matthew E; Shishkova, Evgenia; Chasman, Deborah; Place, Michael; Gitter, Anthony; Coon, Joshua J; Gasch, Audrey P

    2018-05-01

    Cells respond to stressful conditions by coordinating a complex, multi-faceted response that spans many levels of physiology. Much of the response is coordinated by changes in protein phosphorylation. Although the regulators of transcriptome changes during stress are well characterized in Saccharomyces cerevisiae, the upstream regulatory network controlling protein phosphorylation is less well dissected. Here, we developed a computational approach to infer the signaling network that regulates phosphorylation changes in response to salt stress. We developed an approach to link predicted regulators to groups of likely co-regulated phospho-peptides responding to stress, thereby creating new edges in a background protein interaction network. We then use integer linear programming (ILP) to integrate wild type and mutant phospho-proteomic data and predict the network controlling stress-activated phospho-proteomic changes. The network we inferred predicted new regulatory connections between stress-activated and growth-regulating pathways and suggested mechanisms coordinating metabolism, cell-cycle progression, and growth during stress. We confirmed several network predictions with co-immunoprecipitations coupled with mass-spectrometry protein identification and mutant phospho-proteomic analysis. Results show that the cAMP-phosphodiesterase Pde2 physically interacts with many stress-regulated transcription factors targeted by PKA, and that reduced phosphorylation of those factors during stress requires the Rck2 kinase that we show physically interacts with Pde2. Together, our work shows how a high-quality computational network model can facilitate discovery of new pathway interactions during osmotic stress.

  11. Identification of Alternative Splice Variants Using Unique Tryptic Peptide Sequences for Database Searches.

    PubMed

    Tran, Trung T; Bollineni, Ravi C; Strozynski, Margarita; Koehler, Christian J; Thiede, Bernd

    2017-07-07

    Alternative splicing is a mechanism in eukaryotes by which different forms of mRNAs are generated from the same gene. Identification of alternative splice variants requires the identification of peptides specific for alternative splice forms. For this purpose, we generated a human database that contains only unique tryptic peptides specific for alternative splice forms from Swiss-Prot entries. Using this database allows an easy access to splice variant-specific peptide sequences that match to MS data. Furthermore, we combined this database without alternative splice variant-1-specific peptides with human Swiss-Prot. This combined database can be used as a general database for searching of LC-MS data. LC-MS data derived from in-solution digests of two different cell lines (LNCaP, HeLa) and phosphoproteomics studies were analyzed using these two databases. Several nonalternative splice variant-1-specific peptides were found in both cell lines, and some of them seemed to be cell-line-specific. Control and apoptotic phosphoproteomes from Jurkat T cells revealed several nonalternative splice variant-1-specific peptides, and some of them showed clear quantitative differences between the two states.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  14. Interfacial Mechanisms of Water Vapor Sorption into Cellulose Nanofibril Films as Revealed by Quantitative Models.

    PubMed

    Hakalahti, Minna; Faustini, Marco; Boissière, Cédric; Kontturi, Eero; Tammelin, Tekla

    2017-09-11

    Humidity is an efficient instrument for facilitating changes in local architectures of two-dimensional surfaces assembled from nanoscaled biomaterials. Here, complementary surface-sensitive methods are used to collect explicit and precise experimental evidence on the water vapor sorption into (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidized cellulose nanofibril (CNF) thin film over the relative humidity (RH) range from 0 to 97%. Changes in thickness and mass of the film due to water vapor uptake are tracked using spectroscopic ellipsometry and quartz crystal microbalance with dissipation monitoring, respectively. Experimental data is evaluated by the quantitative Langmuir/Flory-Huggins/clustering model and the Brunauer-Emmett-Teller model. The isotherms coupled with the quantitative models unveil distinct regions of predominant sorption modes: specific sorption of water molecules below 10% RH, multilayer build-up between 10 to 75% RH, and clustering of water molecules above 75% RH. The study reveals the sorption mechanisms underlying the well-known water uptake behavior of TEMPO oxidized CNF directly at the gas-solid interface.

  15. Quantitative Dynamics of Proteome, Acetylome, and Succinylome during Stem-Cell Differentiation into Hepatocyte-like Cells.

    PubMed

    Liu, Zekun; Zhang, Qing-Bin; Bu, Chen; Wang, Dawei; Yu, Kai; Gan, Zhixue; Chang, Jianfeng; Cheng, Zhongyi; Liu, Zexian

    2018-06-21

    Stem-cell differentiation is a complex biological process controlled by a series of functional protein clusters and signaling transductions, especially metabolism-related pathways. Although previous studies have quantified the proteome and phosphoproteome for stem-cell differentiation, the investigation of acylation-mediated regulation is still absent. In this study, we quantitatively profiled the proteome, acetylome, and succinylome in pluripotent human embryonic stem cells (hESCs) and differentiated hepatocyte-like cells (HLCs). In total, 3843 proteins, 185 acetylation sites in 103 proteins, and 602 succinylation sites in 391 proteins were quantified. The quantitative proteome showed that in differentiated HLCs the TGF-β, JAK-STAT, and RAS signaling pathways were activated, whereas ECM-related processes such as sulfates and leucine degradation were depressed. Interestingly, it was observed that the acetylation and succinylation were more intensive in hESCs, whereas protein processing in endoplasmic reticulum and the carbon metabolic pathways were especially highly succinylated. Because the metabolism patterns in pluripotent hESCs and the differentiated HLCs were different, we proposed that the dynamic acylations, especially succinylation, might regulate the Warburg-like effect and TCA cycle during differentiation. Taken together, we systematically profiled the protein and acylation levels of regulation in pluripotent hESCs and differentiated HLCs, and the results indicated the important roles of acylation in pluripotency maintenance and differentiation.

  16. Global proteome and phosphoproteome dynamics indicate novel mechanisms of vitamin C induced dormancy in Mycobacterium smegmatis.

    PubMed

    Albeldas, Claudia; Ganief, Naadir; Calder, Bridget; Nakedi, Kehilwe C; Garnett, Shaun; Nel, Andrew J M; Blackburn, Jonathan M; Soares, Nelson C

    2018-05-30

    Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO 2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events. Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic

  17. Comparative Phosphoproteomic Analysis of the Developing Seeds in Two Indica Rice ( Oryza sativa L.) Cultivars with Different Starch Quality.

    PubMed

    Pang, Yuehan; Zhou, Xin; Chen, Yaling; Bao, Jinsong

    2018-03-21

    Protein phosphorylation plays important roles in regulation of various molecular events such as plant growth and seed development. However, its involvement in starch biosynthesis is less understood. Here, a comparative phosphoproteomic analysis of two indica rice cultivars during grain development was performed. A total of 2079 and 2434 phosphopeptides from 1273 and 1442 phosphoproteins were identified, covering 2441 and 2808 phosphosites in indica rice 9311 and Guangluai4 (GLA4), respectively. Comparative analysis identified 303 differentially phosphorylated peptides, and 120 and 258 specifically phosphorylated peptides in 9311 and GLA4, respectively. Phosphopeptides in starch biosynthesis related enzymes such as AGPase, SSIIa, SSIIIa, BEI, BEIIb, PUL, and Pho1were identified. GLA4 and 9311 had different amylose content, pasting viscosities, and gelatinization temperature, suggesting subtle difference in starch biosynthesis and regulation between GLA4 and 9311. Our study will give added impetus to further understanding the regulatory mechanism of starch biosynthesis at the phosphorylation level.

  18. New quantitative approaches reveal the spatial preference of nuclear compartments in mammalian fibroblasts.

    PubMed

    Weston, David J; Russell, Richard A; Batty, Elizabeth; Jensen, Kirsten; Stephens, David A; Adams, Niall M; Freemont, Paul S

    2015-03-06

    The nuclei of higher eukaryotic cells display compartmentalization and certain nuclear compartments have been shown to follow a degree of spatial organization. To date, the study of nuclear organization has often involved simple quantitative procedures that struggle with both the irregularity of the nuclear boundary and the problem of handling replicate images. Such studies typically focus on inter-object distance, rather than spatial location within the nucleus. The concern of this paper is the spatial preference of nuclear compartments, for which we have developed statistical tools to quantitatively study and explore nuclear organization. These tools combine replicate images to generate 'aggregate maps' which represent the spatial preferences of nuclear compartments. We present two examples of different compartments in mammalian fibroblasts (WI-38 and MRC-5) that demonstrate new knowledge of spatial preference within the cell nucleus. Specifically, the spatial preference of RNA polymerase II is preserved across normal and immortalized cells, whereas PML nuclear bodies exhibit a change in spatial preference from avoiding the centre in normal cells to exhibiting a preference for the centre in immortalized cells. In addition, we show that SC35 splicing speckles are excluded from the nuclear boundary and localize throughout the nucleoplasm and in the interchromatin space in non-transformed WI-38 cells. This new methodology is thus able to reveal the effect of large-scale perturbation on spatial architecture and preferences that would not be obvious from single cell imaging.

  19. Quantitative isotope incorporation reveals substrate partitioning in a coastal microbial community.

    PubMed

    Mayali, Xavier; Weber, Peter K

    2018-05-01

    To quantitatively link microbial identity with biogeochemical function, we carried out 14 simultaneous stable isotope probing experiments with organic and inorganic C and N substrates to measure the isotope incorporation by over one hundred co-occurring eukaryotic and prokaryotic populations in a coastal community. We found that nitrate was the most commonly incorporated substrate, and that light-driven carbon fixation was carried out by some bacterial taxa from the Flavobacteriales and OM60 (NOR5) clade, in addition to photoautotrophic phytoplankton. We found that organisms that incorporated starch, maltose, glucose, lactose and bicarbonate were phylogenetically clustered, suggesting that specific bacterial lineages specialized in the incorporation of these substrates. The data further revealed that coastal microorganisms spanned a range of resource utilization strategies from generalists to specialists and demonstrated a high level of substrate partitioning, with two thirds of taxa exhibiting unique substrate incorporation patterns and the remaining third shared by no more than three OTUs each. Specialists exhibited more extreme incorporation levels (high or low), whereas generalists displayed more intermediate activity levels. These results shed valuable insights into the bottom-up ecological strategies enabling the persistence of high microbial diversity in aquatic ecosystems.

  20. Widespread age-related differences in the human brain microstructure revealed by quantitative magnetic resonance imaging.

    PubMed

    Callaghan, Martina F; Freund, Patrick; Draganski, Bogdan; Anderson, Elaine; Cappelletti, Marinella; Chowdhury, Rumana; Diedrichsen, Joern; Fitzgerald, Thomas H B; Smittenaar, Peter; Helms, Gunther; Lutti, Antoine; Weiskopf, Nikolaus

    2014-08-01

    A pressing need exists to disentangle age-related changes from pathologic neurodegeneration. This study aims to characterize the spatial pattern and age-related differences of biologically relevant measures in vivo over the course of normal aging. Quantitative multiparameter maps that provide neuroimaging biomarkers for myelination and iron levels, parameters sensitive to aging, were acquired from 138 healthy volunteers (age range: 19-75 years). Whole-brain voxel-wise analysis revealed a global pattern of age-related degeneration. Significant demyelination occurred principally in the white matter. The observed age-related differences in myelination were anatomically specific. In line with invasive histologic reports, higher age-related differences were seen in the genu of the corpus callosum than the splenium. Iron levels were significantly increased in the basal ganglia, red nucleus, and extensive cortical regions but decreased along the superior occipitofrontal fascicle and optic radiation. This whole-brain pattern of age-associated microstructural differences in the asymptomatic population provides insight into the neurobiology of aging. The results help build a quantitative baseline from which to examine and draw a dividing line between healthy aging and pathologic neurodegeneration. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Widespread age-related differences in the human brain microstructure revealed by quantitative magnetic resonance imaging☆

    PubMed Central

    Callaghan, Martina F.; Freund, Patrick; Draganski, Bogdan; Anderson, Elaine; Cappelletti, Marinella; Chowdhury, Rumana; Diedrichsen, Joern; FitzGerald, Thomas H.B.; Smittenaar, Peter; Helms, Gunther; Lutti, Antoine; Weiskopf, Nikolaus

    2014-01-01

    A pressing need exists to disentangle age-related changes from pathologic neurodegeneration. This study aims to characterize the spatial pattern and age-related differences of biologically relevant measures in vivo over the course of normal aging. Quantitative multiparameter maps that provide neuroimaging biomarkers for myelination and iron levels, parameters sensitive to aging, were acquired from 138 healthy volunteers (age range: 19–75 years). Whole-brain voxel-wise analysis revealed a global pattern of age-related degeneration. Significant demyelination occurred principally in the white matter. The observed age-related differences in myelination were anatomically specific. In line with invasive histologic reports, higher age-related differences were seen in the genu of the corpus callosum than the splenium. Iron levels were significantly increased in the basal ganglia, red nucleus, and extensive cortical regions but decreased along the superior occipitofrontal fascicle and optic radiation. This whole-brain pattern of age-associated microstructural differences in the asymptomatic population provides insight into the neurobiology of aging. The results help build a quantitative baseline from which to examine and draw a dividing line between healthy aging and pathologic neurodegeneration. PMID:24656835

  2. Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation.

    PubMed

    Carruthers, Nicholas J; Stemmer, Paul M; Chen, Ben; Valeriote, Frederick; Gao, Xiaohua; Guatam, Subhash C; Shaw, Jiajiu

    2017-09-15

    UTL-5g is a novel small-molecule TNF-alpha modulator. It reduces cisplatin-induced side effects by protecting kidney, liver, and platelets, thereby increasing tolerance for cisplatin. UTL-5g also reduces radiation-induced acute liver toxicity. The mechanism of action for UTL-5g is not clear at the present time. A phosphoproteomic analysis to a depth of 4943 phosphopeptides and a luminescence-based transcription factor activity assay were used to provide complementary analyses of signaling events that were disrupted by UTL-5g in RAW 264.7 cells. Transcriptional activity downstream of the interferon gamma, IL-6, type 1 Interferon, TGF-β, PKC/Ca 2+ and the glucocorticoid receptor pathways were disrupted by UTL-5g. Phosphoproteomic analysis indicated that hyperphosphorylation of proteins involved in actin remodeling was suppressed by UTL-5g (gene set analysis, FDR < 1%) as was phosphorylation of Stat3, consistent with the IL-6 results in the transcription factor assay. Neither analysis indicated that LPS-induced activation of the NF-kB, cAMP/PKA and JNK signaling pathways were affected by UTL-5g. This global characterization of UTL-5g activity in a macrophage cell line discovered that it disrupts selected aspects of LPS signaling including Stat3 activation and actin remodeling providing new insight on how UTL-5g acts to reduce cisplatin-induced side effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. The Clathrin-dependent Spindle Proteome*

    PubMed Central

    Rao, Sushma R.; Flores-Rodriguez, Neftali; Page, Scott L.; Wong, Chin; Robinson, Phillip J.; Chircop, Megan

    2016-01-01

    The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a “moonlighting” role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. PMID:27174698

  4. The Clathrin-dependent Spindle Proteome.

    PubMed

    Rao, Sushma R; Flores-Rodriguez, Neftali; Page, Scott L; Wong, Chin; Robinson, Phillip J; Chircop, Megan

    2016-08-01

    The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

    PubMed

    Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

    2012-12-27

    L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

  6. Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.

    PubMed

    Tsiatsiani, Liana; Giansanti, Piero; Scheltema, Richard A; van den Toorn, Henk; Overall, Christopher M; Altelaar, A F Maarten; Heck, Albert J R

    2017-02-03

    A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X) n K/R) and LysargiNase (i.e., K/R(X) n ) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.

  7. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sitesmore » whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.« less

  8. Self responses along cingulate cortex reveal quantitative neural phenotype for high functioning autism

    PubMed Central

    Chiu, Pearl H.; Kayali, M. Amin; Kishida, Kenneth T.; Tomlin, Damon; Klinger, Laura G.; Klinger, Mark R.; Montague, P. Read

    2014-01-01

    Summary Attributing behavioral outcomes correctly to oneself or to other agents is essential for all productive social exchange. We approach this issue in high-functioning males with autism spectrum disorder (ASD) using two separate fMRI paradigms. First, using a visual imagery task, we extract a basis set for responses along the cingulate cortex of control subjects that reveals an agent-specific eigenvector (self eigenmode) associated with imagining oneself executing a specific motor act. Second, we show that the same self eigenmode arises during one's own decision (the self phase) in an interpersonal exchange game (iterated trust game). Third, using this exchange game, we show that ASD males exhibit a severely diminished self eigenmode when playing the game with a human partner. This diminished response covaries parametrically with their behaviorally assessed symptom severity suggesting its value as an objective endophenotype. These findings may provide a quantitative assessment tool for high functioning ASD. PMID:18255038

  9. Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

    PubMed

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-12-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

  10. Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions.

    PubMed

    Marumoto, Ariane; Milani, Renato; da Silva, Rodrigo A; da Costa Fernandes, Célio Junior; Granjeiro, José Mauro; Ferreira, Carmen V; Peppelenbosch, Maikel P; Zambuzzi, Willian F

    2017-10-01

    The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions. Copyright © 2017 Elsevier Inc

  11. Phosphoproteomics profiling suggests a role for nuclear βΙPKC in transcription processes of undifferentiated murine embryonic stem cells.

    PubMed

    Costa-Junior, Helio Miranda; Garavello, Nicole Milaré; Duarte, Mariana Lemos; Berti, Denise Aparecida; Glaser, Talita; de Andrade, Alexander; Labate, Carlos A; Ferreira, André Teixeira da Silva; Perales, Jonas Enrique Aguilar; Xavier-Neto, José; Krieger, José Eduardo; Schechtman, Deborah

    2010-12-03

    Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, βIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of βΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one βIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect βΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting βΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express βΙPKC and βΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that βIPKC takes part in the processes that maintain ESCs in their undifferentiated state.

  12. Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging

    PubMed Central

    Yaginuma, Hideyuki; Kawai, Shinnosuke; Tabata, Kazuhito V.; Tomiyama, Keisuke; Kakizuka, Akira; Komatsuzaki, Tamiki; Noji, Hiroyuki; Imamura, Hiromi

    2014-01-01

    Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells. PMID:25283467

  13. Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

    PubMed Central

    Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

    2016-01-01

    Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

  14. Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis1

    PubMed Central

    Chen, Yanmei; Hoehenwarter, Wolfgang

    2015-01-01

    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

  15. What Is "Good" Research? Revealing the Paradigmatic Tensions in Quantitative Criticalist Work

    ERIC Educational Resources Information Center

    Hernández, Ebelia

    2014-01-01

    If quantitative criticalism is thought to be a bridge between positivist epistemologies prevalent in quantitative work and social constructionism often found in critical qualitative work, then this bridge is fraught with challenges and tensions. This chapter examines the methodological issues, questions, and tensions that emerged from a research…

  16. Quantitative Genetic Interactions Reveal Layers of Biological Modularity

    PubMed Central

    Beltrao, Pedro; Cagney, Gerard; Krogan, Nevan J.

    2010-01-01

    In the past, biomedical research has embraced a reductionist approach, primarily focused on characterizing the individual components that comprise a system of interest. Recent technical developments have significantly increased the size and scope of data describing biological systems. At the same time, advances in the field of systems biology have evoked a broader view of how the underlying components are interconnected. In this essay, we discuss how quantitative genetic interaction mapping has enhanced our view of biological systems, allowing a deeper functional interrogation at different biological scales. PMID:20510918

  17. Quantitative proteomics analysis reveals the tolerance of Mirabilis jalapa L. to petroleum contamination.

    PubMed

    Chen, Shuisen; Ma, Hui; Guo, Zhifu; Feng, Yaping; Lin, Jingwei; Zhang, Menghua; Zhong, Ming

    2017-03-01

    Petroleum is not only an important energy resource but is also a major soil pollutant. To gain better insight into the adaptability mechanism of Mirabilis jalapa to petroleum-contaminated soil, the protein profiles of M. jalapa root were investigated using label-free quantitative proteomics technique. After exposing to petroleum-contaminated soil for 24 h, 34 proteins significantly changed their protein abundance and most of the proteins increased in protein abundance (91.18%). Combined with gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses as well as data from previous studies, our results revealed that M. jalapa enhanced tolerance to petroleum by changing antioxidation and detoxification, cell wall organization, amino acid and carbohydrate metabolism, transportation and protein process, and so on. These metabolism alterations could result in the production and secretion of low molecular carbohydrate, amino acid, and functional protein, which enhanced the bioavailability of petroleum and reducing the toxicity of the petroleum. Taken together, these results provided novel information for better understanding of the tolerance of M. jalapa to petroleum stress.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

  20. Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate*

    PubMed Central

    Schwarz, Jennifer Jasmin; Wiese, Heike; Tölle, Regine Charlotte; Zarei, Mostafa; Dengjel, Jörn; Warscheid, Bettina; Thedieck, Kathrin

    2015-01-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation. PMID:25907765

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

    DOE PAGES

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

    2014-05-29

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

  2. Quantitative and Qualitative Differences in Morphological Traits Revealed between Diploid Fragaria Species

    PubMed Central

    SARGENT, DANIEL J.; GEIBEL, M.; HAWKINS, J. A.; WILKINSON, M. J.; BATTEY, N. H.; SIMPSON, D. W.

    2004-01-01

    • Background and Aims The aims of this investigation were to highlight the qualitative and quantitative diversity apparent between nine diploid Fragaria species and produce interspecific populations segregating for a large number of morphological characters suitable for quantitative trait loci analysis. • Methods A qualitative comparison of eight described diploid Fragaria species was performed and measurements were taken of 23 morphological traits from 19 accessions including eight described species and one previously undescribed species. A principal components analysis was performed on 14 mathematically unrelated traits from these accessions, which partitioned the species accessions into distinct morphological groups. Interspecific crosses were performed with accessions of species that displayed significant quantitative divergence and, from these, populations that should segregate for a range of quantitative traits were raised. • Key Results Significant differences between species were observed for all 23 morphological traits quantified and three distinct groups of species accessions were observed after the principal components analysis. Interspecific crosses were performed between these groups, and F2 and backcross populations were raised that should segregate for a range of morphological characters. In addition, the study highlighted a number of distinctive morphological characters in many of the species studied. • Conclusions Diploid Fragaria species are morphologically diverse, yet remain highly interfertile, making the group an ideal model for the study of the genetic basis of phenotypic differences between species through map-based investigation using quantitative trait loci. The segregating interspecific populations raised will be ideal for such investigations and could also provide insights into the nature and extent of genome evolution within this group. PMID:15469944

  3. Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

    PubMed Central

    2013-01-01

    Background Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Results Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. Conclusions The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. PMID:23394617

  4. Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger.

    PubMed

    Manzanares-Miralles, Lara; Sarikaya-Bayram, Özlem; Smith, Elizabeth B; Dolan, Stephen K; Bayram, Özgür; Jones, Gary W; Doyle, Sean

    2016-01-10

    Gliotoxin (GT) is a redox-active metabolite, produced by Aspergillus fumigatus, which inhibits the growth of other fungi. Here we demonstrate how Aspergillus niger responds to GT exposure. Quantitative proteomics revealed that GT dysregulated the abundance of 378 proteins including those involved in methionine metabolism and induced de novo abundance of two S-adenosylmethionine (SAM)-dependent methyltransferases. Increased abundance of enzymes S-adenosylhomocysteinase (p=0.0018) required for homocysteine generation from S-adenosylhomocysteine (SAH), and spermidine synthase (p=0.0068), involved in the recycling of Met, was observed. Analysis of Met-related metabolites revealed significant increases in the levels of Met and adenosine, in correlation with proteomic data. Methyltransferase MT-II is responsible for bisthiobis(methylthio)gliotoxin (BmGT) formation, deletion of MT-II abolished BmGT formation and led to increased GT sensitivity in A. niger. Proteomic analysis also revealed that GT exposure also significantly (p<0.05) increased hydrolytic enzyme abundance, including glycoside hydrolases (n=22) and peptidases (n=16). We reveal that in an attempt to protect against the detrimental affects of GT, methyltransferase-mediated GT thiomethylation alters cellular pathways involving Met and SAM, with consequential dysregulation of hydrolytic enzyme abundance in A. niger. Thus, it provides new opportunities to exploit the response of GT-naïve fungi to GT. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. 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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matsumura, Takayuki; Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480; Oyama, Masaaki

    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 analysismore » 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.« less

  6. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-29

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

  7. Phosphoproteomic Analysis of Signaling Pathways in Head and Neck Squamous Cell Carcinoma Patient Samples

    PubMed Central

    Frederick, Mitchell J.; VanMeter, Amy J.; Gadhikar, Mayur A.; Henderson, Ying C.; Yao, Hui; Pickering, Curtis C.; Williams, Michelle D.; El-Naggar, Adel K.; Sandulache, Vlad; Tarco, Emily; Myers, Jeffrey N.; Clayman, Gary L.; Liotta, Lance A.; Petricoin, Emanuel F.; Calvert, Valerie S.; Fodale, Valentina; Wang, Jing; Weber, Randal S.

    2011-01-01

    Molecular targeted therapy represents a promising new strategy for treating cancers because many small-molecule inhibitors targeting protein kinases have recently become available. Reverse-phase protein microarrays (RPPAs) are a useful platform for identifying dysregulated signaling pathways in tumors and can provide insight into patient-specific differences. In the present study, RPPAs were used to examine 60 protein end points (predominantly phosphoproteins) in matched tumor and nonmalignant biopsy specimens from 23 patients with head and neck squamous cell carcinoma to characterize the cancer phosphoproteome. RPPA identified 18 of 60 analytes globally elevated in tumors versus healthy tissue and 17 of 60 analytes that were decreased. The most significantly elevated analytes in tumor were checkpoint kinase (Chk) 1 serine 345 (S345), Chk 2 S33/35, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) S65, protein kinase C (PKC) ζ/ι threonine 410/412 (T410/T412), LKB1 S334, inhibitor of kappaB alpha (IκB-α) S32, eukaryotic translation initiation factor 4E (eIF4E) S209, Smad2 S465/67, insulin receptor substrate 1 (IRS-1) S612, mitogen-activated ERK kinase 1/2 (MEK1/2) S217/221, and total PKC ι. To our knowledge, this is the first report of elevated PKC ι in head and neck squamous cell carcinoma that may have significance because PKC ι is an oncogene in several other tumor types, including lung cancer. The feasibility of using RPPA for developing theranostic tests to guide personalized therapy is discussed in the context of these data. PMID:21281788

  8. Quantitative Glycoproteomics Analysis Reveals Changes in N-Glycosylation Level Associated with Pancreatic Ductal Adenocarcinoma

    PubMed Central

    2015-01-01

    Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-β, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events. PMID:24471499

  9. Quantitative glycoproteomics analysis reveals changes in N-glycosylation level associated with pancreatic ductal adenocarcinoma.

    PubMed

    Pan, Sheng; Chen, Ru; Tamura, Yasuko; Crispin, David A; Lai, Lisa A; May, Damon H; McIntosh, Martin W; Goodlett, David R; Brentnall, Teresa A

    2014-03-07

    Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-β, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events.

  10. Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W

    PubMed Central

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

    2009-01-01

    To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na+ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H+-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H+-pump activity. PMID:20028841

  11. A Quantitative Model of Motility Reveals Low-Dimensional Variation in Exploratory Behavior Across Multiple Nematode Species

    NASA Astrophysics Data System (ADS)

    Helms, Stephen; Avery, Leon; Stephens, Greg; Shimizu, Tom

    2014-03-01

    Animal behavior emerges from many layers of biological organization--from molecular signaling pathways and neuronal networks to mechanical outputs of muscles. In principle, the large number of interconnected variables at each of these layers could imply dynamics that are complex and hard to control or even tinker with. Yet, for organisms to survive in a competitive, ever-changing environment, behavior must readily adapt. We applied quantitative modeling to identify important aspects of behavior in chromadorean nematodes ranging from the lab strain C. elegans N2 to wild strains and distant species. We revealed subtle yet important features such as speed control and heavy-tailed directional changes. We found that the parameters describing this behavioral model varied among individuals and across species in a correlated way that is consistent with a trade-off between exploratory and exploitative behavior.

  12. Involvement of GABA Transporters in Atropine-Treated Myopic Retina As Revealed by iTRAQ Quantitative Proteomics

    PubMed Central

    2015-01-01

    Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (−15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the

  13. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum—Phytophthora capsici Phytopathosystem

    PubMed Central

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G.; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  14. Transcriptome- Assisted Label-Free Quantitative Proteomics Analysis Reveals Novel Insights into Piper nigrum-Phytophthora capsici Phytopathosystem.

    PubMed

    Mahadevan, Chidambareswaren; Krishnan, Anu; Saraswathy, Gayathri G; Surendran, Arun; Jaleel, Abdul; Sakuntala, Manjula

    2016-01-01

    Black pepper (Piper nigrum L.), a tropical spice crop of global acclaim, is susceptible to Phytophthora capsici, an oomycete pathogen which causes the highly destructive foot rot disease. A systematic understanding of this phytopathosystem has not been possible owing to lack of genome or proteome information. In this study, we explain an integrated transcriptome-assisted label-free quantitative proteomics pipeline to study the basal immune components of black pepper when challenged with P. capsici. We report a global identification of 532 novel leaf proteins from black pepper, of which 518 proteins were functionally annotated using BLAST2GO tool. A label-free quantitation of the protein datasets revealed 194 proteins common to diseased and control protein datasets of which 22 proteins showed significant up-regulation and 134 showed significant down-regulation. Ninety-three proteins were identified exclusively on P. capsici infected leaf tissues and 245 were expressed only in mock (control) infected samples. In-depth analysis of our data gives novel insights into the regulatory pathways of black pepper which are compromised during the infection. Differential down-regulation was observed in a number of critical pathways like carbon fixation in photosynthetic organism, cyano-amino acid metabolism, fructose, and mannose metabolism, glutathione metabolism, and phenylpropanoid biosynthesis. The proteomics results were validated with real-time qRT-PCR analysis. We were also able to identify the complete coding sequences for all the proteins of which few selected genes were cloned and sequence characterized for further confirmation. Our study is the first report of a quantitative proteomics dataset in black pepper which provides convincing evidence on the effectiveness of a transcriptome-based label-free proteomics approach for elucidating the host response to biotic stress in a non-model spice crop like P. nigrum, for which genome information is unavailable. Our dataset

  15. Quantitative habitability.

    PubMed

    Shock, Everett L; Holland, Melanie E

    2007-12-01

    A framework is proposed for a quantitative approach to studying habitability. Considerations of environmental supply and organismal demand of energy lead to the conclusions that power units are most appropriate and that the units for habitability become watts per organism. Extreme and plush environments are revealed to be on a habitability continuum, and extreme environments can be quantified as those where power supply only barely exceeds demand. Strategies for laboratory and field experiments are outlined that would quantify power supplies, power demands, and habitability. An example involving a comparison of various metabolisms pursued by halophiles is shown to be well on the way to a quantitative habitability analysis.

  16. Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica

    DOE PAGES

    Pomraning, Kyle R.; Kim, Young -Mo; Nicora, Carrie D.; ...

    2016-02-25

    Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. Furthermore, while the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield.

  17. Proteome-wide Light/Dark Modulation of Thiol Oxidation in Cyanobacteria Revealed by Quantitative Site-specific Redox Proteomics*

    PubMed Central

    Guo, Jia; Nguyen, Amelia Y.; Dai, Ziyu; Su, Dian; Gaffrey, Matthew J.; Moore, Ronald J.; Jacobs, Jon M.; Monroe, Matthew E.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.; Qian, Wei-Jun

    2014-01-01

    Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of ∼2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for ∼1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover, the redox sensitivity along with the stoichiometric data enabled prediction of potential functional Cys-sites for proteins of interest. The functional significance of redox-sensitive Cys-sites in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin (AhpC/TSA family protein Sll1621), and glucose 6-phosphate dehydrogenase was further confirmed with site-specific mutagenesis and biochemical studies. Together, our findings provide significant insights into the broad redox regulation of photosynthetic organisms. PMID:25118246

  18. QUANTITATIVE PCR ANALYSIS OF HOUSE DUST CAN REVEAL ABNORMAL MOLD CONDITIONS

    EPA Science Inventory

    Indoor mold populations were measured in the dust of homes in Cleveland and Cincinnati, OH, by quantitative PCR (QPCR) and, in Cincinnati, also by culturing. QPCR assays for 82 species (or groups of species) were used to identify and quantify indoor mold populations in moldy home...

  19. Significant and unique changes in phosphorylation levels of four phosphoproteins in two apple rootstock genotypes under drought stress.

    PubMed

    Ren, Jing; Mao, Juan; Zuo, Cunwu; Calderón-Urrea, Alejandro; Dawuda, Mohammed Mujitaba; Zhao, Xin; Li, Xinwen; Chen, Baihong

    2017-12-01

    Drought stress is a major problem around the world and there is still little molecular mechanism about how fruit crops deal with moderate drought stress. Here, the physiological and phosphoproteomic responses of drought-sensitive genotype (M26) and drought-tolerant genotype (MBB) under moderate drought stress were investigated. Our results of the physiology analysis indicated that the MBB genotype could produce more osmosis-regulating substances. Furthermore, phosphoproteins from leaves of both genotypes under moderate drought stress were analyzed using the isobaric tags for relative and absolute quantification technology. A total of 595 unique phosphopeptides, 682 phosphorylated sites, and 446 phosphoproteins were quantitatively analyzed in the two genotypes. Five and thirty-five phosphoproteins with the phosphorylation levels significantly changed (PLSC) were identified in M26 and MBB, respectively. Among these, four PLSC phosphoproteins were common to both genotypes, perhaps indicating a partial overlap of the mechanisms to moderate drought stress. Gene ontology analyses revealed that the PLSC phosphoproteins represent a unique combination of metabolism, transcription, translation, and protein processing, suggesting that the response in apple to moderate drought stress encompasses a new and unique homeostasis of major cellular processes. The basic trend was an increase in protein and organic molecules abundance related to drought. These increases were higher in MBB than in M26. Our study is the first to address the phosphoproteome of apple rootstocks in response to moderate drought stress, and provide insights into the molecular regulation mechanisms of apple rootstock under moderate drought stress.

  20. A quantitative framework for whole-body coordination reveals specific deficits in freely walking ataxic mice

    PubMed Central

    Machado, Ana S; Darmohray, Dana M; Fayad, João; Marques, Hugo G; Carey, Megan R

    2015-01-01

    The coordination of movement across the body is a fundamental, yet poorly understood aspect of motor control. Mutant mice with cerebellar circuit defects exhibit characteristic impairments in locomotor coordination; however, the fundamental features of this gait ataxia have not been effectively isolated. Here we describe a novel system (LocoMouse) for analyzing limb, head, and tail kinematics of freely walking mice. Analysis of visibly ataxic Purkinje cell degeneration (pcd) mice reveals that while differences in the forward motion of individual paws are fully accounted for by changes in walking speed and body size, more complex 3D trajectories and, especially, inter-limb and whole-body coordination are specifically impaired. Moreover, the coordination deficits in pcd are consistent with a failure to predict and compensate for the consequences of movement across the body. These results isolate specific impairments in whole-body coordination in mice and provide a quantitative framework for understanding cerebellar contributions to coordinated locomotion. DOI: http://dx.doi.org/10.7554/eLife.07892.001 PMID:26433022

  1. A Methodological Self-Study of Quantitizing: Negotiating Meaning and Revealing Multiplicity

    ERIC Educational Resources Information Center

    Seltzer-Kelly, Deborah; Westwood, Sean J.; Pena-Guzman, David M.

    2012-01-01

    This inquiry developed during the process of "quantitizing" qualitative data the authors had gathered for a mixed methods curriculum efficacy study. Rather than providing the intended rigor to their data coding process, their use of an intercoder reliability metric prompted their investigation of the multiplicity and messiness that, as they…

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

    PubMed

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

    2015-08-01

    Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Comparative qualitative phosphoproteomics analysis identifies shared phosphorylation motifs and associated biological processes in evolutionary divergent plants.

    PubMed

    Al-Momani, Shireen; Qi, Da; Ren, Zhe; Jones, Andrew R

    2018-06-15

    Phosphorylation is one of the most prevalent post-translational modifications and plays a key role in regulating cellular processes. We carried out a bioinformatics analysis of pre-existing phosphoproteomics data, to profile two model species representing the largest subclasses in flowering plants the dicot Arabidopsis thaliana and the monocot Oryza sativa, to understand the extent to which phosphorylation signaling and function is conserved across evolutionary divergent plants. We identified 6537 phosphopeptides from 3189 phosphoproteins in Arabidopsis and 2307 phosphopeptides from 1613 phosphoproteins in rice. We identified phosphorylation motifs, finding nineteen pS motifs and two pT motifs shared in rice and Arabidopsis. The majority of shared motif-containing proteins were mapped to the same biological processes with similar patterns of fold enrichment, indicating high functional conservation. We also identified shared patterns of crosstalk between phosphoserines with enrichment for motifs pSXpS, pSXXpS and pSXXXpS, where X is any amino acid. Lastly, our results identified several pairs of motifs that are significantly enriched to co-occur in Arabidopsis proteins, indicating cross-talk between different sites, but this was not observed in rice. Our results demonstrate that there are evolutionary conserved mechanisms of phosphorylation-mediated signaling in plants, via analysis of high-throughput phosphorylation proteomics data from key monocot and dicot species: rice and Arabidposis thaliana. The results also suggest that there is increased crosstalk between phosphorylation sites in A. thaliana compared with rice. The results are important for our general understanding of cell signaling in plants, and the ability to use A. thaliana as a general model for plant biology. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Quantitative Analysis Reveals that Actin and Src-Family Kinases Regulate Nuclear YAP1 and Its Export.

    PubMed

    Ege, Nil; Dowbaj, Anna M; Jiang, Ming; Howell, Michael; Hooper, Steven; Foster, Charles; Jenkins, Robert P; Sahai, Erik

    2018-06-08

    The transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts, YAP1 is located in the cytoplasm, while in activated cancer-associated fibroblasts, it is nuclear and promotes the expression of genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photobleaching methods, and mathematical modeling. Imaging of migrating fibroblasts reveals the tight temporal coupling of cell shape change and altered YAP1 localization. Both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear-constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Quantitative Data Analysis--In the Graduate Curriculum

    ERIC Educational Resources Information Center

    Albers, Michael J.

    2017-01-01

    A quantitative research study collects numerical data that must be analyzed to help draw the study's conclusions. Teaching quantitative data analysis is not teaching number crunching, but teaching a way of critical thinking for how to analyze the data. The goal of data analysis is to reveal the underlying patterns, trends, and relationships of a…

  6. Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica.

    PubMed

    Pomraning, Kyle R; Kim, Young-Mo; Nicora, Carrie D; Chu, Rosalie K; Bredeweg, Erin L; Purvine, Samuel O; Hu, Dehong; Metz, Thomas O; Baker, Scott E

    2016-02-25

    Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. While the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to better understand how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield. We found that ribosome structural genes are down-regulated under nitrogen limitation, during which nitrogen containing compounds (alanine, putrescine, spermidine and urea) are depleted and sugar alcohols and TCA cycle intermediates accumulate (citrate, fumarate and malate). We identified 1219 novel phosphorylation sites in Y. lipolytica, 133 of which change in their abundance during nitrogen limitation. Regulatory proteins, including kinases and DNA binding proteins, are particularly enriched for phosphorylation. Within lipid synthesis pathways, we found that ATP-citrate lyase, acetyl-CoA carboxylase and lecithin cholesterol acyl transferase are phosphorylated during nitrogen limitation while many of the proteins involved in β-oxidation are down-regulated, suggesting that storage lipid accumulation may be regulated by phosphorylation of key enzymes. Further, we identified short DNA elements that associate specific transcription factor families with up- and down-regulated genes. Integration of metabolome, proteome and phosphoproteome data identifies lipid accumulation in response to nitrogen limitation as a two-fold result of increased production of acetyl-CoA from excess citrate and decreased capacity for β-oxidation.

  7. Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

    PubMed

    Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

    2016-02-05

    Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

  8. Quantitative evaluation of protocorm growth and fungal colonization in Bletilla striata (Orchidaceae) reveals less-productive symbiosis with a non-native symbiotic fungus.

    PubMed

    Yamamoto, Tatsuki; Miura, Chihiro; Fuji, Masako; Nagata, Shotaro; Otani, Yuria; Yagame, Takahiro; Yamato, Masahide; Kaminaka, Hironori

    2017-02-21

    In nature, orchid plants depend completely on symbiotic fungi for their nutrition at the germination and the subsequent seedling (protocorm) stages. However, only limited quantitative methods for evaluating the orchid-fungus interactions at the protocorm stage are currently available, which greatly constrains our understanding of the symbiosis. Here, we aimed to improve and integrate quantitative evaluations of the growth and fungal colonization in the protocorms of a terrestrial orchid, Blettila striata, growing on a plate medium. We achieved both symbiotic and asymbiotic germinations for the terrestrial orchid B. striata. The protocorms produced by the two germination methods grew almost synchronously for the first three weeks. At week four, however, the length was significantly lower in the symbiotic protocorms. Interestingly, the dry weight of symbiotic protocorms did not significantly change during the growth period, which implies that there was only limited transfer of carbon compounds from the fungus to the protocorms in this relationship. Next, to evaluate the orchid-fungus interactions, we developed an ink-staining method to observe the hyphal coils in protocorms without preparing thin sections. Crushing the protocorm under the coverglass enables us to observe all hyphal coils in the protocorms with high resolution. For this observation, we established a criterion to categorize the stages of hyphal coils, depending on development and degradation. By counting the symbiotic cells within each stage, it was possible to quantitatively evaluate the orchid-fungus symbiosis. We describe a method for quantitative evaluation of orchid-fungus symbiosis by integrating the measurements of plant growth and fungal colonization. The current study revealed that although fungal colonization was observed in the symbiotic protocorms, the weight of the protocorm did not significantly increase, which is probably due to the incompatibility of the fungus in this symbiosis. These

  9. Modeling development and quantitative trait mapping reveal independent genetic modules for leaf size and shape.

    PubMed

    Baker, Robert L; Leong, Wen Fung; Brock, Marcus T; Markelz, R J Cody; Covington, Michael F; Devisetty, Upendra K; Edwards, Christine E; Maloof, Julin; Welch, Stephen; Weinig, Cynthia

    2015-10-01

    Improved predictions of fitness and yield may be obtained by characterizing the genetic controls and environmental dependencies of organismal ontogeny. Elucidating the shape of growth curves may reveal novel genetic controls that single-time-point (STP) analyses do not because, in theory, infinite numbers of growth curves can result in the same final measurement. We measured leaf lengths and widths in Brassica rapa recombinant inbred lines (RILs) throughout ontogeny. We modeled leaf growth and allometry as function valued traits (FVT), and examined genetic correlations between these traits and aspects of phenology, physiology, circadian rhythms and fitness. We used RNA-seq to construct a SNP linkage map and mapped trait quantitative trait loci (QTL). We found genetic trade-offs between leaf size and growth rate FVT and uncovered differences in genotypic and QTL correlations involving FVT vs STPs. We identified leaf shape (allometry) as a genetic module independent of length and width and identified selection on FVT parameters of development. Leaf shape is associated with venation features that affect desiccation resistance. The genetic independence of leaf shape from other leaf traits may therefore enable crop optimization in leaf shape without negative effects on traits such as size, growth rate, duration or gas exchange. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  10. Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

    PubMed

    Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

    2017-10-06

    Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

  11. Quantitative Proteomic Profiling of Prostate Cancer Reveals a Role for miR-128 in Prostate Cancer*

    PubMed Central

    Khan, Amjad P.; Poisson, Laila M.; Bhat, Vadiraja B.; Fermin, Damian; Zhao, Rong; Kalyana-Sundaram, Shanker; Michailidis, George; Nesvizhskii, Alexey I.; Omenn, Gilbert S.; Chinnaiyan, Arul M.; Sreekumar, Arun

    2010-01-01

    Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion. PMID:19955085

  12. Quantitative X-ray Diffraction (QXRD) analysis for revealing thermal transformations of red mud.

    PubMed

    Liao, Chang-Zhong; Zeng, Lingmin; Shih, Kaimin

    2015-07-01

    Red mud is a worldwide environmental problem, and many authorities are trying to find an economic solution for its beneficial application or/and safe disposal. Ceramic production is one of the potential waste-to-resource strategies for using red mud as a raw material. Before implementing such a strategy, an unambiguous understanding of the reaction behavior of red mud under thermal conditions is essential. In this study, the phase compositions and transformation processes were revealed for the Pingguo red mud (PRM) heat-treated at different sintering temperatures. Hematite, perovskite, andradite, cancrinite, kaolinite, diaspore, gibbsite and calcite phases were observed in the samples. However, unlike those red mud samples from the other regions, no TiO2 (rutile or anatase) or quartz were observed. Titanium was found to exist mainly in perovskite and andradite while the iron mainly existed in hematite and andradite. A new silico-ferrite of calcium and aluminum (SFCA) phase was found in samples treated at temperatures above 1100°C, and two possible formation pathways for SFCA were suggested. This is the first SFCA phase to be reported in thermally treated red mud, and this finding may turn PRM waste into a material resource for the iron-making industry. Titanium was found to be enriched in the perovskite phase after 1200°C thermal treatment, and this observation indicated a potential strategy for the recovery of titanium from PRM. In addition to noting these various resource recovery opportunities, this is also the first study to quantitatively summarize the reaction details of PRM phase transformations at various temperatures. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

    PubMed Central

    Rodrigues, Silas P.; Ventura, José A.; Aguilar, Clemente; Nakayasu, Ernesto S.; Choi, HyungWon; Sobreira, Tiago J. P.; Nohara, Lilian L.; Wermelinger, Luciana S.; Almeida, Igor C.; Zingali, Russolina B.; Fernandes, Patricia M. B.

    2012-01-01

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers’ regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

  14. The Impact of Situation-Based Learning to Students’ Quantitative Literacy

    NASA Astrophysics Data System (ADS)

    Latifah, T.; Cahya, E.; Suhendra

    2017-09-01

    Nowadays, the usage of quantities can be seen almost everywhere. There has been an increase of quantitative thinking, such as quantitative reasoning and quantitative literacy, within the context of daily life. However, many people today are still not fully equipped with the knowledge of quantitative thinking. There are still a lot of individuals not having enough quantitative skills to perform well within today’s society. Based on this issue, the research aims to improve students’ quantitative literacy in junior high school. The qualitative analysis of written student work and video observations during the experiment reveal that the impact of situation-based learning affects students’ quantitative literacy.

  15. Identification of ginseng root using quantitative X-ray microtomography.

    PubMed

    Ye, Linlin; Xue, Yanling; Wang, Yudan; Qi, Juncheng; Xiao, Tiqiao

    2017-07-01

    The use of X-ray phase-contrast microtomography for the investigation of Chinese medicinal materials is advantageous for its nondestructive, in situ , and three-dimensional quantitative imaging properties. The X-ray phase-contrast microtomography quantitative imaging method was used to investigate the microstructure of ginseng, and the phase-retrieval method is also employed to process the experimental data. Four different ginseng samples were collected and investigated; these were classified according to their species, production area, and sample growth pattern. The quantitative internal characteristic microstructures of ginseng were extracted successfully. The size and position distributions of the calcium oxalate cluster crystals (COCCs), important secondary metabolites that accumulate in ginseng, are revealed by the three-dimensional quantitative imaging method. The volume and amount of the COCCs in different species of the ginseng are obtained by a quantitative analysis of the three-dimensional microstructures, which shows obvious difference among the four species of ginseng. This study is the first to provide evidence of the distribution characteristics of COCCs to identify four types of ginseng, with regard to species authentication and age identification, by X-ray phase-contrast microtomography quantitative imaging. This method is also expected to reveal important relationships between COCCs and the occurrence of the effective medicinal components of ginseng.

  16. Quantitative proteomics reveals dynamic responses of Synechocystis sp. PCC 6803 to next-generation biofuel butanol.

    PubMed

    Tian, Xiaoxu; Chen, Lei; Wang, Jiangxin; Qiao, Jianjun; Zhang, Weiwen

    2013-01-14

    Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ - LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Comprehensive Proteomics Analysis of Laticifer Latex Reveals New Insights into Ethylene Stimulation of Natural Rubber Production.

    PubMed

    Wang, Xuchu; Wang, Dan; Sun, Yong; Yang, Qian; Chang, Lili; Wang, Limin; Meng, Xueru; Huang, Qixing; Jin, Xiang; Tong, Zheng

    2015-09-08

    Ethylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.

  18. Infrared spectroscopy reveals both qualitative and quantitative differences in equine subchondral bone during maturation

    NASA Astrophysics Data System (ADS)

    Kobrina, Yevgeniya; Isaksson, Hanna; Sinisaari, Miikka; Rieppo, Lassi; Brama, Pieter A.; van Weeren, René; Helminen, Heikki J.; Jurvelin, Jukka S.; Saarakkala, Simo

    2010-11-01

    The collagen phase in bone is known to undergo major changes during growth and maturation. The objective of this study is to clarify whether Fourier transform infrared (FTIR) microspectroscopy, coupled with cluster analysis, can detect quantitative and qualitative changes in the collagen matrix of subchondral bone in horses during maturation and growth. Equine subchondral bone samples (n = 29) from the proximal joint surface of the first phalanx are prepared from two sites subjected to different loading conditions. Three age groups are studied: newborn (0 days old), immature (5 to 11 months old), and adult (6 to 10 years old) horses. Spatial collagen content and collagen cross-link ratio are quantified from the spectra. Additionally, normalized second derivative spectra of samples are clustered using the k-means clustering algorithm. In quantitative analysis, collagen content in the subchondral bone increases rapidly between the newborn and immature horses. The collagen cross-link ratio increases significantly with age. In qualitative analysis, clustering is able to separate newborn and adult samples into two different groups. The immature samples display some nonhomogeneity. In conclusion, this is the first study showing that FTIR spectral imaging combined with clustering techniques can detect quantitative and qualitative changes in the collagen matrix of subchondral bone during growth and maturation.

  19. Comparative Genomics Analyses Reveal Extensive Chromosome Colinearity and Novel Quantitative Trait Loci in Eucalyptus.

    PubMed

    Li, Fagen; Zhou, Changpin; Weng, Qijie; Li, Mei; Yu, Xiaoli; Guo, Yong; Wang, Yu; Zhang, Xiaohong; Gan, Siming

    2015-01-01

    Dense genetic maps, along with quantitative trait loci (QTLs) detected on such maps, are powerful tools for genomics and molecular breeding studies. In the important woody genus Eucalyptus, the recent release of E. grandis genome sequence allows for sequence-based genomic comparison and searching for positional candidate genes within QTL regions. Here, dense genetic maps were constructed for E. urophylla and E. tereticornis using genomic simple sequence repeats (SSR), expressed sequence tag (EST) derived SSR, EST-derived cleaved amplified polymorphic sequence (EST-CAPS), and diversity arrays technology (DArT) markers. The E. urophylla and E. tereticornis maps comprised 700 and 585 markers across 11 linkage groups, totaling at 1,208.2 and 1,241.4 cM in length, respectively. Extensive synteny and colinearity were observed as compared to three earlier DArT-based eucalypt maps (two maps with E. grandis × E. urophylla and one map of E. globulus) and with the E. grandis genome sequence. Fifty-three QTLs for growth (10-56 months of age) and wood density (56 months) were identified in 22 discrete regions on both maps, in which only one colocalizaiton was found between growth and wood density. Novel QTLs were revealed as compared with those previously detected on DArT-based maps for similar ages in Eucalyptus. Eleven to 585 positional candidate genes were obained for a 56-month-old QTL through aligning QTL confidence interval with the E. grandis genome. These results will assist in comparative genomics studies, targeted gene characterization, and marker-assisted selection in Eucalyptus and the related taxa.

  20. Comparative Genomics Analyses Reveal Extensive Chromosome Colinearity and Novel Quantitative Trait Loci in Eucalyptus

    PubMed Central

    Weng, Qijie; Li, Mei; Yu, Xiaoli; Guo, Yong; Wang, Yu; Zhang, Xiaohong; Gan, Siming

    2015-01-01

    Dense genetic maps, along with quantitative trait loci (QTLs) detected on such maps, are powerful tools for genomics and molecular breeding studies. In the important woody genus Eucalyptus, the recent release of E. grandis genome sequence allows for sequence-based genomic comparison and searching for positional candidate genes within QTL regions. Here, dense genetic maps were constructed for E. urophylla and E. tereticornis using genomic simple sequence repeats (SSR), expressed sequence tag (EST) derived SSR, EST-derived cleaved amplified polymorphic sequence (EST-CAPS), and diversity arrays technology (DArT) markers. The E. urophylla and E. tereticornis maps comprised 700 and 585 markers across 11 linkage groups, totaling at 1,208.2 and 1,241.4 cM in length, respectively. Extensive synteny and colinearity were observed as compared to three earlier DArT-based eucalypt maps (two maps with E. grandis × E. urophylla and one map of E. globulus) and with the E. grandis genome sequence. Fifty-three QTLs for growth (10–56 months of age) and wood density (56 months) were identified in 22 discrete regions on both maps, in which only one colocalizaiton was found between growth and wood density. Novel QTLs were revealed as compared with those previously detected on DArT-based maps for similar ages in Eucalyptus. Eleven to 585 positional candidate genes were obained for a 56-month-old QTL through aligning QTL confidence interval with the E. grandis genome. These results will assist in comparative genomics studies, targeted gene characterization, and marker-assisted selection in Eucalyptus and the related taxa. PMID:26695430

  1. Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators

    PubMed Central

    Scheibe, Marion; Arnoult, Nausica; Kappei, Dennis; Buchholz, Frank; Decottignies, Anabelle; Butter, Falk; Mann, Matthias

    2013-01-01

    Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)–based proteomics to obtain a high-confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pull-down experiment and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtained many candidates reported in these two studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knockdown of 10 genes encoding candidate proteins significantly affected total cellular levels of TERRA, and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/ARS2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knockdown of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation. PMID:23921659

  2. Quantitative Proteomic Analysis Reveals That Anti-Cancer Effects of Selenium-Binding Protein 1 In Vivo Are Associated with Metabolic Pathways

    PubMed Central

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M.; Lu, Zhaoxin; Yang, Wancai; Bie, Xiaomei

    2015-01-01

    Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways. PMID:25974208

  3. Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

    PubMed Central

    Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

    2016-01-01

    Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

  4. A Quantitative Chemotherapy Genetic Interaction Map Reveals Factors Associated with PARP Inhibitor Resistance.

    PubMed

    Hu, Hsien-Ming; Zhao, Xin; Kaushik, Swati; Robillard, Lilliane; Barthelet, Antoine; Lin, Kevin K; Shah, Khyati N; Simmons, Andy D; Raponi, Mitch; Harding, Thomas C; Bandyopadhyay, Sourav

    2018-04-17

    Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy. This quantitative map is predictive of interactions maintained in other cell lines, identifies DNA-repair factors, predicts cancer cell line responses to therapy, and prioritizes synergistic drug combinations. We identify that ARID1A loss confers resistance to PARP inhibitors in cells and ovarian cancer patients and that loss of GPBP1 causes resistance to cisplatin and PARP inhibitors through the regulation of genes involved in homologous recombination. This map helps navigate patient genomic data and optimize chemotherapeutic regimens by delineating factors involved in the response to specific types of DNA damage. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes.

    PubMed

    Adav, Sunil S; Li, An A; Manavalan, Arulmani; Punt, Peter; Sze, Siu Kwan

    2010-08-06

    The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

  6. Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

    PubMed

    Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

    2012-06-18

    Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Context influences on TALE–DNA binding revealed by quantitative profiling

    PubMed Central

    Rogers, Julia M.; Barrera, Luis A.; Reyon, Deepak; Sander, Jeffry D.; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L.

    2015-01-01

    Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE–DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000–20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE–DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design. PMID:26067805

  8. Context influences on TALE-DNA binding revealed by quantitative profiling.

    PubMed

    Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

    2015-06-11

    Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

  9. Quantitative Proteomic Analysis Reveals Populus cathayana Females Are More Sensitive and Respond More Sophisticatedly to Iron Deficiency than Males.

    PubMed

    Zhang, Sheng; Zhang, Yunxiang; Cao, Yanchun; Lei, Yanbao; Jiang, Hao

    2016-03-04

    Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder to nitrogen and phosphorus deficiencies, but little is known about the sex-specific differences in responses to iron deficiency. In this study, the effects of iron deficiency on the morphology, physiology, and proteome of P. cathayana males and females were investigated. The results showed that iron deficiency (25 days) significantly decreased height growth, photosynthetic rate, chlorophyll content, and tissue iron concentration in both sexes. A comparison between the sexes indicated that iron-deficient males had less height inhibition and photosynthesis system II or chloroplast ultrastructural damage than iron-deficient females. iTRAQ-based quantitative proteomic analysis revealed that 144 and 68 proteins were decreased in abundance (e.g., proteins involved in photosynthesis, carbohydrate and energy metabolism, and gene expression regulation) and 78 and 39 proteins were increased in abundance (e.g., proteins involved in amino acid metabolism and stress response) according to the criterion of ratio ≥1.5 in females and males, respectively. A comparison between the sexes indicated that iron-deficient females exhibited a greater change in the proteins involved in photosynthesis, carbon and energy metabolism, the redox system, and stress responsive proteins. This study reveals females are more sensitive and have a more sophisticated response to iron deficiency compared with males and provides new insights into differential sexual responses to nutrient deficiency.

  10. Quantitative analysis of single-molecule superresolution images

    PubMed Central

    Coltharp, Carla; Yang, Xinxing; Xiao, Jie

    2014-01-01

    This review highlights the quantitative capabilities of single-molecule localization-based superresolution imaging methods. In addition to revealing fine structural details, the molecule coordinate lists generated by these methods provide the critical ability to quantify the number, clustering, and colocalization of molecules with 10 – 50 nm resolution. Here we describe typical workflows and precautions for quantitative analysis of single-molecule superresolution images. These guidelines include potential pitfalls and essential control experiments, allowing critical assessment and interpretation of superresolution images. PMID:25179006

  11. Quantitative Metabolomics Reveals an Epigenetic Blueprint for Iron Acquisition in Uropathogenic Escherichia coli

    PubMed Central

    Henderson, Jeffrey P.; Crowley, Jan R.; Pinkner, Jerome S.; Walker, Jennifer N.; Tsukayama, Pablo; Stamm, Walter E.; Hooton, Thomas M.; Hultgren, Scott J.

    2009-01-01

    Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies. PMID:19229321

  12. Quantitative Proteomics Reveal Distinct Protein Regulations Caused by Aggregatibacter actinomycetemcomitans within Subgingival Biofilms

    PubMed Central

    Bao, Kai; Bostanci, Nagihan; Selevsek, Nathalie; Thurnheer, Thomas; Belibasakis, Georgios N.

    2015-01-01

    Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species “subgingival” biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute

  13. Quantitative proteomics reveals molecular mechanism of gamabufotalin and its potential inhibition on Hsp90 in lung cancer.

    PubMed

    Zhang, Liyuan; Yu, Zhenlong; Wang, Yan; Wang, Xiaobo; Zhang, Lianru; Wang, Chao; Yue, Qingxi; Wang, Xun; Deng, Sa; Huo, Xiaokui; Tian, Xiangge; Huang, Shanshan; Zhang, Baojing; Ma, Xiaochi

    2016-11-22

    Gamabufotalin (CS-6) is a major bufadienolide of Chansu, which shows desirable metabolic stability and less adverse effect in cancer therapy. CS-6 treatment inhibited the proliferation of NSCLC in a nanomolar range. And CS-6 could induce G2/M cell cycle arrest and apoptosis in A549 cells. However, its molecular mechanism in antitumor activity remains poorly understood. We employed a quantitative proteomics approach to identify the potential cellular targets of CS-6, and found 38 possible target-related proteins. Among them, 31 proteins were closely related in the protein-protein interaction network. One of the regulatory nodes in key pathways was occupied by Hsp90. Molecular docking revealed that CS-6 interacted with the ATP-binding sites of Hsp90. In addition, CS-6 inhibited the chaperone function of Hsp90 and reduced expression of Hsp90-dependent client proteins. Moreover, CS-6 markedly down-regulated the protein level of Hsp90 in tumor tissues of the xenograft mice. Taken together, our results suggest that CS-6 might be a novel inhibitor of Hsp90, and the possible network associated with CS-6 target-related proteins was constructed, which provided experimental evidence for the preclinical value of using CS-6 as an effective antitumor agent in treatment of NSCLC.

  14. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  15. Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

    PubMed

    Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

    2014-10-01

    Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

  16. SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

    PubMed Central

    Edelmann, Mariola J.; Shack, Leslie A.; Naske, Caitlin D.; Walters, Keisha B.; Nanduri, Bindu

    2014-01-01

    Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level. PMID:25470785

  17. Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

    PubMed

    Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

    2018-01-08

    For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

  18. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    PubMed

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-02

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  19. Comparing quantitative analysis on revealed comparative advantages of aquatic products trade of china and ASEAN based on 21st century maritime silk road

    NASA Astrophysics Data System (ADS)

    Luo, X. F.; Han, Y. H.; Li, Z. W.

    2017-11-01

    As the world’s leading aquaculture, aquatic production and trading country, China’s development of aquatic products trade with ASEAN is facing a historic opportunity in the favourable circumstances of construction of the 21st century Maritime Silk Road. In order to make guidance of the product selection and transformation for corresponding export enterprises, this article makes a quantitative analysis the Revealed Comparative Advantage of aquatic products trade from China and ASEAN respectively based on the HS classification and thoroughly compares the RCA indices. The comparison results show that the international competitiveness of aquatic products structures of China and ASEAN are quite different with few overlaps of strong competitive products, and there is a great gap between the two areas in many kinds of products.

  20. QUANTITATIVE DECISION TOOLS AND MANAGEMENT DEVELOPMENT PROGRAMS.

    ERIC Educational Resources Information Center

    BYARS, LLOYD L.; NUNN, GEOFFREY E.

    THIS ARTICLE OUTLINED THE CURRENT STATUS OF QUANTITATIVE METHODS AND OPERATIONS RESEARCH (OR), SKETCHED THE STRENGTHS OF TRAINING EFFORTS AND ISOLATED WEAKNESSES, AND FORMULATED WORKABLE CRITERIA FOR EVALUATING SUCCESS OF OPERATIONS RESEARCH TRAINING PROGRAMS. A SURVEY OF 105 COMPANIES REVEALED THAT PERT, INVENTORY CONTROL THEORY AND LINEAR…

  1. Quantitative proteomics reveals a role of JAZ7 in plant defense response to Pseudomonas syringae DC3000.

    PubMed

    Zhang, Tong; Meng, Li; Kong, Wenwen; Yin, Zepeng; Wang, Yang; Schneider, Jacqueline D; Chen, Sixue

    2018-03-20

    Jasmonate ZIM-domain (JAZ) proteins are key transcriptional repressors regulating various biological processes. Although many studies have studied JAZ proteins by genetic and biochemical analyses, little is known about JAZ7-associated global protein networks and how JAZ7 contributes to bacterial pathogen defense. In this study, we aim to fill this knowledge gap by conducting unbiased large-scale quantitative proteomics using tandem mass tags (TMT). We compared the proteomes of a JAZ7 knock-out line, a JAZ7 overexpression line, as well as the wild type Arabidopsis plants in the presence and absence of Pseudomonas syringae DC3000 infection. Both pairwise comparison and multi-factor analysis of variance reveal that differential proteins are enriched in biological processes such as primary and secondary metabolism, redox regulation, and response to stress. The differential regulation in these pathways may account for the alterations in plant size, redox homeostasis and accumulation of glucosinolates. In addition, possible interplay between genotype and environment is suggested as the abundance of seven proteins is influenced by the interaction of the two factors. Collectively, we demonstrate a role of JAZ7 in pathogen defense and provide a list of proteins that are uniquely responsive to genetic disruption, pathogen infection, or the interaction between genotypes and environmental factors. We report proteomic changes as a result of genetic perturbation of JAZ7, and the contribution of JAZ7 in plant immunity. Specifically, the similarity between the proteomes of a JAZ7 knockout mutant and the wild type plants confirmed the functional redundancy of JAZs. In contrast, JAZ7 overexpression plants were much different, and proteomic analysis of the JAZ7 overexpression plants under Pst DC3000 infection revealed that JAZ7 may regulate plant immunity via ROS modulation, energy balance and glucosinolate biosynthesis. Multiple variate analysis for this two-factor proteomics

  2. Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation.

    PubMed

    Xu, Gege; Davis, Jasmine Cc; Goonatilleke, Elisha; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B

    2017-01-01

    The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards. The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers. This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of α(1,2)-fucosylated HMOs were used to determine the mothers' phenotypic secretor status with high sensitivity and specificity. We used Friedman's test and Wilcoxon's signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student's t test was used to compare secretors and nonsecretors. A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05). HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the health of breastfed infants in order to investigate the protective

  3. Novel phosphorylation states of the yeast spindle pole body.

    PubMed

    Fong, Kimberly K; Zelter, Alex; Graczyk, Beth; Hoyt, Jill M; Riffle, Michael; Johnson, Richard; MacCoss, Michael J; Davis, Trisha N

    2018-06-14

    Phosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of enriched Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature sensitive mutation, with particular focus on phosphorylation events on the γ-tubulin small complex (γ-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites that influence microtubule formation. © 2018. Published by The Company of Biologists Ltd.

  4. Reconstruction of the temporal signaling network in Salmonella-infected human cells.

    PubMed

    Budak, Gungor; Eren Ozsoy, Oyku; Aydin Son, Yesim; Can, Tolga; Tuncbag, Nurcan

    2015-01-01

    Salmonella enterica is a bacterial pathogen that usually infects its host through food sources. Translocation of the pathogen proteins into the host cells leads to changes in the signaling mechanism either by activating or inhibiting the host proteins. Given that the bacterial infection modifies the response network of the host, a more coherent view of the underlying biological processes and the signaling networks can be obtained by using a network modeling approach based on the reverse engineering principles. In this work, we have used a published temporal phosphoproteomic dataset of Salmonella-infected human cells and reconstructed the temporal signaling network of the human host by integrating the interactome and the phosphoproteomic dataset. We have combined two well-established network modeling frameworks, the Prize-collecting Steiner Forest (PCSF) approach and the Integer Linear Programming (ILP) based edge inference approach. The resulting network conserves the information on temporality, direction of interactions, while revealing hidden entities in the signaling, such as the SNARE binding, mTOR signaling, immune response, cytoskeleton organization, and apoptosis pathways. Targets of the Salmonella effectors in the host cells such as CDC42, RHOA, 14-3-3δ, Syntaxin family, Oxysterol-binding proteins were included in the reconstructed signaling network although they were not present in the initial phosphoproteomic data. We believe that integrated approaches, such as the one presented here, have a high potential for the identification of clinical targets in infectious diseases, especially in the Salmonella infections.

  5. Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action.

    PubMed

    Rabiee, Atefeh; Krüger, Marcus; Ardenkjær-Larsen, Jacob; Kahn, C Ronald; Emanuelli, Brice

    2018-07-01

    Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1 -/- and IRS-2 -/- mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Quantitative metagenomics reveals unique gut microbiome biomarkers in ankylosing spondylitis.

    PubMed

    Wen, Chengping; Zheng, Zhijun; Shao, Tiejuan; Liu, Lin; Xie, Zhijun; Le Chatelier, Emmanuelle; He, Zhixing; Zhong, Wendi; Fan, Yongsheng; Zhang, Linshuang; Li, Haichang; Wu, Chunyan; Hu, Changfeng; Xu, Qian; Zhou, Jia; Cai, Shunfeng; Wang, Dawei; Huang, Yun; Breban, Maxime; Qin, Nan; Ehrlich, Stanislav Dusko

    2017-07-27

    The assessment and characterization of the gut microbiome has become a focus of research in the area of human autoimmune diseases. Ankylosing spondylitis is an inflammatory autoimmune disease and evidence showed that ankylosing spondylitis may be a microbiome-driven disease. To investigate the relationship between the gut microbiome and ankylosing spondylitis, a quantitative metagenomics study based on deep shotgun sequencing was performed, using gut microbial DNA from 211 Chinese individuals. A total of 23,709 genes and 12 metagenomic species were shown to be differentially abundant between ankylosing spondylitis patients and healthy controls. Patients were characterized by a form of gut microbial dysbiosis that is more prominent than previously reported cases with inflammatory bowel disease. Specifically, the ankylosing spondylitis patients demonstrated increases in the abundance of Prevotella melaninogenica, Prevotella copri, and Prevotella sp. C561 and decreases in Bacteroides spp. It is noteworthy that the Bifidobacterium genus, which is commonly used in probiotics, accumulated in the ankylosing spondylitis patients. Diagnostic algorithms were established using a subset of these gut microbial biomarkers. Alterations of the gut microbiome are associated with development of ankylosing spondylitis. Our data suggest biomarkers identified in this study might participate in the pathogenesis or development process of ankylosing spondylitis, providing new leads for the development of new diagnostic tools and potential treatments.

  7. The integration of quantitative genetics, paleontology, and neontology reveals genetic underpinnings of primate dental evolution.

    PubMed

    Hlusko, Leslea J; Schmitt, Christopher A; Monson, Tesla A; Brasil, Marianne F; Mahaney, Michael C

    2016-08-16

    Developmental genetics research on mice provides a relatively sound understanding of the genes necessary and sufficient to make mammalian teeth. However, mouse dentitions are highly derived compared with human dentitions, complicating the application of these insights to human biology. We used quantitative genetic analyses of data from living nonhuman primates and extensive osteological and paleontological collections to refine our assessment of dental phenotypes so that they better represent how the underlying genetic mechanisms actually influence anatomical variation. We identify ratios that better characterize the output of two dental genetic patterning mechanisms for primate dentitions. These two newly defined phenotypes are heritable with no measurable pleiotropic effects. When we consider how these two phenotypes vary across neontological and paleontological datasets, we find that the major Middle Miocene taxonomic shift in primate diversity is characterized by a shift in these two genetic outputs. Our results build on the mouse model by combining quantitative genetics and paleontology, and thereby elucidate how genetic mechanisms likely underlie major events in primate evolution.

  8. Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection.

    PubMed

    de la Fuente, Cynthia; Pinkham, Chelsea; Dabbagh, Deemah; Beitzel, Brett; Garrison, Aura; Palacios, Gustavo; Hodge, Kimberley Alex; Petricoin, Emanuel F; Schmaljohn, Connie; Campbell, Catherine E; Narayanan, Aarthi; Kehn-Hall, Kylene

    2018-01-01

    Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.

  9. 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

  10. Relationship between Plaque Echo, Thickness and Neovascularization Assessed by Quantitative and Semi-quantitative Contrast-Enhanced Ultrasonography in Different Stenosis Groups.

    PubMed

    Song, Yan; Feng, Jun; Dang, Ying; Zhao, Chao; Zheng, Jie; Ruan, Litao

    2017-12-01

    The aim of this study was to determine the relationship between plaque echo, thickness and neovascularization in different stenosis groups using quantitative and semi-quantitative contrast-enhanced ultrasound (CEUS) in patients with carotid atherosclerosis plaque. A total of 224 plaques were divided into mild stenosis (<50%; 135 plaques, 60.27%), moderate stenosis (50%-69%; 39 plaques, 17.41%) and severe stenosis (70%-99%; 50 plaques, 22.32%) groups. Quantitative and semi-quantitative methods were used to assess plaque neovascularization and determine the relationship between plaque echo, thickness and neovascularization. Correlation analysis revealed no relationship of neovascularization with plaque echo in the groups using either quantitative or semi-quantitative methods. Furthermore, there was no correlation of neovascularization with plaque thickness using the semi-quantitative method. The ratio of areas under the curve (RAUC) was negatively correlated with plaque thickness (r = -0.317, p = 0.001) in the mild stenosis group. With the quartile method, plaque thickness of the mild stenosis group was divided into four groups, with significant differences between the 1.5-2.2 mm and ≥3.5 mm groups (p = 0.002), 2.3-2.8 mm and ≥3.5 mm groups (p <0.001) and 2.9-3.4 mm and ≥3.5 mm groups (p <0.001). Both semi-quantitative and quantitative CEUS methods characterizing neovascularization of plaque are equivalent with respect to assessing relationships between neovascularization, echogenicity and thickness. However, the quantitative method could fail for plaque <3.5 mm because of motion artifacts. Copyright © 2017 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.

  11. Global Phosphoproteomics Identifies a Major Role for AKT and 14-3-3 in Regulating EDC3*

    PubMed Central

    Larance, Mark; Rowland, Alexander F.; Hoehn, Kyle L.; Humphreys, David T.; Preiss, Thomas; Guilhaus, Michael; James, David E.

    2010-01-01

    Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization. PMID:20051463

  12. Embedding Quantitative Methods by Stealth in Political Science: Developing a Pedagogy for Psephology

    ERIC Educational Resources Information Center

    Gunn, Andrew

    2017-01-01

    Student evaluations of quantitative methods courses in political science often reveal they are characterised by aversion, alienation and anxiety. As a solution to this problem, this paper describes a pedagogic research project with the aim of embedding quantitative methods by stealth into the first-year undergraduate curriculum. This paper…

  13. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

    PubMed

    Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

    2012-12-01

    Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

  14. Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

    PubMed

    Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

    2017-02-15

    Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

  15. Strigolactone-regulated proteins revealed by iTRAQ-based quantitative proteomics in Arabidopsis.

    PubMed

    Li, Zhou; Czarnecki, Olaf; Chourey, Karuna; Yang, Jun; Tuskan, Gerald A; Hurst, Gregory B; Pan, Chongle; Chen, Jin-Gui

    2014-03-07

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. A quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found that SLs regulate the expression of about three dozen proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  16. qSR: a quantitative super-resolution analysis tool reveals the cell-cycle dependent organization of RNA Polymerase I in live human cells.

    PubMed

    Andrews, J O; Conway, W; Cho, W -K; Narayanan, A; Spille, J -H; Jayanth, N; Inoue, T; Mullen, S; Thaler, J; Cissé, I I

    2018-05-09

    We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell.

  17. Quantitative flux analysis reveals folate-dependent NADPH production

    NASA Astrophysics Data System (ADS)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  18. Quantitative imaging reveals heterogeneous growth dynamics and treatment-dependent residual tumor distributions in a three-dimensional ovarian cancer model

    NASA Astrophysics Data System (ADS)

    Celli, Jonathan P.; Rizvi, Imran; Evans, Conor L.; Abu-Yousif, Adnan O.; Hasan, Tayyaba

    2010-09-01

    Three-dimensional tumor models have emerged as valuable in vitro research tools, though the power of such systems as quantitative reporters of tumor growth and treatment response has not been adequately explored. We introduce an approach combining a 3-D model of disseminated ovarian cancer with high-throughput processing of image data for quantification of growth characteristics and cytotoxic response. We developed custom MATLAB routines to analyze longitudinally acquired dark-field microscopy images containing thousands of 3-D nodules. These data reveal a reproducible bimodal log-normal size distribution. Growth behavior is driven by migration and assembly, causing an exponential decay in spatial density concomitant with increasing mean size. At day 10, cultures are treated with either carboplatin or photodynamic therapy (PDT). We quantify size-dependent cytotoxic response for each treatment on a nodule by nodule basis using automated segmentation combined with ratiometric batch-processing of calcein and ethidium bromide fluorescence intensity data (indicating live and dead cells, respectively). Both treatments reduce viability, though carboplatin leaves micronodules largely structurally intact with a size distribution similar to untreated cultures. In contrast, PDT treatment disrupts micronodular structure, causing punctate regions of toxicity, shifting the distribution toward smaller sizes, and potentially increasing vulnerability to subsequent chemotherapeutic treatment.

  19. Comprehensive Quantitative Analysis on Privacy Leak Behavior

    PubMed Central

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

    2013-01-01

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

  20. Comprehensive quantitative analysis on privacy leak behavior.

    PubMed

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

    2013-01-01

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

  1. Clusters of Insomnia Disorder: An Exploratory Cluster Analysis of Objective Sleep Parameters Reveals Differences in Neurocognitive Functioning, Quantitative EEG, and Heart Rate Variability

    PubMed Central

    Miller, Christopher B.; Bartlett, Delwyn J.; Mullins, Anna E.; Dodds, Kirsty L.; Gordon, Christopher J.; Kyle, Simon D.; Kim, Jong Won; D'Rozario, Angela L.; Lee, Rico S.C.; Comas, Maria; Marshall, Nathaniel S.; Yee, Brendon J.; Espie, Colin A.; Grunstein, Ronald R.

    2016-01-01

    Study Objectives: To empirically derive and evaluate potential clusters of Insomnia Disorder through cluster analysis from polysomnography (PSG). We hypothesized that clusters would differ on neurocognitive performance, sleep-onset measures of quantitative (q)-EEG and heart rate variability (HRV). Methods: Research volunteers with Insomnia Disorder (DSM-5) completed a neurocognitive assessment and overnight PSG measures of total sleep time (TST), wake time after sleep onset (WASO), and sleep onset latency (SOL) were used to determine clusters. Results: From 96 volunteers with Insomnia Disorder, cluster analysis derived at least two clusters from objective sleep parameters: Insomnia with normal objective sleep duration (I-NSD: n = 53) and Insomnia with short sleep duration (I-SSD: n = 43). At sleep onset, differences in HRV between I-NSD and I-SSD clusters suggest attenuated parasympathetic activity in I-SSD (P < 0.05). Preliminary work suggested three clusters by retaining the I-NSD and splitting the I-SSD cluster into two: I-SSD A (n = 29): defined by high WASO and I-SSD B (n = 14): a second I-SSD cluster with high SOL and medium WASO. The I-SSD B cluster performed worse than I-SSD A and I-NSD for sustained attention (P ≤ 0.05). In an exploratory analysis, q-EEG revealed reduced spectral power also in I-SSD B before (Delta, Alpha, Beta-1) and after sleep-onset (Beta-2) compared to I-SSD A and I-NSD (P ≤ 0.05). Conclusions: Two insomnia clusters derived from cluster analysis differ in sleep onset HRV. Preliminary data suggest evidence for three clusters in insomnia with differences for sustained attention and sleep-onset q-EEG. Clinical Trial Registration: Insomnia 100 sleep study: Australia New Zealand Clinical Trials Registry (ANZCTR) identification number 12612000049875. URL: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=347742. Citation: Miller CB, Bartlett DJ, Mullins AE, Dodds KL, Gordon CJ, Kyle SD, Kim JW, D'Rozario AL, Lee RS, Comas

  2. Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation123

    PubMed Central

    Xu, Gege; Davis, Jasmine CC; Goonatilleke, Elisha; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B

    2017-01-01

    Background: The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards. Objective: The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers. Methods: This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of α(1,2)-fucosylated HMOs were used to determine the mothers’ phenotypic secretor status with high sensitivity and specificity. We used Friedman’s test and Wilcoxon’s signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student’s t test was used to compare secretors and nonsecretors. Results: A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05). Conclusions: HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the

  3. Power-Law Modeling of Cancer Cell Fates Driven by Signaling Data to Reveal Drug Effects

    PubMed Central

    Zhang, Fan; Wu, Min; Kwoh, Chee Keong; Zheng, Jie

    2016-01-01

    Extracellular signals are captured and transmitted by signaling proteins inside a cell. An important type of cellular responses to the signals is the cell fate decision, e.g., apoptosis. However, the underlying mechanisms of cell fate regulation are still unclear, thus comprehensive and detailed kinetic models are not yet available. Alternatively, data-driven models are promising to bridge signaling data with the phenotypic measurements of cell fates. The traditional linear model for data-driven modeling of signaling pathways has its limitations because it assumes that the a cell fate is proportional to the activities of signaling proteins, which is unlikely in the complex biological systems. Therefore, we propose a power-law model to relate the activities of all the measured signaling proteins to the probabilities of cell fates. In our experiments, we compared our nonlinear power-law model with the linear model on three cancer datasets with phosphoproteomics and cell fate measurements, which demonstrated that the nonlinear model has superior performance on cell fates prediction. By in silico simulation of virtual protein knock-down, the proposed model is able to reveal drug effects which can complement traditional approaches such as binding affinity analysis. Moreover, our model is able to capture cell line specific information to distinguish one cell line from another in cell fate prediction. Our results show that the power-law data-driven model is able to perform better in cell fate prediction and provide more insights into the signaling pathways for cancer cell fates than the linear model. PMID:27764199

  4. A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila.

    PubMed

    Hoermann, Astrid; Cicin-Sain, Damjan; Jaeger, Johannes

    2016-03-15

    Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

    PubMed Central

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

    2015-01-01

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

  6. Proteogenomics connects somatic mutations to signalling in breast cancer.

    PubMed

    Mertins, Philipp; Mani, D R; Ruggles, Kelly V; Gillette, Michael A; Clauser, Karl R; Wang, Pei; Wang, Xianlong; Qiao, Jana W; Cao, Song; Petralia, Francesca; Kawaler, Emily; Mundt, Filip; Krug, Karsten; Tu, Zhidong; Lei, Jonathan T; Gatza, Michael L; Wilkerson, Matthew; Perou, Charles M; Yellapantula, Venkata; Huang, Kuan-lin; Lin, Chenwei; McLellan, Michael D; Yan, Ping; Davies, Sherri R; Townsend, R Reid; Skates, Steven J; Wang, Jing; Zhang, Bing; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Ding, Li; Paulovich, Amanda G; Fenyö, David; Ellis, Matthew J; Carr, Steven A

    2016-06-02

    Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

  7. Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

    PubMed

    Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

    2017-07-14

    The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells.

    PubMed

    Na, Yi Rang; Hong, Ji Hye; Lee, Min Yong; Jung, Jae Hun; Jung, Daun; Kim, Young Won; Son, Dain; Choi, Murim; Kim, Kwang Pyo; Seok, Seung Hyeok

    2015-10-01

    Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism

    PubMed Central

    Sychev, Zoi E.; Hu, Alex; Lagunoff, Michael

    2017-01-01

    Kaposi’s Sarcoma associated Herpesvirus (KSHV), an oncogenic, human gamma-herpesvirus, is the etiological agent of Kaposi’s Sarcoma the most common tumor of AIDS patients world-wide. KSHV is predominantly latent in the main KS tumor cell, the spindle cell, a cell of endothelial origin. KSHV modulates numerous host cell-signaling pathways to activate endothelial cells including major metabolic pathways involved in lipid metabolism. To identify the underlying cellular mechanisms of KSHV alteration of host signaling and endothelial cell activation, we identified changes in the host proteome, phosphoproteome and transcriptome landscape following KSHV infection of endothelial cells. A Steiner forest algorithm was used to integrate the global data sets and, together with transcriptome based predicted transcription factor activity, cellular networks altered by latent KSHV were predicted. Several interesting pathways were identified, including peroxisome biogenesis. To validate the predictions, we showed that KSHV latent infection increases the number of peroxisomes per cell. Additionally, proteins involved in peroxisomal lipid metabolism of very long chain fatty acids, including ABCD3 and ACOX1, are required for the survival of latently infected cells. In summary, novel cellular pathways altered during herpesvirus latency that could not be predicted by a single systems biology platform, were identified by integrated proteomics and transcriptomics data analysis and when correlated with our metabolomics data revealed that peroxisome lipid metabolism is essential for KSHV latent infection of endothelial cells. PMID:28257516

  10. Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pan, Xiao; Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824; Whitten, Douglas A.

    Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤ 30 min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250 ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at bothmore » known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment. - Highlights: ► Mycotoxin deoxynivalenol (DON) induces immunotoxicity via ribotoxic stress response. ► SILAC

  11. Comparative mapping reveals quantitative trait loci that affect spawning time in coho salmon (Oncorhynchus kisutch)

    PubMed Central

    Araneda, Cristian; Díaz, Nelson F.; Gomez, Gilda; López, María Eugenia; Iturra, Patricia

    2012-01-01

    Spawning time in salmonids is a sex-limited quantitative trait that can be modified by selection. In rainbow trout (Oncorhynchus mykiss), various quantitative trait loci (QTL) that affect the expression of this trait have been discovered. In this study, we describe four microsatellite loci associated with two possible spawning time QTL regions in coho salmon (Oncorhynchus kisutch). The four loci were identified in females from two populations (early and late spawners) produced by divergent selection from the same base population. Three of the loci (OmyFGT34TUF, One2ASC and One19ASC) that were strongly associated with spawning time in coho salmon (p < 0.0002) were previously associated with QTL for the same trait in rainbow trout; a fourth loci (Oki10) with a suggestive association (p = 0.00035) mapped 10 cM from locus OmyFGT34TUF in rainbow trout. The changes in allelic frequency observed after three generations of selection were greater than expected because of genetic drift. This work shows that comparing information from closely-related species is a valid strategy for identifying QTLs for marker-assisted selection in species whose genomes are poorly characterized or lack a saturated genetic map. PMID:22888302

  12. SILAC-based quantitative proteomic analysis reveals widespread molecular alterations in human skin keratinocytes upon chronic arsenic exposure.

    PubMed

    Mir, Sartaj Ahmad; Pinto, Sneha M; Paul, Somnath; Raja, Remya; Nanjappa, Vishalakshi; Syed, Nazia; Advani, Jayshree; Renuse, Santosh; Sahasrabuddhe, Nandini A; Prasad, T S Keshava; Giri, Ashok K; Gowda, Harsha; Chatterjee, Aditi

    2017-03-01

    Chronic exposure to arsenic is associated with dermatological and nondermatological disorders. Consumption of arsenic-contaminated drinking water results in accumulation of arsenic in liver, spleen, kidneys, lungs, and gastrointestinal tract. Although arsenic is cleared from these sites, a substantial amount of residual arsenic is left in keratin-rich tissues including skin. Epidemiological studies suggest the association of skin cancer upon arsenic exposure, however, the mechanism of arsenic-induced carcinogenesis is not completely understood. We developed a cell line based model to understand the molecular mechanisms involved in arsenic-mediated toxicity and carcinogenicity. Human skin keratinocyte cell line, HaCaT, was chronically exposed to 100 nM sodium arsenite over a period of 6 months. We observed an increase in basal ROS levels in arsenic-exposed cells. SILAC-based quantitative proteomics approach resulted in identification of 2111 proteins of which 42 proteins were found to be overexpressed and 54 downregulated (twofold) upon chronic arsenic exposure. Our analysis revealed arsenic-induced overexpression of aldo-keto reductase family 1 member C2 (AKR1C2), aldo-keto reductase family 1 member C3 (AKR1C3), glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) among others. We observed downregulation of several members of the plakin family including periplakin (PPL), envoplakin (EVPL), and involucrin (IVL) that are essential for terminal differentiation of keratinocytes. MRM and Western blot analysis confirmed differential expression of several candidate proteins. Our study provides insights into molecular alterations upon chronic arsenic exposure on skin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast

    PubMed Central

    Dephoure, Noah; Hwang, Sunyoung; O'Sullivan, Ciara; Dodgson, Stacie E; Gygi, Steven P; Amon, Angelika; Torres, Eduardo M

    2014-01-01

    Aneuploidy causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. Here, we examined the consequences of aneuploidy on the proteome of aneuploid budding yeast strains. We show that although protein levels largely scale with gene copy number, subunits of multi-protein complexes are notable exceptions. Posttranslational mechanisms attenuate their expression when their encoding genes are in excess. Our proteomic analyses further revealed a novel aneuploidy-associated protein expression signature characteristic of altered metabolism and redox homeostasis. Indeed aneuploid cells harbor increased levels of reactive oxygen species (ROS). Interestingly, increased protein turnover attenuates ROS levels and this novel aneuploidy-associated signature and improves the fitness of most aneuploid strains. Our results show that aneuploidy causes alterations in metabolism and redox homeostasis. Cells respond to these alterations through both transcriptional and posttranscriptional mechanisms. DOI: http://dx.doi.org/10.7554/eLife.03023.001 PMID:25073701

  14. High-resolution mapping of a fruit firmness-related quantitative trait locus in tomato reveals epistatic interactions associated with a complex combinatorial locus.

    PubMed

    Chapman, Natalie H; Bonnet, Julien; Grivet, Laurent; Lynn, James; Graham, Neil; Smith, Rebecca; Sun, Guiping; Walley, Peter G; Poole, Mervin; Causse, Mathilde; King, Graham J; Baxter, Charles; Seymour, Graham B

    2012-08-01

    Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Fir(s.p.)QTL2.1 to Fir(s.p.)QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Fir(s.p.)QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Fir(s.p.)QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

  15. Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance.

    PubMed

    Lai, Shu-Jung; Tu, I-Fan; Wu, Wan-Ling; Yang, Jhih-Tian; Luk, Louis Y P; Lai, Mei-Chin; Tsai, Yu-Hsuan; Wu, Shih-Hsiung

    2017-05-25

    Phosphorylation of amino acid residues on proteins is an important and common post-translational modification in both eukaryotes and prokaryotes. Most research work has been focused on phosphorylation of serine, threonine or tyrosine residues, whereas phosphorylation of other amino acids are significantly less clear due to the controversy on their stability under standard bioanalytical conditions. Here we applied a shotgun strategy to analyze the histidine and aspartate phosphorylations in different microbes. Our results collectively indicate that histidine and aspartate phosphorylations frequently occur also in proteins that are not part of the two-component systems. Noticeably, a number of the modified proteins are pathogenesis-related or essential for survival in host. These include the zinc ion periplasmic transporter ZnuA in Acinetobacter baumannii SK17, the multidrug and toxic compound extrusion (MATE) channel YeeO in Klebsiella pneumoniae NTUH-K2044, branched amino acid transporter AzlC in Vibrio vulnificus and the RNA-modifying pseudouridine synthase in Helicobacter pylori. In summary, histidine and aspartate phosphorylation is likely to be ubiquitous and to take place in proteins of various functions. This work also sheds light into how these functionally important proteins and potential drug targets might be regulated at a post-translational level.

  16. Physiological frailty index (PFI): quantitative in-life estimate of individual biological age in mice.

    PubMed

    Antoch, Marina P; Wrobel, Michelle; Kuropatwinski, Karen K; Gitlin, Ilya; Leonova, Katerina I; Toshkov, Ilia; Gleiberman, Anatoli S; Hutson, Alan D; Chernova, Olga B; Gudkov, Andrei V

    2017-03-19

    The development of healthspan-extending pharmaceuticals requires quantitative estimation of age-related progressive physiological decline. In humans, individual health status can be quantitatively assessed by means of a frailty index (FI), a parameter which reflects the scale of accumulation of age-related deficits. However, adaptation of this methodology to animal models is a challenging task since it includes multiple subjective parameters. Here we report a development of a quantitative non-invasive procedure to estimate biological age of an individual animal by creating physiological frailty index (PFI). We demonstrated the dynamics of PFI increase during chronological aging of male and female NIH Swiss mice. We also demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity and provide a tool for its quantitative assessment. Additionally, we showed that PFI could reveal anti-aging effect of mTOR inhibitor rapatar (bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors. Together, these data introduce PFI as a reliable, non-invasive, quantitative tool suitable for testing potential anti-aging pharmaceuticals in pre-clinical studies.

  17. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer

    PubMed Central

    Chen, I-Hsuan; Xue, Liang; Hsu, Chuan-Chih; Paez, Juan Sebastian Paez; Pan, Li; Andaluz, Hillary; Wendt, Michael K.; Iliuk, Anton B.; Tao, W. Andy

    2017-01-01

    The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring. PMID:28270605

  18. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer.

    PubMed

    Chen, I-Hsuan; Xue, Liang; Hsu, Chuan-Chih; Paez, Juan Sebastian Paez; Pan, Li; Andaluz, Hillary; Wendt, Michael K; Iliuk, Anton B; Zhu, Jian-Kang; Tao, W Andy

    2017-03-21

    The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

  19. Quantitative Differential Expression Analysis Reveals Mir-7 As Major Islet MicroRNA

    PubMed Central

    Bravo-Egana, Valia; Rosero, Samuel; Molano, R. Damaris; Pileggi, Antonello; Ricordi, Camillo; Domínguez-Bendala, Juan; Pastori, Ricardo L.

    2008-01-01

    MicroRNAs (miRNAs) are non-coding gene products that regulate gene expression through specific binding to target mRNAs. Cell-specific patterns of miRNAs are associated with the acquisition and maintenance of a given phenotype, such as endocrine pancreas (islets). We hypothesized that a subset of miRNAs could be differentially expressed in the islets. Using miRNA microarray technology and quantitative RT-PCR we identified a subset of miRNAs that are the most differentially expressed islet miRNAs (ratio islet/acinar >150-fold), mir-7 being the most abundant. A similarly high ratio for mir-7 was observed in human islets. The ratio islet/acinar for mir-375, a previously described islet miRNA, was <10, and is 2.5X more abundant in the islets than mir-7. Therefore, we conclude that mir-7 is the most abundant endocrine miRNA in islets while mir-375 is the most abundant intra-islet miRNA. Our results may offer new insights into regulatory pathways of islet gene expression. PMID:18086561

  20. Proteomic Analysis to Identify Functional Molecules in Drug Resistance Caused by E-Cadherin Knockdown in 3D-Cultured Colorectal Cancer Models

    DTIC Science & Technology

    2014-09-01

    total number of 538 phosphopeptides were identified, among which 350 phosphopeptides had been identified with the first round of TiO2 enrichment and 430...year research and the collection of proteomic and phosphoproteomic data is still in process. PRODUCTS Manuscripts: Yue XS , Hummon AB. Combining...of IMAC and TiO2 enrichment methods to increase phosphoproteomic identifications, manuscript in preparation. Yue XS , Hummon AB. Proteomic and

  1. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

    PubMed

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

    2015-03-30

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

  2. LymPHOS 2.0: an update of a phosphosite database of primary human T cells

    PubMed Central

    Nguyen, Tien Dung; Vidal-Cortes, Oriol; Gallardo, Oscar; Abian, Joaquin; Carrascal, Montserrat

    2015-01-01

    LymPHOS is a web-oriented database containing peptide and protein sequences and spectrometric information on the phosphoproteome of primary human T-Lymphocytes. Current release 2.0 contains 15 566 phosphorylation sites from 8273 unique phosphopeptides and 4937 proteins, which correspond to a 45-fold increase over the original database description. It now includes quantitative data on phosphorylation changes after time-dependent treatment with activators of the TCR-mediated signal transduction pathway. Sequence data quality has also been improved with the use of multiple search engines for database searching. LymPHOS can be publicly accessed at http://www.lymphos.org. Database URL: http://www.lymphos.org. PMID:26708986

  3. Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis

    PubMed Central

    Tsujikawa, Takahiro; Kumar, Sushil; Borkar, Rohan N.; Azimi, Vahid; Thibault, Guillaume; Chang, Young Hwan; Balter, Ariel; Kawashima, Rie; Choe, Gina; Sauer, David; El Rassi, Edward; Clayburgh, Daniel R.; Kulesz-Martin, Molly F.; Lutz, Eric R.; Zheng, Lei; Jaffee, Elizabeth M.; Leyshock, Patrick; Margolin, Adam A.; Mori, Motomi; Gray, Joe W.; Flint, Paul W.; Coussens, Lisa M.

    2017-01-01

    SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment. PMID:28380359

  4. Proof of the quantitative potential of immunofluorescence by mass spectrometry.

    PubMed

    Toki, Maria I; Cecchi, Fabiola; Hembrough, Todd; Syrigos, Konstantinos N; Rimm, David L

    2017-03-01

    Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R 2 =0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

  5. Quantitative mass spectrometry reveals changes in SNAP-25 isoforms in schizophrenia

    PubMed Central

    Barakauskas, Vilte E; Moradian, Annie; Barr, Alasdair M.; Beasley, Clare L; Rosoklija, Gorazd; Mann, J John; Ilievski, Boro; Stankov, Aleksandar; Dwork, Andrew J; Falkai, Peter; Morin, Gregg B; Honer, William G

    2016-01-01

    SNAP-25 and syntaxin are presynaptic terminal SNARE proteins altered in amount and function in schizophrenia. In the ventral caudate, we observed 32% lower SNAP-25 and 26% lower syntaxin, but greater interaction between the two proteins using an in vitro assay. SNAP-25 has two isoforms, SNAP-25A and B, differing by only 9 amino acids, but with different effects on neurotransmission. A quantitative mass spectrometry assay was developed to measure total SNAP-25, and proportions of SNAP-25A and B. The assay had a good linear range (50- to 150-fold) and coefficient of variation (4.5%). We studied ventral caudate samples from patients with schizophrenia (n=15) previously reported to have lower total SNAP-25 than controls (n=13). We confirmed 27% lower total SNAP-25 in schizophrenia, and observed 31% lower SNAP-25A (P = 0.002) with 20% lower SNAP-25B amounts (P = 0.10). Lower SNAP-25A amount correlated with greater SNAP-25-syntaxin protein-protein interactions (r = -0.41, P = 0.03); the level of SNAP-25B did not. Administration of haloperidol or clozapine to rats did not mimic the changes found in schizophrenia. The findings suggest that lower levels of SNAP-25 in schizophrenia may represent a greater effect of the illness on the SNAP-25A isoform. This in turn could contribute to the greater interaction between SNAP25 and syntaxin, and possibly disturb neurotransmission in the illness. PMID:26971072

  6. Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco.

    PubMed

    Stanislas, Thomas; Bouyssie, David; Rossignol, Michel; Vesa, Simona; Fromentin, Jérôme; Morel, Johanne; Pichereaux, Carole; Monsarrat, Bernard; Simon-Plas, Françoise

    2009-09-01

    A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used (14)N/(15)N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in

  7. Quantitative Proteomics Reveals a Dynamic Association of Proteins to Detergent-resistant Membranes upon Elicitor Signaling in Tobacco*

    PubMed Central

    Stanislas, Thomas; Bouyssie, David; Rossignol, Michel; Vesa, Simona; Fromentin, Jérôme; Morel, Johanne; Pichereaux, Carole; Monsarrat, Bernard; Simon-Plas, Françoise

    2009-01-01

    A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used 14N/15N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the

  8. Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

    PubMed

    Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan

    2016-01-01

    Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular

  9. Quantitative Correlation between Infectivity and Gp120 Density on HIV-1 Virions Revealed by Optical Trapping Virometry*

    PubMed Central

    DeSantis, Michael C.; Kim, Jin H.; Song, Hanna; Klasse, Per Johan

    2016-01-01

    The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves 1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface, 2) site-specific labeling of Env-specific antibody Fab with a fluorophore at high efficiency, and 3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with a Hill coefficient of 2.4 ± 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection. PMID:27129237

  10. LmaPA2G4, a Homolog of Human Ebp1, Is an Essential Gene and Inhibits Cell Proliferation in L. major

    PubMed Central

    Joyce, Michelle V.; Morales, Miguel A.

    2014-01-01

    We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I∶C)] as shown in an electrophoretic mobility shift assay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved in translation initiation and elongation. Significantly, we found a strong reduction of de novo protein biosynthesis in transgenic parasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentially implicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention. PMID:24421916

  11. Linking the proteins--elucidation of proteome-scale networks using mass spectrometry.

    PubMed

    Pflieger, Delphine; Gonnet, Florence; de la Fuente van Bentem, Sergio; Hirt, Heribert; de la Fuente, Alberto

    2011-01-01

    Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks. Copyright © 2010 Wiley Periodicals, Inc.

  12. Quantitative genetics

    USDA-ARS?s Scientific Manuscript database

    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, ...

  13. Quantitative filter forensics for indoor particle sampling.

    PubMed

    Haaland, D; Siegel, J A

    2017-03-01

    Filter forensics is a promising indoor air investigation technique involving the analysis of dust which has collected on filters in central forced-air heating, ventilation, and air conditioning (HVAC) or portable systems to determine the presence of indoor particle-bound contaminants. In this study, we summarize past filter forensics research to explore what it reveals about the sampling technique and the indoor environment. There are 60 investigations in the literature that have used this sampling technique for a variety of biotic and abiotic contaminants. Many studies identified differences between contaminant concentrations in different buildings using this technique. Based on this literature review, we identified a lack of quantification as a gap in the past literature. Accordingly, we propose an approach to quantitatively link contaminants extracted from HVAC filter dust to time-averaged integrated air concentrations. This quantitative filter forensics approach has great potential to measure indoor air concentrations of a wide variety of particle-bound contaminants. Future studies directly comparing quantitative filter forensics to alternative sampling techniques are required to fully assess this approach, but analysis of past research suggests the enormous possibility of this approach. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Expression Quantitative Trait Locus Mapping across Water Availability Environments Reveals Contrasting Associations with Genomic Features in Arabidopsis[C][W][OPEN

    PubMed Central

    Lowry, David B.; Logan, Tierney L.; Santuari, Luca; Hardtke, Christian S.; Richards, James H.; DeRose-Wilson, Leah J.; McKay, John K.; Sen, Saunak; Juenger, Thomas E.

    2013-01-01

    The regulation of gene expression is crucial for an organism’s development and response to stress, and an understanding of the evolution of gene expression is of fundamental importance to basic and applied biology. To improve this understanding, we conducted expression quantitative trait locus (eQTL) mapping in the Tsu-1 (Tsushima, Japan) × Kas-1 (Kashmir, India) recombinant inbred line population of Arabidopsis thaliana across soil drying treatments. We then used genome resequencing data to evaluate whether genomic features (promoter polymorphism, recombination rate, gene length, and gene density) are associated with genes responding to the environment (E) or with genes with genetic variation (G) in gene expression in the form of eQTLs. We identified thousands of genes that responded to soil drying and hundreds of main-effect eQTLs. However, we identified very few statistically significant eQTLs that interacted with the soil drying treatment (GxE eQTL). Analysis of genome resequencing data revealed associations of several genomic features with G and E genes. In general, E genes had lower promoter diversity and local recombination rates. By contrast, genes with eQTLs (G) had significantly greater promoter diversity and were located in genomic regions with higher recombination. These results suggest that genomic architecture may play an important a role in the evolution of gene expression. PMID:24045022

  15. Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

    PubMed

    Oliver, David M; Bird, Clare; Burd, Emmy; Wyman, Michael

    2016-09-06

    The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.

  16. Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

    PubMed Central

    Zheng, Peng; Xiong, Qian; Wu, Ying; Chen, Ying; Chen, Zhuo; Fleming, Joy; Gao, Ding; Bi, Lijun; Ge, Feng

    2015-01-01

    Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs. PMID:25762744

  17. Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

    PubMed Central

    Megger, Dominik A.; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

    2017-01-01

    Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus. PMID:28959263

  18. Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

    PubMed

    Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

    2017-01-01

    Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

  19. Quantitative Analysis of the Human Milk Whey Proteome Reveals Developing Milk and Mammary-Gland Functions across the First Year of Lactation

    PubMed Central

    Zhang, Qiang; Cundiff, Judy K.; Maria, Sarah D.; McMahon, Robert J.; Woo, Jessica G.; Davidson, Barbara S.; Morrow, Ardythe L.

    2013-01-01

    In-depth understanding of the changing functions of human milk (HM) proteins and the corresponding physiological adaptions of the lactating mammary gland has been inhibited by incomplete knowledge of the HM proteome. We analyzed the HM whey proteome (n = 10 women with samples at 1 week and 1, 3, 6, 9 and 12 months) using a quantitative proteomic approach. One thousand three hundred and thirty three proteins were identified with 615 being quantified. Principal component analysis revealed a transition in the HM whey proteome-throughout the first year of lactation. Abundance changes in IgG, sIgA and sIgM display distinct features during the first year. Complement components and other acute-phase proteins are generally at higher levels in early lactation. Proteomic analysis further suggests that the sources of milk fatty acids (FA) shift from more direct blood influx to more de novo mammary synthesis over lactation. The abundances of the majority of glycoproteins decline over lactation, which is consistent with increased enzyme expression in glycoprotein degradation and decreased enzyme expression in glycoprotein synthesis. Cellular detoxification machinery may be transformed as well, thereby accommodating increased metabolic activities in late lactation. The multiple developing functions of HM proteins and the corresponding mammary adaption become more apparent from this study. PMID:28250401

  20. Expression quantitative trait loci and genetic regulatory network analysis reveals that Gabra2 is involved in stress responses in the mouse.

    PubMed

    Dai, Jiajuan; Wang, Xusheng; Chen, Ying; Wang, Xiaodong; Zhu, Jun; Lu, Lu

    2009-11-01

    Previous studies have revealed that the subunit alpha 2 (Gabra2) of the gamma-aminobutyric acid receptor plays a critical role in the stress response. However, little is known about the gentetic regulatory network for Gabra2 and the stress response. We combined gene expression microarray analysis and quantitative trait loci (QTL) mapping to characterize the genetic regulatory network for Gabra2 expression in the hippocampus of BXD recombinant inbred (RI) mice. Our analysis found that the expression level of Gabra2 exhibited much variation in the hippocampus across the BXD RI strains and between the parental strains, C57BL/6J, and DBA/2J. Expression QTL (eQTL) mapping showed three microarray probe sets of Gabra2 to have highly significant linkage likelihood ratio statistic (LRS) scores. Gene co-regulatory network analysis showed that 10 genes, including Gria3, Chka, Drd3, Homer1, Grik2, Odz4, Prkag2, Grm5, Gabrb1, and Nlgn1 are directly or indirectly associated with stress responses. Eleven genes were implicated as Gabra2 downstream genes through mapping joint modulation. The genetical genomics approach demonstrates the importance and the potential power of the eQTL studies in identifying genetic regulatory networks that contribute to complex traits, such as stress responses.

  1. Love me tender: an Omics window on the bovine meat tenderness network.

    PubMed

    D'Alessandro, Angelo; Rinalducci, Sara; Marrocco, Cristina; Zolla, Valerio; Napolitano, Francesco; Zolla, Lello

    2012-07-19

    Meat tenderness prediction is a challenging task, especially in Maremmana, an Italian autochtonous and highly appreciated beef breed. In the present study we reported an integrated proteomics, phosphoproteomics and metabolomics overview of meat tenderness in longissimus dorsi from 15 male Maremmana individuals, through the discrimination of tender and tough groups via standard meat tenderness indicators (WBS, MFI(4 h), MFI(10 days), sarcomere length) and their correlation with results from Omics analyses. Results revealed that the tender meat group was characterized by higher levels of glycolytic enzymes, which were less phosphorylated and overall more active (lactate accumulation was higher in the tender group), than in tough counterparts. Additionally, we could observe a higher level of oxidative stress in the tender group. No proteomics nor phosphoproteomics result hinted at the widely accepted role of calpains and cathepsins, except for the indication of calcium homeostasis dysregulation. Nevertheless, myofibrillar degradation was monitored and related to structural protein fragmentations. Fragmentation of structural proteins and activities of glycolytic enzymes were inversely related to their phosphorylation levels, suggesting that PTMs might add further levels of complexity in the frame of meat tenderness. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Quantitative prediction of drug side effects based on drug-related features.

    PubMed

    Niu, Yanqing; Zhang, Wen

    2017-09-01

    Unexpected side effects of drugs are great concern in the drug development, and the identification of side effects is an important task. Recently, machine learning methods are proposed to predict the presence or absence of interested side effects for drugs, but it is difficult to make the accurate prediction for all of them. In this paper, we transform side effect profiles of drugs as their quantitative scores, by summing up their side effects with weights. The quantitative scores may measure the dangers of drugs, and thus help to compare the risk of different drugs. Here, we attempt to predict quantitative scores of drugs, namely the quantitative prediction. Specifically, we explore a variety of drug-related features and evaluate their discriminative powers for the quantitative prediction. Then, we consider several feature combination strategies (direct combination, average scoring ensemble combination) to integrate three informative features: chemical substructures, targets, and treatment indications. Finally, the average scoring ensemble model which produces the better performances is used as the final quantitative prediction model. Since weights for side effects are empirical values, we randomly generate different weights in the simulation experiments. The experimental results show that the quantitative method is robust to different weights, and produces satisfying results. Although other state-of-the-art methods cannot make the quantitative prediction directly, the prediction results can be transformed as the quantitative scores. By indirect comparison, the proposed method produces much better results than benchmark methods in the quantitative prediction. In conclusion, the proposed method is promising for the quantitative prediction of side effects, which may work cooperatively with existing state-of-the-art methods to reveal dangers of drugs.

  3. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

    PubMed Central

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-01-01

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

  4. Multimodal quantitative phase and fluorescence imaging of cell apoptosis

    NASA Astrophysics Data System (ADS)

    Fu, Xinye; Zuo, Chao; Yan, Hao

    2017-06-01

    Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

  5. Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

    PubMed

    Song, Jun; Du, Lina; Li, Li; Kalt, Wilhelmina; Palmer, Leslie Campbell; Fillmore, Sherry; Zhang, Ying; Zhang, ZhaoQi; Li, XiHong

    2015-06-03

    To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides

  6. Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

    DOE PAGES

    Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.; ...

    2015-12-23

    Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Here, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO 2, Fe 3O 4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observemore » that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe 3O 4 > SiO 2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.« less

  7. Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.

    Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Here, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO 2, Fe 3O 4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observemore » that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe 3O 4 > SiO 2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.« less

  8. Cardiovascular and pulmonary dynamics by quantitative imaging

    NASA Technical Reports Server (NTRS)

    Wood, E. H.

    1976-01-01

    The accuracy and range of studies on cardiovascular and pulmonary functions can be greatly facilitated if the motions of the underlying organ systems throughout individual cycles can be directly visualized and readily measured with minimum or preferably no effect on these motions. Achievement of this objective requires development of techniques for quantitative noninvasive or minimally invasive dynamic and stop-action imaging of the organ systems. A review of advances in dynamic quantitative imaging of moving organs reveals that the revolutionary value of cross-sectional and three-dimensional images produced by various types of radiant energy such as X-rays and gamma rays, positrons, electrons, protons, light, and ultrasound for clinical diagnostic and biomedical research applications is just beginning to be realized. The fabrication of a clinically useful cross-section reconstruction device with sensing capabilities for both anatomical structural composition and chemical composition may be possible and awaits future development.

  9. Quantitative iTRAQ LC-MS/MS proteomics reveals metabolic responses to biofuel ethanol in cyanobacterial Synechocystis sp. PCC 6803.

    PubMed

    Qiao, Jianjun; Wang, Jiangxin; Chen, Lei; Tian, Xiaoxu; Huang, Siqiang; Ren, Xiaoyue; Zhang, Weiwen

    2012-11-02

    Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC-MS/MS technologies. The resulting high-quality proteomic data set consisted of 24,887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.

  10. Quantitative analysis of the polarization characteristics of atherosclerotic plaques

    NASA Astrophysics Data System (ADS)

    Gubarkova, Ekaterina V.; Kirillin, Michail Y.; Dudenkova, Varvara V.; Kiseleva, Elena B.; Moiseev, Alexander A.; Gelikonov, Grigory V.; Timofeeva, Lidia B.; Fiks, Ilya I.; Feldchtein, Felix I.; Gladkova, Natalia D.

    2016-04-01

    In this study we demonstrate the capability of cross-polarization optical coherence tomography (CP OCT) to assess collagen and elastin fibers condition in atherosclerotic plaques basing on ratio of the OCT signal levels in cross- and co- polarizations. We consider the depolarization factor (DF) and the effective birefringence (Δn) as quantitative characteristics of CP OCT images. We revealed that calculation of both DF and Δn in the region of interest (fibrous cap) yields a statistically significant difference between stable and unstable plaques (0.46+/-0.21 vs 0.09+/-0.04 for IDF; (4.7+/-1.0)•10-4 vs (2.5+/-0.7)•10-4 for Δn p<0.05). In parallel with CP OCT we used the nonlinear microscopy for analysis of thin cross-section of atherosclerotic plaque, revealing the different average isotropy index of collagen and elastin fibers for stable and unstable plaques (0.30 +/- 0.10 vs 0.70 +/- 0.08; p<0.001). The proposed approach for quantitative assessment of CP OCT images allows cross-scattering and birefringence characterization of stable and unstable atherosclerotic plaques.

  11. Magnetoresistive biosensors for quantitative proteomics

    NASA Astrophysics Data System (ADS)

    Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

    2017-08-01

    Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

  12. Quantitative Peptidomics with Five-plex Reductive Methylation labels

    NASA Astrophysics Data System (ADS)

    Tashima, Alexandre K.; Fricker, Lloyd D.

    2017-12-01

    Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

  13. Quantitative Peptidomics with Five-plex Reductive Methylation labels

    NASA Astrophysics Data System (ADS)

    Tashima, Alexandre K.; Fricker, Lloyd D.

    2018-05-01

    Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

  14. Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia.

    PubMed

    Alikhani, Mehdi; Mirzaei, Mehdi; Sabbaghian, Marjan; Parsamatin, Pouria; Karamzadeh, Razieh; Adib, Samane; Sodeifi, Niloofar; Gilani, Mohammad Ali Sadighi; Zabet-Moghaddam, Masoud; Parker, Lindsay; Wu, Yunqi; Gupta, Vivek; Haynes, Paul A; Gourabi, Hamid; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2017-06-06

    Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction. The obtained data showed a drastic suppression of proteins involved in spliceosome, cell cycle and proteasome proteins, as well as energy and metabolic production in Sertoli cell only syndrome testis tissue, and to a lesser extent in maturation arrest samples. Moreover, we identified new transcription factors that are highly down-regulated in SCOS and MA patients, thus helping to understand the molecular complexity of spermatogenesis in male infertility. Our findings provide novel candidate protein targets associated

  15. A Quantitative Chemotherapy Genetic Interaction Map Reveals Factors Associated with PARP Inhibitor Resistance. | Office of Cancer Genomics

    Cancer.gov

    Chemotherapy is used to treat most cancer patients, yet our understanding of factors that dictate response and resistance to such drugs remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells charting the impact of the knockdown of 625 genes related to cancer and DNA repair on sensitivity to 29 drugs, covering all classes of chemotherapy.

  16. SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin

    PubMed Central

    Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

    2017-01-01

    Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin. PMID:28402964

  17. SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

    PubMed

    Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

    2017-06-20

    Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

  18. Proteome Analysis Using Isobaric Tags for Relative and Absolute Analysis Quantitation (iTRAQ) Reveals Alterations in Stress-Induced Dysfunctional Chicken Muscle.

    PubMed

    Xing, Tong; Wang, Chong; Zhao, Xue; Dai, Chen; Zhou, Guanghong; Xu, Xinglian

    2017-04-05

    The current study was designed to investigate changes in the protein profiles of pale, soft, and exudative (PSE)-like muscles of broilers subjected to transportation under high-temperature conditions, using isobaric tags for relative and absolute analysis quantitation (iTRAQ). Arbor Acres chickens (n = 112) were randomly divided into two treatments: unstressed control (CON) and 0.5 h of transport (T). Birds were transported according to a designed protocol. Pectoralis major (PM) muscle samples in the T group were collected and classified as normal (T-NOR) or PSE-like (T-PSE). Plasma activities of stress indicators, muscle microstructure, and proteome were measured. Results indicated that broilers in the T-PSE group exhibited higher activities of plasma stress indicators. The microstructure of T-PSE group showed a looser network and larger intercellular spaces in comparison to the other groups. Proteomic analysis, based on iTRAQ, revealed 29 differentially expressed proteins in the T-NOR and T-PSE groups that were involved in protein turnover, signal transduction, stress and defense, calcium handling, cell structure, and metabolism. In particular, proteins relating to the glycolysis pathway, calcium signaling, and molecular chaperones exhibited significant differences that may contribute to the inferior post-mortem meat quality. Overall, the proteomic results provide a further understanding of the mechanism of meat quality changes in response to stress.

  19. Quantitative methods for analysing cumulative effects on fish migration success: a review.

    PubMed

    Johnson, J E; Patterson, D A; Martins, E G; Cooke, S J; Hinch, S G

    2012-07-01

    It is often recognized, but seldom addressed, that a quantitative assessment of the cumulative effects, both additive and non-additive, of multiple stressors on fish survival would provide a more realistic representation of the factors that influence fish migration. This review presents a compilation of analytical methods applied to a well-studied fish migration, a more general review of quantitative multivariable methods, and a synthesis on how to apply new analytical techniques in fish migration studies. A compilation of adult migration papers from Fraser River sockeye salmon Oncorhynchus nerka revealed a limited number of multivariable methods being applied and the sub-optimal reliance on univariable methods for multivariable problems. The literature review of fisheries science, general biology and medicine identified a large number of alternative methods for dealing with cumulative effects, with a limited number of techniques being used in fish migration studies. An evaluation of the different methods revealed that certain classes of multivariable analyses will probably prove useful in future assessments of cumulative effects on fish migration. This overview and evaluation of quantitative methods gathered from the disparate fields should serve as a primer for anyone seeking to quantify cumulative effects on fish migration survival. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

  20. A Fan-tastic Quantitative Exploration of Ohm's Law

    NASA Astrophysics Data System (ADS)

    Mitchell, Brandon; Ekey, Robert; McCullough, Roy; Reitz, William

    2018-02-01

    Teaching simple circuits and Ohm's law to students in the introductory classroom has been extensively investigated through the common practice of using incandescent light bulbs to help students develop a conceptual foundation before moving on to quantitative analysis. However, the bulb filaments' resistance has a large temperature dependence, which makes them less suitable as a tool for quantitative analysis. Some instructors show that light bulbs do not obey Ohm's law either outright or through inquiry-based laboratory experiments. Others avoid the subject altogether by using bulbs strictly for qualitative purposes and then later switching to resistors for a numerical analysis, or by changing the operating conditions of the bulb so that it is "barely" glowing. It seems incongruous to develop a conceptual basis for the behavior of simple circuits using bulbs only to later reveal that they do not follow Ohm's law. Recently, small computer fans were proposed as a suitable replacement of bulbs for qualitative analysis of simple circuits where the current is related to the rotational speed of the fans. In this contribution, we demonstrate that fans can also be used for quantitative measurements and provide suggestions for successful classroom implementation.

  1. Data-Independent Acquisition-Based Quantitative Proteomic Analysis Reveals Potential Biomarkers of Kidney Cancer.

    PubMed

    Song, Yimeng; Zhong, Lijun; Zhou, Juntuo; Lu, Min; Xing, Tianying; Ma, Lulin; Shen, Jing

    2017-12-01

    Renal cell carcinoma (RCC) is a malignant and metastatic cancer with 95% mortality, and clear cell RCC (ccRCC) is the most observed among the five major subtypes of RCC. Specific biomarkers that can distinguish cancer tissues from adjacent normal tissues should be developed to diagnose this disease in early stages and conduct a reliable prognostic evaluation. Data-independent acquisition (DIA) strategy has been widely employed in proteomic analysis because of various advantages, including enhanced protein coverage and reliable data acquisition. In this study, a DIA workflow is constructed on a quadrupole-Orbitrap LC-MS platform to reveal dysregulated proteins between ccRCC and adjacent normal tissues. More than 4000 proteins are identified, 436 of these proteins are dysregulated in ccRCC tissues. Bioinformatic analysis reveals that multiple pathways and Gene Ontology items are strongly associated with ccRCC. The expression levels of L-lactate dehydrogenase A chain, annexin A4, nicotinamide N-methyltransferase, and perilipin-2 examined through RT-qPCR, Western blot, and immunohistochemistry confirm the validity of the proteomic analysis results. The proposed DIA workflow yields optimum time efficiency and data reliability and provides a good choice for proteomic analysis in biological and clinical studies, and these dysregulated proteins might be potential biomarkers for ccRCC diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Quantitative and Sensitive Detection of Chloramphenicol by Surface-Enhanced Raman Scattering

    PubMed Central

    Ding, Yufeng; Yin, Hongjun; Meng, Qingyun; Zhao, Yongmei; Liu, Luo; Wu, Zhenglong; Xu, Haijun

    2017-01-01

    We used surface-enhanced Raman scattering (SERS) for the quantitative and sensitive detection of chloramphenicol (CAP). Using 30 nm colloidal Au nanoparticles (NPs), a low detection limit for CAP of 10−8 M was obtained. The characteristic Raman peak of CAP centered at 1344 cm−1 was used for the rapid quantitative detection of CAP in three different types of CAP eye drops, and the accuracy of the measurement result was verified by high-performance liquid chromatography (HPLC). The experimental results reveal that the SERS technique based on colloidal Au NPs is accurate and sensitive, and can be used for the rapid detection of various antibiotics. PMID:29261161

  3. Novel Autism Subtype-Dependent Genetic Variants Are Revealed by Quantitative Trait and Subphenotype Association Analyses of Published GWAS Data

    PubMed Central

    Hu, Valerie W.; Addington, Anjene; Hyman, Alexander

    2011-01-01

    The heterogeneity of symptoms associated with autism spectrum disorders (ASDs) has presented a significant challenge to genetic analyses. Even when associations with genetic variants have been identified, it has been difficult to associate them with a specific trait or characteristic of autism. Here, we report that quantitative trait analyses of ASD symptoms combined with case-control association analyses using distinct ASD subphenotypes identified on the basis of symptomatic profiles result in the identification of highly significant associations with 18 novel single nucleotide polymorphisms (SNPs). The symptom categories included deficits in language usage, non-verbal communication, social development, and play skills, as well as insistence on sameness or ritualistic behaviors. Ten of the trait-associated SNPs, or quantitative trait loci (QTL), were associated with more than one subtype, providing partial replication of the identified QTL. Notably, none of the novel SNPs is located within an exonic region, suggesting that these hereditary components of ASDs are more likely related to gene regulatory processes (or gene expression) than to structural or functional changes in gene products. Seven of the QTL reside within intergenic chromosomal regions associated with rare copy number variants that have been previously reported in autistic samples. Pathway analyses of the genes associated with the QTL identified in this study implicate neurological functions and disorders associated with autism pathophysiology. This study underscores the advantage of incorporating both quantitative traits as well as subphenotypes into large-scale genome-wide analyses of complex disorders. PMID:21556359

  4. Sample normalization methods in quantitative metabolomics.

    PubMed

    Wu, Yiman; Li, Liang

    2016-01-22

    To reveal metabolomic changes caused by a biological event in quantitative metabolomics, it is critical to use an analytical tool that can perform accurate and precise quantification to examine the true concentration differences of individual metabolites found in different samples. A number of steps are involved in metabolomic analysis including pre-analytical work (e.g., sample collection and storage), analytical work (e.g., sample analysis) and data analysis (e.g., feature extraction and quantification). Each one of them can influence the quantitative results significantly and thus should be performed with great care. Among them, the total sample amount or concentration of metabolites can be significantly different from one sample to another. Thus, it is critical to reduce or eliminate the effect of total sample amount variation on quantification of individual metabolites. In this review, we describe the importance of sample normalization in the analytical workflow with a focus on mass spectrometry (MS)-based platforms, discuss a number of methods recently reported in the literature and comment on their applicability in real world metabolomics applications. Sample normalization has been sometimes ignored in metabolomics, partially due to the lack of a convenient means of performing sample normalization. We show that several methods are now available and sample normalization should be performed in quantitative metabolomics where the analyzed samples have significant variations in total sample amounts. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Quantitative Susceptibility Mapping Reveals an Association between Brain Iron Load and Depression Severity

    PubMed Central

    Yao, Shun; Zhong, Yi; Xu, Yuhao; Qin, Jiasheng; Zhang, Ningning; Zhu, Xiaolan; Li, Yuefeng

    2017-01-01

    Previous studies have detected abnormal serum ferritin levels in patients with depression; however, the results have been inconsistent. This study used quantitative susceptibility mapping (QSM) for the first time to examine brain iron concentration in depressed patients and evaluated whether it is related to severity. We included three groups of age- and gender-matched participants: 30 patients with mild-moderate depression (MD), 14 patients with major depression disorder (MDD) and 20 control subjects. All participants underwent MR scans with a 3D gradient-echo sequence reconstructing for QSM and performed the 17-item Hamilton Depression Rating Scale (HDRS) test. In MDD, the susceptibility value in the bilateral putamen was significantly increased compared with MD or control subjects. In addition, a significant difference was also observed in the left thalamus in MDD patients compared with controls. However, the susceptibility values did not differ between MD patients and controls. The susceptibility values positively correlated with the severity of depression as indicated by the HDRS scores. Our results provide evidence that brain iron deposition may be associated with depression and may even be a biomarker for investigating the pathophysiological mechanism of depression. PMID:28900391

  6. Quantitative nephelometry

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003545.htm Quantitative nephelometry test To use the sharing features on this page, please enable JavaScript. Quantitative nephelometry is a lab test to quickly and ...

  7. Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera L. ssp. sativa) berry: a quantitative trait locus to quantitative trait nucleotide integrated study.

    PubMed

    Fournier-Level, Alexandre; Le Cunff, Loïc; Gomez, Camila; Doligez, Agnès; Ageorges, Agnès; Roux, Catherine; Bertrand, Yves; Souquet, Jean-Marc; Cheynier, Véronique; This, Patrice

    2009-11-01

    The combination of QTL mapping studies of synthetic lines and association mapping studies of natural diversity represents an opportunity to throw light on the genetically based variation of quantitative traits. With the positional information provided through quantitative trait locus (QTL) mapping, which often leads to wide intervals encompassing numerous genes, it is now feasible to directly target candidate genes that are likely to be responsible for the observed variation in completely sequenced genomes and to test their effects through association genetics. This approach was performed in grape, a newly sequenced genome, to decipher the genetic architecture of anthocyanin content. Grapes may be either white or colored, ranging from the lightest pink to the darkest purple tones according to the amount of anthocyanin accumulated in the berry skin, which is a crucial trait for both wine quality and human nutrition. Although the determinism of the white phenotype has been fully identified, the genetic bases of the quantitative variation of anthocyanin content in berry skin remain unclear. A single QTL responsible for up to 62% of the variation in the anthocyanin content was mapped on a Syrah x Grenache F(1) pseudo-testcross. Among the 68 unigenes identified in the grape genome within the QTL interval, a cluster of four Myb-type genes was selected on the basis of physiological evidence (VvMybA1, VvMybA2, VvMybA3, and VvMybA4). From a core collection of natural resources (141 individuals), 32 polymorphisms revealed significant association, and extended linkage disequilibrium was observed. Using a multivariate regression method, we demonstrated that five polymorphisms in VvMybA genes except VvMybA4 (one retrotransposon, three single nucleotide polymorphisms and one 2-bp insertion/deletion) accounted for 84% of the observed variation. All these polymorphisms led to either structural changes in the MYB proteins or differences in the VvMybAs promoters. We concluded that

  8. [Application of target restoration space quantity and quantitative relation in precise esthetic prosthodontics].

    PubMed

    Haiyang, Yu; Tian, Luo

    2016-06-01

    Target restoration space (TRS) is the most precise space required for designing optimal prosthesis. TRS consists of an internal or external tooth space to confirm the esthetics and function of the final restoration. Therefore, assisted with quantitive analysis transfer, TRS quantitative analysis is a significant improvement for minimum tooth preparation. This article presents TRS quantity-related measurement, analysis, transfer, and internal relevance of three TR. classifications. Results reveal the close bond between precision and minimally invasive treatment. This study can be used to improve the comprehension and execution of precise esthetic prosthodontics.

  9. Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

    PubMed Central

    Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A.; Liu, Jie; Kirkpatrick, Beth D.; Chowdhury, Anwarul H.; Jamil, Khondoker M.; Haque, Rashidul; Petri, William A.; Houpt, Eric R.

    2014-01-01

    Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture+/qPCR+ and culture−/qPCR+ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

  10. Quantitative proteomics of seed filling in castor: comparison with soybean and rapeseed reveals differences between photosynthetic and nonphotosynthetic seed metabolism.

    PubMed

    Houston, Norma L; Hajduch, Martin; Thelen, Jay J

    2009-10-01

    Seed maturation or seed filling is a phase of development that plays a major role in the storage reserve composition of a seed. In many plant seeds photosynthesis plays a major role in this process, although oilseeds, such as castor (Ricinus communis), are capable of accumulating oil without the benefit of photophosphorylation to augment energy demands. To characterize seed filling in castor, a systematic quantitative proteomics study was performed. Two-dimensional gel electrophoresis was used to resolve and quantify Cy-dye-labeled proteins expressed at 2, 3, 4, 5, and 6 weeks after flowering in biological triplicate. Expression profiles for 660 protein spot groups were established, and of these, 522 proteins were confidently identified by liquid chromatography-tandem mass spectrometry by mining against the castor genome. Identified proteins were classified according to function, and the most abundant groups of proteins were involved in protein destination and storage (34%), energy (19%), and metabolism (15%). Carbon assimilatory pathways in castor were compared with previous studies of photosynthetic oilseeds, soybean (Glycine max) and rapeseed (Brassica napus). These comparisons revealed differences in abundance and number of protein isoforms at numerous steps in glycolysis. One such difference was the number of enolase isoforms and their sum abundance; castor had approximately six times as many isoforms as soy and rapeseed. Furthermore, Rubisco was 11-fold less prominent in castor compared to rapeseed. These and other differences suggest some aspects of carbon flow, carbon recapture, as well as ATP and NADPH production in castor differs from photosynthetic oilseeds.

  11. Quantitative microbiome profiling links gut community variation to microbial load.

    PubMed

    Vandeputte, Doris; Kathagen, Gunter; D'hoe, Kevin; Vieira-Silva, Sara; Valles-Colomer, Mireia; Sabino, João; Wang, Jun; Tito, Raul Y; De Commer, Lindsey; Darzi, Youssef; Vermeire, Séverine; Falony, Gwen; Raes, Jeroen

    2017-11-23

    Current sequencing-based analyses of faecal microbiota quantify microbial taxa and metabolic pathways as fractions of the sample sequence library generated by each analysis. Although these relative approaches permit detection of disease-associated microbiome variation, they are limited in their ability to reveal the interplay between microbiota and host health. Comparative analyses of relative microbiome data cannot provide information about the extent or directionality of changes in taxa abundance or metabolic potential. If microbial load varies substantially between samples, relative profiling will hamper attempts to link microbiome features to quantitative data such as physiological parameters or metabolite concentrations. Saliently, relative approaches ignore the possibility that altered overall microbiota abundance itself could be a key identifier of a disease-associated ecosystem configuration. To enable genuine characterization of host-microbiota interactions, microbiome research must exchange ratios for counts. Here we build a workflow for the quantitative microbiome profiling of faecal material, through parallelization of amplicon sequencing and flow cytometric enumeration of microbial cells. We observe up to tenfold differences in the microbial loads of healthy individuals and relate this variation to enterotype differentiation. We show how microbial abundances underpin both microbiota variation between individuals and covariation with host phenotype. Quantitative profiling bypasses compositionality effects in the reconstruction of gut microbiota interaction networks and reveals that the taxonomic trade-off between Bacteroides and Prevotella is an artefact of relative microbiome analyses. Finally, we identify microbial load as a key driver of observed microbiota alterations in a cohort of patients with Crohn's disease, here associated with a low-cell-count Bacteroides enterotype (as defined through relative profiling).

  12. Clock Work: How Tools for Time Mediate Problem Solving and Reveal Understanding

    ERIC Educational Resources Information Center

    Earnest, Darrell

    2017-01-01

    This article reports on elementary students' understanding of time in the context of common classroom manipulatives and notational systems. Students in Grades 2 (n = 72) and 4 (n = 72) participated in problem-solving interviews involving different clocks. Quantitative results revealed that students' performances were significantly different as a…

  13. A powerful approach reveals numerous expression quantitative trait haplotypes in multiple tissues.

    PubMed

    Ying, Dingge; Li, Mulin Jun; Sham, Pak Chung; Li, Miaoxin

    2018-04-26

    Recently many studies showed single nucleotide polymorphisms (SNPs) affect gene expression and contribute to development of complex traits/diseases in a tissue context-dependent manner. However, little is known about haplotype's influence on gene expression and complex traits, which reflects the interaction effect between SNPs. In the present study, we firstly proposed a regulatory region guided eQTL haplotype association analysis approach, and then systematically investigate the expression quantitative trait loci (eQTL) haplotypes in 20 different tissues by the approach. The approach has a powerful design of reducing computational burden by the utilization of regulatory predictions for candidate SNP selection and multiple testing corrections on non-independent haplotypes. The application results in multiple tissues showed that haplotype-based eQTLs not only increased the number of eQTL genes in a tissue specific manner, but were also enriched in loci that associated with complex traits in a tissue-matched manner. In addition, we found that tag SNPs of eQTL haplotypes from whole blood were selectively enriched in certain combination of regulatory elements (e.g. promoters and enhancers) according to predicted chromatin states. In summary, this eQTL haplotype detection approach, together with the application results, shed insights into synergistic effect of sequence variants on gene expression and their susceptibility to complex diseases. The executable application "eHaplo" is implemented in Java and is publicly available at http://grass.cgs.hku.hk/limx/ehaplo/. jonsonfox@gmail.com, limiaoxin@mail.sysu.edu.cn. Supplementary data are available at Bioinformatics online.

  14. Stiffness and evolution of interfacial micropancakes revealed by AFM quantitative nanomechanical imaging.

    PubMed

    Zhao, Binyu; Wang, Xingya; Song, Yang; Hu, Jun; Lü, Junhong; Zhou, Xingfei; Tai, Renzhong; Zhang, Xuehua; Zhang, Lijuan

    2015-05-28

    Micropancakes are quasi-two-dimensional micron-sized domains on crystalline substrates (e.g. highly oriented pyrolytic graphite (HOPG)) immersed in water. They are only a few nanometers thick, and are suspected to come from the accumulation of dissolved air at the solid-water interface. However, the exact chemical nature and basic physical properties of micropancakes have been under debate ever since their first observation, primarily due to the lack of a suitable characterization technique. In this study, the stiffness of micropancakes at the interface between HOPG and ethanol-water solutions was investigated by using PeakForce Quantitative NanoMechanics (PF-QNM) mode Atomic Force Microscopy (AFM). Our measurements showed that micropancakes were stiffer than nanobubbles, and for bilayer micropancakes, the bottom layer in contact with the substrate was stiffer than the top one. Interestingly, the micropancakes became smaller and softer with an increase in the ethanol concentration in the solution, and were undetectable by AFM above a critical concentration of ethanol. But they re-appeared after the ethanol concentration in the solution was reduced. Clearly the evolution and stiffness of the micropancakes were dependent on the chemical composition in the solution, which could be attributed to the correlation of the mechanical properties of the micropancakes with the surface tension of the liquid phase. Based on the "go-and-come" behaviors of micropancakes with the ethanol concentration, we found that the micropancakes could actually tolerate the ethanol concentration much higher than 5%, a value reported in the literature. The results from this work may be helpful in alluding the chemical nature of micropancakes.

  15. Quantitative genetic-interaction mapping in mammalian cells

    PubMed Central

    Roguev, Assen; Talbot, Dale; Negri, Gian Luca; Shales, Michael; Cagney, Gerard; Bandyopadhyay, Sourav; Panning, Barbara; Krogan, Nevan J

    2013-01-01

    Mapping genetic interactions (GIs) by simultaneously perturbing pairs of genes is a powerful tool for understanding complex biological phenomena. Here we describe an experimental platform for generating quantitative GI maps in mammalian cells using a combinatorial RNA interference strategy. We performed ~11,000 pairwise knockdowns in mouse fibroblasts, focusing on 130 factors involved in chromatin regulation to create a GI map. Comparison of the GI and protein-protein interaction (PPI) data revealed that pairs of genes exhibiting positive GIs and/or similar genetic profiles were predictive of the corresponding proteins being physically associated. The mammalian GI map identified pathways and complexes but also resolved functionally distinct submodules within larger protein complexes. By integrating GI and PPI data, we created a functional map of chromatin complexes in mouse fibroblasts, revealing that the PAF complex is a central player in the mammalian chromatin landscape. PMID:23407553

  16. Quantitative somatosensory testing of the penis: optimizing the clinical neurological examination.

    PubMed

    Bleustein, Clifford B; Eckholdt, Haftan; Arezzo, Joseph C; Melman, Arnold

    2003-06-01

    Quantitative somatosensory testing, including vibration, pressure, spatial perception and thermal thresholds of the penis, has demonstrated neuropathy in patients with a history of erectile dysfunction of all etiologies. We evaluated which measurement of neurological function of the penis was best at predicting erectile dysfunction and examined the impact of location on the penis for quantitative somatosensory testing measurements. A total of 107 patients were evaluated. All patients were required to complete the erectile function domain of the International Index of Erectile Function (IIEF) questionnaire, of whom 24 had no complaints of erectile dysfunction and scored within the "normal" range on the IIEF. Patients were subsequently tested on ventral middle penile shaft, proximal dorsal midline penile shaft and glans penis (with foreskin retracted) for vibration, pressure, spatial perception, and warm and cold thermal thresholds. Mixed models repeated measures analysis of variance controlling for age, diabetes and hypertension revealed that method of measurement (quantitative somatosensory testing) was predictive of IIEF score (F = 209, df = 4,1315, p <0.001), while site of measurement on the penis was not. To determine the best method of measurement, we used hierarchical regression, which revealed that warm temperature was the best predictor of erectile dysfunction with pseudo R(2) = 0.19, p <0.0007. There was no significant improvement in predicting erectile dysfunction when another test was added. Using 37C and greater as the warm thermal threshold yielded a sensitivity of 88.5%, specificity 70.0% and positive predictive value 85.5%. Quantitative somatosensory testing using warm thermal threshold measurements taken at the glans penis can be used alone to assess the neurological status of the penis. Warm thermal thresholds alone offer a quick, noninvasive accurate method of evaluating penile neuropathy in an office setting.

  17. A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

    PubMed

    Belmont, Judson; Gu, Tao; Mudd, Ashley; Salomon, Arthur R

    2017-08-04

    Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca 2+ signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr 192 phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr 192 phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.

  18. Quantitative analysis of professionally trained versus untrained voices.

    PubMed

    Siupsinskiene, Nora

    2003-01-01

    The aim of this study was to compare healthy trained and untrained voices as well as healthy and dysphonic trained voices in adults using combined voice range profile and aerodynamic tests, to define the normal range limiting values of quantitative voice parameters and to select the most informative quantitative voice parameters for separation between healthy and dysphonic trained voices. Three groups of persons were evaluated. One hundred eighty six healthy volunteers were divided into two groups according to voice training: non-professional speakers group consisted of 106 untrained voices persons (36 males and 70 females) and professional speakers group--of 80 trained voices persons (21 males and 59 females). Clinical group consisted of 103 dysphonic professional speakers (23 males and 80 females) with various voice disorders. Eighteen quantitative voice parameters from combined voice range profile (VRP) test were analyzed: 8 of voice range profile, 8 of speaking voice, overall vocal dysfunction degree and coefficient of sound, and aerodynamic maximum phonation time. Analysis showed that healthy professional speakers demonstrated expanded vocal abilities in comparison to healthy non-professional speakers. Quantitative voice range profile parameters- pitch range, high frequency limit, area of high frequencies and coefficient of sound differed significantly between healthy professional and non-professional voices, and were more informative than speaking voice or aerodynamic parameters in showing the voice training. Logistic stepwise regression revealed that VRP area in high frequencies was sufficient to discriminate between healthy and dysphonic professional speakers for male subjects (overall discrimination accuracy--81.8%) and combination of three quantitative parameters (VRP high frequency limit, maximum voice intensity and slope of speaking curve) for female subjects (overall model discrimination accuracy--75.4%). We concluded that quantitative voice assessment

  19. Quantitative radiomic profiling of glioblastoma represents transcriptomic expression.

    PubMed

    Kong, Doo-Sik; Kim, Junhyung; Ryu, Gyuha; You, Hye-Jin; Sung, Joon Kyung; Han, Yong Hee; Shin, Hye-Mi; Lee, In-Hee; Kim, Sung-Tae; Park, Chul-Kee; Choi, Seung Hong; Choi, Jeong Won; Seol, Ho Jun; Lee, Jung-Il; Nam, Do-Hyun

    2018-01-19

    Quantitative imaging biomarkers have increasingly emerged in the field of research utilizing available imaging modalities. We aimed to identify good surrogate radiomic features that can represent genetic changes of tumors, thereby establishing noninvasive means for predicting treatment outcome. From May 2012 to June 2014, we retrospectively identified 65 patients with treatment-naïve glioblastoma with available clinical information from the Samsung Medical Center data registry. Preoperative MR imaging data were obtained for all 65 patients with primary glioblastoma. A total of 82 imaging features including first-order statistics, volume, and size features, were semi-automatically extracted from structural and physiologic images such as apparent diffusion coefficient and perfusion images. Using commercially available software, NordicICE, we performed quantitative imaging analysis and collected the dataset composed of radiophenotypic parameters. Unsupervised clustering methods revealed that the radiophenotypic dataset was composed of three clusters. Each cluster represented a distinct molecular classification of glioblastoma; classical type, proneural and neural types, and mesenchymal type. These clusters also reflected differential clinical outcomes. We found that extracted imaging signatures does not represent copy number variation and somatic mutation. Quantitative radiomic features provide a potential evidence to predict molecular phenotype and treatment outcome. Radiomic profiles represents transcriptomic phenotypes more well.

  20. Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes in Jurkat T-cells treated with HIV-1 Tat protein.

    PubMed

    Liao, Wenting; Tan, Guangguo; Zhu, Zhenyu; Chen, Qiuli; Lou, Ziyang; Dong, Xin; Zhang, Wei; Pan, Wei; Chai, Yifeng

    2012-11-02

    HIV-1 Tat protein is released by infected cells and can affect bystander uninfected T cells and induce numerous biological responses which contribute to its pathogenesis. To elucidate the complex pathogenic mechanism, we conducted a comprehensive investigation on Tat protein-related extracellular and intracellular metabolic changes in Jurkat T-cells using combined gas chromatography-mass spectrometry (GC-MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS)-based metabonomics approach. Quantitative real-time PCR (qRT-PCR) analyses were further employed to measure expressions of several relevant enzymes together with perturbed metabolic pathways. Combined metabonomic and qRT-PCR analyses revealed that HIV-1 Tat caused significant and comprehensive metabolic changes, as represented by significant changes of 37 metabolites and 10 relevant enzymes in HIV-1 Tat-treated cells. Using MetaboAnalyst 2.0, it was found that 11 pathways (Impact-value >0.10) among the regulated pathways were acutely perturbed, including sphingolipid metabolism, glycine, serine and threonine metabolism, pyruvate metabolism, inositol phosphate metabolism, arginine and proline metabolism, citrate cycle, phenylalanine metabolism, tryptophan metabolism, pentose phosphate pathway, glycerophospholipid metabolism, glycolysis or gluconeogenesis. These results provide metabolic evidence of the complex pathogenic mechanism of HIV-1 Tat protein as a "viral toxin", and would help obligate Tat protein as "an important target" for therapeutic intervention and vaccine development.

  1. Cloning of quantitative trait genes from rice reveals conservation and divergence of photoperiod flowering pathways in Arabidopsis and rice

    PubMed Central

    Matsubara, Kazuki; Hori, Kiyosumi; Ogiso-Tanaka, Eri; Yano, Masahiro

    2014-01-01

    Flowering time in rice (Oryza sativa L.) is determined primarily by daylength (photoperiod), and natural variation in flowering time is due to quantitative trait loci involved in photoperiodic flowering. To date, genetic analysis of natural variants in rice flowering time has resulted in the positional cloning of at least 12 quantitative trait genes (QTGs), including our recently cloned QTGs, Hd17, and Hd16. The QTGs have been assigned to specific photoperiodic flowering pathways. Among them, 9 have homologs in the Arabidopsis genome, whereas it was evident that there are differences in the pathways between rice and Arabidopsis, such that the rice Ghd7–Ehd1–Hd3a/RFT1 pathway modulated by Hd16 is not present in Arabidopsis. In this review, we describe QTGs underlying natural variation in rice flowering time. Additionally, we discuss the implications of the variation in adaptive divergence and its importance in rice breeding. PMID:24860584

  2. Untargeted Metabolic Quantitative Trait Loci Analyses Reveal a Relationship between Primary Metabolism and Potato Tuber Quality1[W][OA

    PubMed Central

    Carreno-Quintero, Natalia; Acharjee, Animesh; Maliepaard, Chris; Bachem, Christian W.B.; Mumm, Roland; Bouwmeester, Harro; Visser, Richard G.F.; Keurentjes, Joost J.B.

    2012-01-01

    Recent advances in -omics technologies such as transcriptomics, metabolomics, and proteomics along with genotypic profiling have permitted dissection of the genetics of complex traits represented by molecular phenotypes in nonmodel species. To identify the genetic factors underlying variation in primary metabolism in potato (Solanum tuberosum), we have profiled primary metabolite content in a diploid potato mapping population, derived from crosses between S. tuberosum and wild relatives, using gas chromatography-time of flight-mass spectrometry. In total, 139 polar metabolites were detected, of which we identified metabolite quantitative trait loci for approximately 72% of the detected compounds. In order to obtain an insight into the relationships between metabolic traits and classical phenotypic traits, we also analyzed statistical associations between them. The combined analysis of genetic information through quantitative trait locus coincidence and the application of statistical learning methods provide information on putative indicators associated with the alterations in metabolic networks that affect complex phenotypic traits. PMID:22223596

  3. Comparison of Quantitative and Qualitative Research Traditions: Epistemological, Theoretical, and Methodological Differences

    ERIC Educational Resources Information Center

    Yilmaz, Kaya

    2013-01-01

    There has been much discussion about quantitative and qualitative approaches to research in different disciplines. In the behavioural and social sciences, these two paradigms are compared to reveal their relative strengths and weaknesses. But the debate about both traditions has commonly taken place in academic books. It is hard to find an article…

  4. Developing Quantitative Reasoning: Will Taking Traditional Math Courses Suffice? An Empirical Study

    ERIC Educational Resources Information Center

    Agustin, Ma Zenia; Agustin, Marcus; Brunkow, Paul; Thomas, Susan

    2012-01-01

    Southern Illinois University Edwardsville (SIUE) is a postbaccalaureate comprehensive university in the Midwest. In 2005, SIUE embarked on a challenging journey of general education reform. A review of the current general education program revealed that it is possible for a student to graduate from SIUE without taking a quantitative course. Hence,…

  5. Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq

    PubMed Central

    Shepard, Peter J.; Choi, Eun-A; Lu, Jente; Flanagan, Lisa A.; Hertel, Klemens J.; Shi, Yongsheng

    2011-01-01

    Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation. PMID:21343387

  6. A quantitative chemotherapy genetic interaction map reveals new factors associated with PARP inhibitor resistance | Office of Cancer Genomics

    Cancer.gov

    Nearly every cancer patient is treated with chemotherapy yet our understanding of factors that dictate response and resistance to such agents remains limited. We report the generation of a quantitative chemical-genetic interaction map in human mammary epithelial cells that charts the impact of knockdown of 625 cancer and DNA repair related genes on sensitivity to 29 drugs, covering all classes of cancer chemotherapeutics.

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

    DTIC Science & Technology

    2012-03-01

    with Remotely Piloted Aircraft (RPA) has resulted in the need of a platform to evaluate interface design. The Vigilant Spirit Control Station ( VSCS ...Spirit interface. A modified version of the HCI Index was successfully applied to perform a quantitative analysis of the baseline VSCS interface and...time of the original VSCS interface. These results revealed the effectiveness of the tool and demonstrated in the design of future generation

  8. DISQOVER the Landcover - R based tools for quantitative vegetation reconstruction

    NASA Astrophysics Data System (ADS)

    Theuerkauf, Martin; Couwenberg, John; Kuparinen, Anna; Liebscher, Volkmar

    2016-04-01

    Quantitative methods have gained increasing attention in the field of vegetation reconstruction over the past decade. The DISQOVER package implements key tools in the R programming environment for statistical computing. This implementation has three main goals: 1) Provide a user-friendly, transparent, and open implementation of the methods 2) Provide full flexibility in all parameters (including the underlying pollen dispersal model) 3) Provide a sandbox for testing the sensitivity of the methods. We illustrate the possibilities of the package with tests of the REVEALS model and of the extended downscaling approach (EDA). REVEALS (Sugita 2007) is designed to translate pollen data from large lakes into regional vegetation composition. We applied REVEALSinR on pollen data from Lake Tiefer See (NE-Germany) and validated the results with historic landcover data. The results clearly show that REVEALS is sensitive to the underlying pollen dispersal model; REVEALS performs best when applied with the state of the art Lagrangian stochastic dispersal model. REVEALS applications with the conventional Gauss model can produce realistic results, but only if unrealistic pollen productivity estimates are used. The EDA (Theuerkauf et al. 2014) employs pollen data from many sites across a landscape to explore whether species distributions in the past were related to know stable patterns in the landscape, e.g. the distribution of soil types. The approach had so far only been implemented in simple settings with few taxa. Tests with EDAinR show that it produces sharp results in complex settings with many taxa as well. The DISQOVER package is open source software, available from disqover.uni-greifswald.de. This website can be used as a platform to discuss and improve quantitative methods in vegetation reconstruction. To introduce the tool we plan a short course in autumn of this year. This study is a contribution to the Virtual Institute of Integrated Climate and Landscape Evolution

  9. Connecting qualitative observation and quantitative measurement for enhancing quantitative literacy in plant anatomy course

    NASA Astrophysics Data System (ADS)

    Nuraeni, E.; Rahmat, A.

    2018-05-01

    Forming of cognitive schemes of plant anatomy concepts is performed by processing of qualitative and quantitative data obtained from microscopic observations. To enhancing student’s quantitative literacy, strategy of plant anatomy course was modified by adding the task to analyze quantitative data produced by quantitative measurement of plant anatomy guided by material course. Participant in this study was 24 biology students and 35 biology education students. Quantitative Literacy test, complex thinking in plant anatomy test and questioner used to evaluate the course. Quantitative literacy capability data was collected by quantitative literacy test with the rubric from the Association of American Colleges and Universities, Complex thinking in plant anatomy by test according to Marzano and questioner. Quantitative literacy data are categorized according to modified Rhodes and Finley categories. The results showed that quantitative literacy of biology education students is better than biology students.

  10. Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics.

    PubMed

    Boone, Cory H T; Grove, Ryan A; Adamcova, Dana; Braga, Camila P; Adamec, Jiri

    2016-07-01

    Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Quantitative Proteomics Reveals Ecophysiological Effects of Light and Silver Stress on the Mixotrophic Protist Poterioochromonas malhamensis

    PubMed Central

    Kaschani, Farnusch; Graupner, Nadine; Grossmann, Lars; Jensen, Manfred; Ninck, Sabrina; Schulz, Florian; Rahmann, Sven; Boenigk, Jens; Kaiser, Markus

    2017-01-01

    Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to a direct

  12. Quantitatively in Situ Imaging Silver Nanowire Hollowing Kinetics

    DOE PAGES

    Yu, Le; Yan, Zhongying; Cai, Zhonghou; ...

    2016-09-28

    We report the in-situ investigation of the morphological evolution of silver nanowires to hollow silver oxide nanotubes using transmission x-ray microscopy (TXM). Complex silver diffusion kinetics and hollowing process via the Kirkendall effect have been captured in real time. Further quantitative x-ray absorption analysis reveals the difference between the longitudinal and radial diffusions. In conclusion, the diffusion coefficient of silver in its oxide nanoshell is, for the first time, calculated to be 1.2 × 10 -13 cm 2/s from the geometrical parameters extracted from the TXM images.

  13. Evaluation of acute ischemic stroke using quantitative EEG: a comparison with conventional EEG and CT scan.

    PubMed

    Murri, L; Gori, S; Massetani, R; Bonanni, E; Marcella, F; Milani, S

    1998-06-01

    The sensitivity of quantitative electroencephalogram (EEG) was compared with that of conventional EEG in patients with acute ischaemic stroke. In addition, a correlation between quantitative EEG data and computerized tomography (CT) scan findings was carried out for all the areas of lesion in order to reassess the actual role of EEG in the evaluation of stroke. Sixty-five patients were tested with conventional and quantitative EEG within 24 h from the onset of neurological symptoms, whereas CT scan was performed within 4 days from the onset of stroke. EEG was recorded from 19 electrodes placed upon the scalp according to the International 10-20 System. Spectral analysis was carried out on 30 artefact-free 4-sec epochs. For each channel absolute and relative power were calculated for the delta, theta, alpha and beta frequency bands and such data were successively represented in colour-coded maps. Ten patients with extensive lesions documented by CT scan were excluded. The results indicated that conventional EEG revealed abnormalities in 40 of 55 cases, while EEG mapping showed abnormalities in 46 of 55 cases: it showed focal abnormalities in five cases and nonfocal abnormalities in one of six cases which had appeared to be normal according to visual inspection of EEG. In a further 11 cases, where the conventional EEG revealed abnormalities in one hemisphere, the quantitative EEG and maps allowed to further localize abnormal activity in a more localized way. The sensitivity of both methods was higher for frontocentral, temporal and parieto-occipital cortical-subcortical infarctions than for basal ganglia and internal capsule lesions; however, quantitative EEG was more efficient for all areas of lesion in detecting cases that had appeared normal by visual inspection and was clearly superior in revealing focal abnormalities. When we considered the electrode related to which the maximum power of the delta frequency band is recorded, a fairly close correlation was found

  14. A Quantitative Study: Enhancing the Productivity of the Emotionally Challenged High School Students

    ERIC Educational Resources Information Center

    Mammen, John

    2013-01-01

    This quantitative, causal-comparative study examined the degree of influence the parent teacher relationship can make on the grade point averages and graduation rates of students in an alternative school setting. Findings of this study revealed that the active parent teacher communication had direct relationship with the success rate of…

  15. Qualitative and Quantitative Analyses of Glycogen in Human Milk.

    PubMed

    Matsui-Yatsuhashi, Hiroko; Furuyashiki, Takashi; Takata, Hiroki; Ishida, Miyuki; Takumi, Hiroko; Kakutani, Ryo; Kamasaka, Hiroshi; Nagao, Saeko; Hirose, Junko; Kuriki, Takashi

    2017-02-22

    Identification as well as a detailed analysis of glycogen in human milk has not been shown yet. The present study confirmed that glycogen is contained in human milk by qualitative and quantitative analyses. High-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography with a multiangle laser light scattering detector (HPSEC-MALLS) were used for qualitative analysis of glycogen in human milk. Quantitative analysis was carried out by using samples obtained from the individual milks. The result revealed that the concentration of human milk glycogen varied depending on the mother's condition-such as the period postpartum and inflammation. The amounts of glycogen in human milk collected at 0 and 1-2 months postpartum were higher than in milk collected at 3-14 months postpartum. In the milk from mothers with severe mastitis, the concentration of glycogen was about 40 times higher than that in normal milk.

  16. Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress*

    PubMed Central

    Kültz, Dietmar; Li, Johnathon; Gardell, Alison; Sacchi, Romina

    2013-01-01

    A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into

  17. Quantitative study of flavonoids in leaves of citrus plants.

    PubMed

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M; Koizumi, M; Ito, C; Furukawa, H

    2000-09-01

    Leaf flavonoids were quantitatively determined in 68 representative or economically important Citrus species, cultivars, and near-Citrus relatives. Contents of 23 flavonoids including 6 polymethoxylated flavones were analyzed by means of reversed phase HPLC analysis. Principal component analysis revealed that the 7 associations according to Tanaka's classification were observed, but some do overlap each other. Group VII species could be divided into two different subgroups, namely, the first-10-species class and the last-19-species class according to Tanaka's classification numbers.

  18. Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis.

    PubMed

    Stoletov, Konstantin; Willetts, Lian; Paproski, Robert J; Bond, David J; Raha, Srijan; Jovel, Juan; Adam, Benjamin; Robertson, Amy E; Wong, Francis; Woolner, Emma; Sosnowski, Deborah L; Bismar, Tarek A; Wong, Gane Ka-Shu; Zijlstra, Andries; Lewis, John D

    2018-06-14

    Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.

  19. Identification of common coexpression modules based on quantitative network comparison.

    PubMed

    Jo, Yousang; Kim, Sanghyeon; Lee, Doheon

    2018-06-13

    Finding common molecular interactions from different samples is essential work to understanding diseases and other biological processes. Coexpression networks and their modules directly reflect sample-specific interactions among genes. Therefore, identification of common coexpression network or modules may reveal the molecular mechanism of complex disease or the relationship between biological processes. However, there has been no quantitative network comparison method for coexpression networks and we examined previous methods for other networks that cannot be applied to coexpression network. Therefore, we aimed to propose quantitative comparison methods for coexpression networks and to find common biological mechanisms between Huntington's disease and brain aging by the new method. We proposed two similarity measures for quantitative comparison of coexpression networks. Then, we performed experiments using known coexpression networks. We showed the validity of two measures and evaluated threshold values for similar coexpression network pairs from experiments. Using these similarity measures and thresholds, we quantitatively measured the similarity between disease-specific and aging-related coexpression modules and found similar Huntington's disease-aging coexpression module pairs. We identified similar Huntington's disease-aging coexpression module pairs and found that these modules are related to brain development, cell death, and immune response. It suggests that up-regulated cell signalling related cell death and immune/ inflammation response may be the common molecular mechanisms in the pathophysiology of HD and normal brain aging in the frontal cortex.

  20. Quantitative research.

    PubMed

    Watson, Roger

    2015-04-01

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

  1. Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos.

    PubMed

    Zhao, Ziqing W; White, Melanie D; Bissiere, Stephanie; Levi, Valeria; Plachta, Nicolas

    2016-12-23

    Probing dynamic processes occurring within the cell nucleus at the quantitative level has long been a challenge in mammalian biology. Advances in bio-imaging techniques over the past decade have enabled us to directly visualize nuclear processes in situ with unprecedented spatial and temporal resolution and single-molecule sensitivity. Here, using transcription as our primary focus, we survey recent imaging studies that specifically emphasize the quantitative understanding of nuclear dynamics in both time and space. These analyses not only inform on previously hidden physical parameters and mechanistic details, but also reveal a hierarchical organizational landscape for coordinating a wide range of transcriptional processes shared by mammalian systems of varying complexity, from single cells to whole embryos.

  2. A Quantitative Features Analysis of Recommended No- and Low-Cost Preschool E-Books

    ERIC Educational Resources Information Center

    Parette, Howard P.; Blum, Craig; Luthin, Katie

    2015-01-01

    In recent years, recommended e-books have drawn increasing attention from early childhood education professionals. This study applied a quantitative descriptive features analysis of cost (n = 70) and no-cost (n = 60) e-books recommended by the Texas Computer Education Association. While t tests revealed no statistically significant differences…

  3. iTRAQ-based quantitative proteomic analysis reveals potential factors associated with the enhancement of phenazine-1-carboxamide production in Pseudomonas chlororaphis P3

    PubMed Central

    Jin, Xue-Jie; Peng, Hua-Song; Hu, Hong-Bo; Huang, Xian-Qing; Wang, Wei; Zhang, Xue-Hong

    2016-01-01

    Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains. PMID:27273243

  4. Comparison of different approaches to quantitative adenovirus detection in stool specimens of hematopoietic stem cell transplant recipients.

    PubMed

    Kosulin, K; Dworzak, S; Lawitschka, A; Matthes-Leodolter, S; Lion, T

    2016-12-01

    Adenoviruses almost invariably proliferate in the gastrointestinal tract prior to dissemination, and critical threshold concentrations in stool correlate with the risk of viremia. Monitoring of adenovirus loads in stool may therefore be important for timely initiation of treatment in order to prevent invasive infection. Comparison of a manual DNA extraction kit in combination with a validated in-house PCR assay with automated extraction on the NucliSENS-EasyMAG device coupled with the Adenovirus R-gene kit (bioMérieux) for quantitative adenovirus analysis in stool samples. Stool specimens spiked with adenovirus concentrations in a range from 10E2-10E11 copies/g and 32 adenovirus-positive clinical stool specimens from pediatric stem cell transplant recipients were tested along with appropriate negative controls. Quantitative analysis of viral load in adenovirus-positive stool specimens revealed a median difference of 0.5 logs (range 0.1-2.2) between the detection systems tested and a difference of 0.3 logs (range 0.0-1.7) when the comparison was restricted to the PCR assays only. Spiking experiments showed a detection limit of 10 2 -10 3 adenovirus copies/g stool revealing a somewhat higher sensitivity offered by the automated extraction. The dynamic range of accurate quantitative analysis by both systems investigated was between 10 3 and 10 8 virus copies/g. The differences in quantitative analysis of adenovirus copy numbers between the systems tested were primarily attributable to the DNA extraction method used, while the qPCR assays revealed a high level of concordance. Both systems showed adequate performance for detection and monitoring of adenoviral load in stool specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

    PubMed

    Awasthi, Shivangi; Maity, Tapan; Oyler, Benjamin L; Qi, Yue; Zhang, Xu; Goodlett, David R; Guha, Udayan

    2018-04-13

    Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR L858R mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR L858R/T790M mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and

  6. A quantitative approach to painting styles

    NASA Astrophysics Data System (ADS)

    Vieira, Vilson; Fabbri, Renato; Sbrissa, David; da Fontoura Costa, Luciano; Travieso, Gonzalo

    2015-01-01

    This research extends a method previously applied to music and philosophy (Vilson Vieira et al., 2012), representing the evolution of art as a time-series where relations like dialectics are measured quantitatively. For that, a corpus of paintings of 12 well-known artists from baroque and modern art is analyzed. A set of 99 features is extracted and the features which most contributed to the classification of painters are selected. The projection space obtained provides the basis to the analysis of measurements. These quantitative measures underlie revealing observations about the evolution of painting styles, specially when compared with other humanity fields already analyzed: while music evolved along a master-apprentice tradition (high dialectics) and philosophy by opposition, painting presents another pattern: constant increasing skewness, low opposition between members of the same movement and opposition peaks in the transition between movements. Differences between baroque and modern movements are also observed in the projected "painting space": while baroque paintings are presented as an overlapped cluster, the modern paintings present minor overlapping and are disposed more widely in the projection than the baroque counterparts. This finding suggests that baroque painters shared aesthetics while modern painters tend to "break rules" and develop their own style.

  7. Integrative transcriptome, proteome, phosphoproteome and genetic mapping reveals new aspects in a fiberless mutant of cotton

    PubMed Central

    Ma, Qi-Feng; Wu, Chun-Hui; Wu, Man; Pei, Wen-Feng; Li, Xing-Li; Wang, Wen-Kui; Zhang, Jinfa; Yu, Ji-Wen; Yu, Shu-Xun

    2016-01-01

    To investigate the molecular mechanisms of fiber initiation in cotton (Gossypium spp.), an integrated approach combining transcriptome, iTRAQ-based proteome and genetic mapping was taken to compare the ovules of the Xuzhou 142 wild type (WT) with its fuzzless-lintless (fl) mutant at −3 and 0 day post-anthesis. A total of 1,953 mRNAs, 187 proteins, and 131 phosphoproteins were differentially expressed (DE) between WT and fl, and the levels of transcripts and their encoded proteins and phosphoproteins were highly congruent. A functional analysis suggested that the abundance of proteins were mainly involved in amino sugar, nucleotide sugar and fatty acid metabolism, one carbon pool for folate metabolism and flavonoid biosynthesis. qRT-PCR, Western blotting, and enzymatic assays were performed to confirm the regulation of these transcripts and proteins. A molecular mapping located the lintless gene li3 in the fl mutant on chromosome 26 for the first time. A further in-silico physical mapping of DE genes with sequence variations between fl and WT identified one and four candidate genes in the li3 and n2 regions, respectively. Taken together, the transcript abundance, phosphorylation status of proteins at the fiber initiation stage and candidate genes have provided insights into regulatory processes underlying cotton fiber initiation. PMID:27075604

  8. On sweat analysis for quantitative estimation of dehydration during physical exercise.

    PubMed

    Ring, Matthias; Lohmueller, Clemens; Rauh, Manfred; Eskofier, Bjoern M

    2015-08-01

    Quantitative estimation of water loss during physical exercise is of importance because dehydration can impair both muscular strength and aerobic endurance. A physiological indicator for deficit of total body water (TBW) might be the concentration of electrolytes in sweat. It has been shown that concentrations differ after physical exercise depending on whether water loss was replaced by fluid intake or not. However, to the best of our knowledge, this fact has not been examined for its potential to quantitatively estimate TBW loss. Therefore, we conducted a study in which sweat samples were collected continuously during two hours of physical exercise without fluid intake. A statistical analysis of these sweat samples revealed significant correlations between chloride concentration in sweat and TBW loss (r = 0.41, p <; 0.01), and between sweat osmolality and TBW loss (r = 0.43, p <; 0.01). A quantitative estimation of TBW loss resulted in a mean absolute error of 0.49 l per estimation. Although the precision has to be improved for practical applications, the present results suggest that TBW loss estimation could be realizable using sweat samples.

  9. In silico quantitative structure-toxicity relationship study of aromatic nitro compounds.

    PubMed

    Pasha, Farhan Ahmad; Neaz, Mohammad Morshed; Cho, Seung Joo; Ansari, Mohiuddin; Mishra, Sunil Kumar; Tiwari, Sharvan

    2009-05-01

    Small molecules often have toxicities that are a function of molecular structural features. Minor variations in structural features can make large difference in such toxicity. Consequently, in silico techniques may be used to correlate such molecular toxicities with their structural features. Relative to nine different sets of aromatic nitro compounds having known observed toxicities against different targets, we developed ligand-based 2D quantitative structure-toxicity relationship models using 20 selected topological descriptors. The topological descriptors have several advantages such as conformational independency, facile and less time-consuming computation to yield good results. Multiple linear regression analysis was used to correlate variations of toxicity with molecular properties. The information index on molecular size, lopping centric index and Kier flexibility index were identified as fundamental descriptors for different kinds of toxicity, and further showed that molecular size, branching and molecular flexibility might be particularly important factors in quantitative structure-toxicity relationship analysis. This study revealed that topological descriptor-guided quantitative structure-toxicity relationship provided a very useful, cost and time-efficient, in silico tool for describing small-molecule toxicities.

  10. PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

    PubMed

    Chaudhuri, Rima; Sadrieh, Arash; Hoffman, Nolan J; Parker, Benjamin L; Humphrey, Sean J; Stöckli, Jacqueline; Hill, Adam P; James, David E; Yang, Jean Yee Hwa

    2015-08-19

    Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community. Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites

  11. Dynamic quantitative photothermal monitoring of cell death of individual human red blood cells upon glucose depletion

    NASA Astrophysics Data System (ADS)

    Vasudevan, Srivathsan; Chen, George Chung Kit; Andika, Marta; Agarwal, Shuchi; Chen, Peng; Olivo, Malini

    2010-09-01

    Red blood cells (RBCs) have been found to undergo ``programmed cell death,'' or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.

  12. Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast

    PubMed Central

    Pang, Wei; Coghill, George M.

    2015-01-01

    In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. PMID:25864377

  13. Rigour in quantitative research.

    PubMed

    Claydon, Leica Sarah

    2015-07-22

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

  14. Discovery of phosphorylation motif mixtures in phosphoproteomics data

    PubMed Central

    Ritz, Anna; Shakhnarovich, Gregory; Salomon, Arthur R.; Raphael, Benjamin J.

    2009-01-01

    Motivation: Modification of proteins via phosphorylation is a primary mechanism for signal transduction in cells. Phosphorylation sites on proteins are determined in part through particular patterns, or motifs, present in the amino acid sequence. Results: We describe an algorithm that simultaneously discovers multiple motifs in a set of peptides that were phosphorylated by several different kinases. Such sets of peptides are routinely produced in proteomics experiments.Our motif-finding algorithm uses the principle of minimum description length to determine a mixture of sequence motifs that distinguish a foreground set of phosphopeptides from a background set of unphosphorylated peptides. We show that our algorithm outperforms existing motif-finding algorithms on synthetic datasets consisting of mixtures of known phosphorylation sites. We also derive a motif specificity score that quantifies whether or not the phosphoproteins containing an instance of a motif have a significant number of known interactions. Application of our motif-finding algorithm to recently published human and mouse proteomic studies recovers several known phosphorylation motifs and reveals a number of novel motifs that are enriched for interactions with a particular kinase or phosphatase. Our tools provide a new approach for uncovering the sequence specificities of uncharacterized kinases or phosphatases. Availability: Software is available at http:/cs.brown.edu/people/braphael/software.html. Contact: aritz@cs.brown.edu; braphael@cs.brown.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18996944

  15. A Quantitative Analysis of the Increase in Public School Segregation in Delaware: 1989-2006

    ERIC Educational Resources Information Center

    Glenn, William J.

    2011-01-01

    This study analyzes the increase in school segregation in Delaware from a quantitative perspective. The article tests the hypothesis that the declaration of unitary status that released the Wilmington area school districts from their desegregation order caused the increase in segregation. The research reveals that the declaration of unitary status…

  16. iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis

    PubMed Central

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

    2016-01-01

    Background Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. Results The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Conclusions

  17. iTRAQ-Based Quantitative Proteomics Analysis of Black Rice Grain Development Reveals Metabolic Pathways Associated with Anthocyanin Biosynthesis.

    PubMed

    Chen, Linghua; Huang, Yining; Xu, Ming; Cheng, Zuxin; Zhang, Dasheng; Zheng, Jingui

    2016-01-01

    Black rice (Oryza sativa L.), whose pericarp is rich in anthocyanins (ACNs), is considered as a healthier alternative to white rice. Molecular species of ACNs in black rice have been well documented in previous studies; however, information about the metabolic mechanisms underlying ACN biosynthesis during black rice grain development is unclear. The aim of the present study was to determine changes in the metabolic pathways that are involved in the dynamic grain proteome during the development of black rice indica cultivar, (Oryza sativa L. indica var. SSP). Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were employed to identify statistically significant alterations in the grain proteome. Approximately 928 proteins were detected, of which 230 were differentially expressed throughout 5 successive developmental stages, starting from 3 to 20 days after flowering (DAF). The greatest number of differentially expressed proteins was observed on 7 and 10 DAF, including 76 proteins that were upregulated and 39 that were downregulated. The biological process analysis of gene ontology revealed that the 230 differentially expressed proteins could be sorted into 14 functional groups. Proteins in the largest group were related to metabolic process, which could be integrated into multiple biochemical pathways. Specifically, proteins with a role in ACN biosynthesis, sugar synthesis, and the regulation of gene expression were upregulated, particularly from the onset of black rice grain development and during development. In contrast, the expression of proteins related to signal transduction, redox homeostasis, photosynthesis and N-metabolism decreased during grain maturation. Finally, 8 representative genes encoding different metabolic proteins were verified via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, these genes had differed in transcriptional and translational expression during grain development. Expression analyses of

  18. Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors.

    PubMed

    Klajic, Jovana; Fleischer, Thomas; Dejeux, Emelyne; Edvardsen, Hege; Warnberg, Fredrik; Bukholm, Ida; Lønning, Per Eystein; Solvang, Hiroko; Børresen-Dale, Anne-Lise; Tost, Jörg; Kristensen, Vessela N

    2013-10-05

    Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.

  19. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    PubMed

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  20. Quantitative photoacoustic characterization of blood clot in blood: A mechanobiological assessment through spectral information

    NASA Astrophysics Data System (ADS)

    Biswas, Deblina; Vasudevan, Srivathsan; Chen, George C. K.; Sharma, Norman

    2017-02-01

    Formation of blood clots, called thrombus, can happen due to hyper-coagulation of blood. Thrombi, while moving through blood vessels can impede blood flow, an important criterion for many critical diseases like deep vein thrombosis and heart attacks. Understanding mechanical properties of clot formation is vital for assessment of severity of thrombosis and proper treatment. However, biomechanics of thrombus is less known to clinicians and not very well investigated. Photoacoustic (PA) spectral response, a non-invasive technique, is proposed to investigate the mechanism of formation of blood clots through elasticity and also differentiate clots from blood. Distinct shift (increase in frequency) of the PA response dominant frequency during clot formation is reported. In addition, quantitative differentiation of blood clots from blood has been achieved through parameters like dominant frequency and spectral energy of PA spectral response. Nearly twofold increases in dominant frequency in blood clots compared to blood were found in the PA spectral response. Significant changes in energy also help in quantitatively differentiating clots from blood, in the blood. Our results reveal that increase in density during clot formation is reflected in the PA spectral response, a significant step towards understanding the mechanobiology of thrombus formation. Hence, the proposed tool, in addition to detecting thrombus formation, could reveal mechanical properties of the sample through quantitative photoacoustic spectral parameters.

  1. Quantitative rates of brain glucose metabolism distinguish minimally conscious from vegetative state patients.

    PubMed

    Stender, Johan; Kupers, Ron; Rodell, Anders; Thibaut, Aurore; Chatelle, Camille; Bruno, Marie-Aurélie; Gejl, Michael; Bernard, Claire; Hustinx, Roland; Laureys, Steven; Gjedde, Albert

    2015-01-01

    The differentiation of the vegetative or unresponsive wakefulness syndrome (VS/UWS) from the minimally conscious state (MCS) is an important clinical issue. The cerebral metabolic rate of glucose (CMRglc) declines when consciousness is lost, and may reveal the residual cognitive function of these patients. However, no quantitative comparisons of cerebral glucose metabolism in VS/UWS and MCS have yet been reported. We calculated the regional and whole-brain CMRglc of 41 patients in the states of VS/UWS (n=14), MCS (n=21) or emergence from MCS (EMCS, n=6), and healthy volunteers (n=29). Global cortical CMRglc in VS/UWS and MCS averaged 42% and 55% of normal, respectively. Differences between VS/UWS and MCS were most pronounced in the frontoparietal cortex, at 42% and 60% of normal. In brainstem and thalamus, metabolism declined equally in the two conditions. In EMCS, metabolic rates were indistinguishable from those of MCS. Ordinal logistic regression predicted that patients are likely to emerge into MCS at CMRglc above 45% of normal. Receiver-operating characteristics showed that patients in MCS and VS/UWS can be differentiated with 82% accuracy, based on cortical metabolism. Together these results reveal a significant correlation between whole-brain energy metabolism and level of consciousness, suggesting that quantitative values of CMRglc reveal consciousness in severely brain-injured patients.

  2. Race and Older Mothers’ Differentiation: A Sequential Quantitative and Qualitative Analysis

    PubMed Central

    Sechrist, Jori; Suitor, J. Jill; Riffin, Catherine; Taylor-Watson, Kadari; Pillemer, Karl

    2011-01-01

    The goal of this paper is to demonstrate a process by which qualitative and quantitative approaches are combined to reveal patterns in the data that are unlikely to be detected and confirmed by either method alone. Specifically, we take a sequential approach to combining qualitative and quantitative data to explore race differences in how mothers differentiate among their adult children. We began with a standard multivariate analysis examining race differences in mothers’ differentiation among their adult children regarding emotional closeness and confiding. Finding no race differences in this analysis, we conducted an in-depth comparison of the Black and White mothers’ narratives to determine whether there were underlying patterns that we had been unable to detect in our first analysis. Using this method, we found that Black mothers were substantially more likely than White mothers to emphasize interpersonal relationships within the family when describing differences among their children. In our final step, we developed a measure of familism based on the qualitative data and conducted a multivariate analysis to confirm the patterns revealed by the in-depth comparison of the mother’s narratives. We conclude that using such a sequential mixed methods approach to data analysis has the potential to shed new light on complex family relations. PMID:21967639

  3. Use of MRI in Differentiation of Papillary Renal Cell Carcinoma Subtypes: Qualitative and Quantitative Analysis.

    PubMed

    Doshi, Ankur M; Ream, Justin M; Kierans, Andrea S; Bilbily, Matthew; Rusinek, Henry; Huang, William C; Chandarana, Hersh

    2016-03-01

    The purpose of this study was to determine whether qualitative and quantitative MRI feature analysis is useful for differentiating type 1 from type 2 papillary renal cell carcinoma (PRCC). This retrospective study included 21 type 1 and 17 type 2 PRCCs evaluated with preoperative MRI. Two radiologists independently evaluated various qualitative features, including signal intensity, heterogeneity, and margin. For the quantitative analysis, a radiology fellow and a medical student independently drew 3D volumes of interest over the entire tumor on T2-weighted HASTE images, apparent diffusion coefficient parametric maps, and nephrographic phase contrast-enhanced MR images to derive first-order texture metrics. Qualitative and quantitative features were compared between the groups. For both readers, qualitative features with greater frequency in type 2 PRCC included heterogeneous enhancement, indistinct margin, and T2 heterogeneity (all, p < 0.035). Indistinct margins and heterogeneous enhancement were independent predictors (AUC, 0.822). Quantitative analysis revealed that apparent diffusion coefficient, HASTE, and contrast-enhanced entropy were greater in type 2 PRCC (p < 0.05; AUC, 0.682-0.716). A combined quantitative and qualitative model had an AUC of 0.859. Qualitative features within the model had interreader concordance of 84-95%, and the quantitative data had intraclass coefficients of 0.873-0.961. Qualitative and quantitative features can help discriminate between type 1 and type 2 PRCC. Quantitative analysis may capture useful information that complements the qualitative appearance while benefiting from high interobserver agreement.

  4. Critical Quantitative Inquiry in Context

    ERIC Educational Resources Information Center

    Stage, Frances K.; Wells, Ryan S.

    2014-01-01

    This chapter briefly traces the development of the concept of critical quantitative inquiry, provides an expanded conceptualization of the tasks of critical quantitative research, offers theoretical explanation and justification for critical research using quantitative methods, and previews the work of quantitative criticalists presented in this…

  5. Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast.

    PubMed

    Pang, Wei; Coghill, George M

    2015-05-01

    In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  6. Quantitative dispersion microscopy

    PubMed Central

    Fu, Dan; Choi, Wonshik; Sung, Yongjin; Yaqoob, Zahid; Dasari, Ramachandra R.; Feld, Michael

    2010-01-01

    Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples. PMID:21113234

  7. Reproducibility and quantitation of amplicon sequencing-based detection

    PubMed Central

    Zhou, Jizhong; Wu, Liyou; Deng, Ye; Zhi, Xiaoyang; Jiang, Yi-Huei; Tu, Qichao; Xie, Jianping; Van Nostrand, Joy D; He, Zhili; Yang, Yunfeng

    2011-01-01

    To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative

  8. Systematic inference of functional phosphorylation events in yeast metabolism.

    PubMed

    Chen, Yu; Wang, Yonghong; Nielsen, Jens

    2017-07-01

    Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  9. Qualitative and quantitative studies of chemical composition of sandarac resin by GC-MS.

    PubMed

    Kononenko, I; de Viguerie, L; Rochut, S; Walter, Ph

    2017-01-01

    The chemical composition of sandarac resin was investigated qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS). Six compounds with labdane and pimarane skeletons were identified in the resin. The obtained mass spectra were interpreted and the mass spectrometric behaviour of these diterpenoids under EI conditions was described. Quantitative analysis by the method of internal standard revealed that identified diterpenoids represent only 10-30% of the analysed sample. The sandarac resin from different suppliers was analysed (from Kremer, Okhra, Color Rare, La Marchande de Couleurs, L'Atelier Montessori, Hevea). The analysis of different lumps of resins showed that the chemical composition differs from one lump to another, varying mainly in the relative distributions of the components.

  10. Quantitative proteomics analysis by iTRAQ revealed underlying changes in thermotolerance of Arthrospira platensis.

    PubMed

    Chang, Rong; Lv, Bingxin; Li, Bosheng

    2017-08-08

    Growth temperature is a critical factor that affects cultivation of Arthrospira platensis which is a type of cyanobacterium widely known as Spirulina that has significant commercial value. To investigate the molecular mechanism underlying the thermotolerance of Spirulina, differential protein expression profiling was carried out using iTRAQ-based quantitative proteomic analysis. This study only analyzed changes in thylakoids. Among the 2085 proteins quantified, 43 differentially expressed proteins were selected based on the fold change cutoff scores of ≥2 or ≤0.5 for up-regulation or down-regulation, respectively. An analysis of these 43 proteins found that 23% of them are photosynthetic system proteins which include photosynthetic enzymes and pigment proteins. The dynamic change of these proteins indicates that photosynthetic system functions were profoundly affected under heat stress and the light-dependent reactions were probably the most sensitive to temperature changes. Meanwhile, to cope with the low energy production due to impaired photosynthesis there was a considerable down-shift in protein synthesis which is a very energy demanding process. The impaired photosynthesis led to low energy generation that was compensated by a down-shift in translation (the most energy-demanding process) and an up-shift of glycolysis. The reduction of many ribosome proteins may lead to a loss in translation efficiency; therefore, Spirulina may adopted a different mechanism to increase translational elongation under heat stress to compensate for this loss, such as elevate L7/L12 proteins. Changes were also found in the classical heat shock proteins, the ROS scavenging system, DNA-binding proteins, and some membrane proteins. In conclusion, this research demonstrate that heat stress induces profound changes in cellular physiology and shed light on the mechanism of the heat stress response and thermotolerance of Arthrospira platensis. Arthrospira platensis, widely known as

  11. Quantitative and Qualitative Evaluations of the Enhanced Logo-autobiography Program for Korean-American Women.

    PubMed

    Sung, Kyung Mi; Bernstein, Kunsook

    2017-12-01

    This study extends Bernstein et al.'s (2016) investigation of the effects of the Enhanced Logo-autobiography Program on Korean-American women's depressive symptoms, coping strategies, purpose in life, and posttraumatic growth by analyzing quantitative and qualitative data. This study's participants significantly improved on quantitative measures of depression, coping strategies, purpose in life, and post-traumatic growth at eight weeks post-intervention and follow-up. The qualitative content analysis revealed 17 themes with five essential themes. The program's activity to promote purpose in life through posttraumatic growth facilitated participants' recovery from traumatic experiences. Standardized guidelines are needed to conduct this program in Korean community centers.

  12. Quantitative characterization of mechanically indented in vivo human skin in adults and infants using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Huang, Pin-Chieh; Pande, Paritosh; Shelton, Ryan L.; Joa, Frank; Moore, Dave; Gillman, Elisa; Kidd, Kimberly; Nolan, Ryan M.; Odio, Mauricio; Carr, Andrew; Boppart, Stephen A.

    2017-03-01

    Influenced by both the intrinsic viscoelasticity of the tissue constituents and the time-evolved redistribution of fluid within the tissue, the biomechanical response of skin can reflect not only localized pathology but also systemic physiology of an individual. While clinical diagnosis of skin pathologies typically relies on visual inspection and manual palpation, a more objective and quantitative approach for tissue characterization is highly desirable. Optical coherence tomography (OCT) is an interferometry-based imaging modality that enables in vivo assessment of cross-sectional tissue morphology with micron-scale resolution, which surpasses those of most standard clinical imaging tools, such as ultrasound imaging and magnetic resonance imaging. This pilot study investigates the feasibility of characterizing the biomechanical response of in vivo human skin using OCT. OCT-based quantitative metrics were developed and demonstrated on the human subject data, where a significant difference between deformed and nondeformed skin was revealed. Additionally, the quantified postindentation recovery results revealed differences between aged (adult) and young (infant) skin. These suggest that OCT has the potential to quantitatively assess the mechanically perturbed skin as well as distinguish different physiological conditions of the skin, such as changes with age or disease.

  13. Direct visualization reveals kinetics of meiotic chromosome synapsis

    DOE PAGES

    Rog, Ofer; Dernburg, Abby  F.

    2015-03-17

    The synaptonemal complex (SC) is a conserved protein complex that stabilizes interactions along homologous chromosomes (homologs) during meiosis. The SC regulates genetic exchanges between homologs, thereby enabling reductional division and the production of haploid gametes. Here, we directly observe SC assembly (synapsis) by optimizing methods for long-term fluorescence recording in C. elegans. We report that synapsis initiates independently on each chromosome pair at or near pairing centers—specialized regions required for homolog associations. Once initiated, the SC extends rapidly and mostly irreversibly to chromosome ends. Quantitation of SC initiation frequencies and extension rates reveals that initiation is a rate-limiting step inmore » homolog interactions. Eliminating the dynein-driven chromosome movements that accompany synapsis severely retards SC extension, revealing a new role for these conserved motions. This work provides the first opportunity to directly observe and quantify key aspects of meiotic chromosome interactions and will enable future in vivo analysis of germline processes.« less

  14. Toward Quantitative Small Animal Pinhole SPECT: Assessment of Quantitation Accuracy Prior to Image Compensations

    PubMed Central

    Chen, Chia-Lin; Wang, Yuchuan; Lee, Jason J. S.; Tsui, Benjamin M. W.

    2011-01-01

    Purpose We assessed the quantitation accuracy of small animal pinhole single photon emission computed tomography (SPECT) under the current preclinical settings, where image compensations are not routinely applied. Procedures The effects of several common image-degrading factors and imaging parameters on quantitation accuracy were evaluated using Monte-Carlo simulation methods. Typical preclinical imaging configurations were modeled, and quantitative analyses were performed based on image reconstructions without compensating for attenuation, scatter, and limited system resolution. Results Using mouse-sized phantom studies as examples, attenuation effects alone degraded quantitation accuracy by up to −18% (Tc-99m or In-111) or −41% (I-125). The inclusion of scatter effects changed the above numbers to −12% (Tc-99m or In-111) and −21% (I-125), respectively, indicating the significance of scatter in quantitative I-125 imaging. Region-of-interest (ROI) definitions have greater impacts on regional quantitation accuracy for small sphere sources as compared to attenuation and scatter effects. For the same ROI, SPECT acquisitions using pinhole apertures of different sizes could significantly affect the outcome, whereas the use of different radii-of-rotation yielded negligible differences in quantitation accuracy for the imaging configurations simulated. Conclusions We have systematically quantified the influence of several factors affecting the quantitation accuracy of small animal pinhole SPECT. In order to consistently achieve accurate quantitation within 5% of the truth, comprehensive image compensation methods are needed. PMID:19048346

  15. The Development of Mathematical Knowledge for Teaching for Quantitative Reasoning Using Video-Based Instruction

    NASA Astrophysics Data System (ADS)

    Walters, Charles David

    Quantitative reasoning (P. W. Thompson, 1990, 1994) is a powerful mathematical tool that enables students to engage in rich problem solving across the curriculum. One way to support students' quantitative reasoning is to develop prospective secondary teachers' (PSTs) mathematical knowledge for teaching (MKT; Ball, Thames, & Phelps, 2008) related to quantitative reasoning. However, this may prove challenging, as prior to entering the classroom, PSTs often have few opportunities to develop MKT by examining and reflecting on students' thinking. Videos offer one avenue through which such opportunities are possible. In this study, I report on the design of a mini-course for PSTs that featured a series of videos created as part of a proof-of-concept NSF-funded project. These MathTalk videos highlight the ways in which the quantitative reasoning of two high school students developed over time. Using a mixed approach to grounded theory, I analyzed pre- and postinterviews using an extant coding scheme based on the Silverman and Thompson (2008) framework for the development of MKT. This analysis revealed a shift in participants' affect as well as three distinct shifts in their MKT around quantitative reasoning with distances, including shifts in: (a) quantitative reasoning; (b) point of view (decentering); and (c) orientation toward problem solving. Using the four-part focusing framework (Lobato, Hohensee, & Rhodehamel, 2013), I analyzed classroom data to account for how participants' noticing was linked with the shifts in MKT. Notably, their increased noticing of aspects of MKT around quantitative reasoning with distances, which features prominently in the MathTalk videos, seemed to contribute to the emergence of the shifts in MKT. Results from this study link elements of the learning environment to the development of specific facets of MKT around quantitative reasoning with distances. These connections suggest that vicarious experiences with two students' quantitative

  16. Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

    PubMed Central

    Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

    2016-01-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  17. Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

    PubMed

    Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

    2016-11-01

    The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Quantitative modelling in cognitive ergonomics: predicting signals passed at danger.

    PubMed

    Moray, Neville; Groeger, John; Stanton, Neville

    2017-02-01

    This paper shows how to combine field observations, experimental data and mathematical modelling to produce quantitative explanations and predictions of complex events in human-machine interaction. As an example, we consider a major railway accident. In 1999, a commuter train passed a red signal near Ladbroke Grove, UK, into the path of an express. We use the Public Inquiry Report, 'black box' data, and accident and engineering reports to construct a case history of the accident. We show how to combine field data with mathematical modelling to estimate the probability that the driver observed and identified the state of the signals, and checked their status. Our methodology can explain the SPAD ('Signal Passed At Danger'), generate recommendations about signal design and placement and provide quantitative guidance for the design of safer railway systems' speed limits and the location of signals. Practitioner Summary: Detailed ergonomic analysis of railway signals and rail infrastructure reveals problems of signal identification at this location. A record of driver eye movements measures attention, from which a quantitative model for out signal placement and permitted speeds can be derived. The paper is an example of how to combine field data, basic research and mathematical modelling to solve ergonomic design problems.

  19. Quantitative sonoelastography for the in vivo assessment of skeletal muscle viscoelasticity

    NASA Astrophysics Data System (ADS)

    Hoyt, Kenneth; Kneezel, Timothy; Castaneda, Benjamin; Parker, Kevin J.

    2008-08-01

    A novel quantitative sonoelastography technique for assessing the viscoelastic properties of skeletal muscle tissue was developed. Slowly propagating shear wave interference patterns (termed crawling waves) were generated using a two-source configuration vibrating normal to the surface. Theoretical models predict crawling wave displacement fields, which were validated through phantom studies. In experiments, a viscoelastic model was fit to dispersive shear wave speed sonoelastographic data using nonlinear least-squares techniques to determine frequency-independent shear modulus and viscosity estimates. Shear modulus estimates derived using the viscoelastic model were in agreement with that obtained by mechanical testing on phantom samples. Preliminary sonoelastographic data acquired in healthy human skeletal muscles confirm that high-quality quantitative elasticity data can be acquired in vivo. Studies on relaxed muscle indicate discernible differences in both shear modulus and viscosity estimates between different skeletal muscle groups. Investigations into the dynamic viscoelastic properties of (healthy) human skeletal muscles revealed that voluntarily contracted muscles exhibit considerable increases in both shear modulus and viscosity estimates as compared to the relaxed state. Overall, preliminary results are encouraging and quantitative sonoelastography may prove clinically feasible for in vivo characterization of the dynamic viscoelastic properties of human skeletal muscle.

  20. Verbal Rehearsal and Memory in Children with Closed Head Injury: A Quantitative and Qualitative Analysis.

    ERIC Educational Resources Information Center

    Harris, Jessica R.

    1996-01-01

    Nine closed head injured (CHI) children (mean age 11 years) with post-onset intervals of 7 months to 8 years were given an overt free recall task. Quantitative analysis suggested inefficient passive rehearsal strategy by severely injured subjects. Qualitative analysis revealed differences between CHI children and controls in rehearsal strategies,…

  1. Deficiencies in Jasmonate-Mediated Plant Defense Reveal Quantitative Variation in Botrytis cinerea Pathogenesis

    PubMed Central

    Rowe, Heather C.; Walley, Justin W.; Corwin, Jason; Chan, Eva K.-F.; Dehesh, Katayoon; Kliebenstein, Daniel J.

    2010-01-01

    Despite the described central role of jasmonate signaling in plant defense against necrotrophic pathogens, the existence of intraspecific variation in pathogen capacity to activate or evade plant jasmonate-mediated defenses is rarely considered. Experimental infection of jasmonate-deficient and jasmonate-insensitive Arabidopsis thaliana with diverse isolates of the necrotrophic fungal pathogen Botrytis cinerea revealed pathogen variation for virulence inhibition by jasmonate-mediated plant defenses and induction of plant defense metabolites. Comparison of the transcriptional effects of infection by two distinct B. cinerea isolates showed only minor differences in transcriptional responses of wild-type plants, but notable isolate-specific transcript differences in jasmonate-insensitive plants. These transcriptional differences suggest B. cinerea activation of plant defenses that require plant jasmonate signaling for activity in response to only one of the two B. cinerea isolates tested. Thus, similar infection phenotypes observed in wild-type plants result from different signaling interactions with the plant that are likely integrated by jasmonate signaling. PMID:20419157

  2. Visualization and Quantitative Analysis of Crack-Tip Plastic Zone in Pure Nickel

    NASA Astrophysics Data System (ADS)

    Kelton, Randall; Sola, Jalal Fathi; Meletis, Efstathios I.; Huang, Haiying

    2018-05-01

    Changes in surface morphology have long been thought to be associated with crack propagation in metallic materials. We have studied areal surface texture changes around crack tips in an attempt to understand the correlations between surface texture changes and crack growth behavior. Detailed profiling of the fatigue sample surface was carried out at short fatigue intervals. An image processing algorithm was developed to calculate the surface texture changes. Quantitative analysis of the crack-tip plastic zone, crack-arrested sites near triple points, and large surface texture changes associated with crack release from arrested locations was carried out. The results indicate that surface texture imaging enables visualization of the development of plastic deformation around a crack tip. Quantitative analysis of the surface texture changes reveals the effects of local microstructures on the crack growth behavior.

  3. Quantitative Literacy: Geosciences and Beyond

    NASA Astrophysics Data System (ADS)

    Richardson, R. M.; McCallum, W. G.

    2002-12-01

    Quantitative literacy seems like such a natural for the geosciences, right? The field has gone from its origin as a largely descriptive discipline to one where it is hard to imagine failing to bring a full range of mathematical tools to the solution of geological problems. Although there are many definitions of quantitative literacy, we have proposed one that is analogous to the UNESCO definition of conventional literacy: "A quantitatively literate person is one who, with understanding, can both read and represent quantitative information arising in his or her everyday life." Central to this definition is the concept that a curriculum for quantitative literacy must go beyond the basic ability to "read and write" mathematics and develop conceptual understanding. It is also critical that a curriculum for quantitative literacy be engaged with a context, be it everyday life, humanities, geoscience or other sciences, business, engineering, or technology. Thus, our definition works both within and outside the sciences. What role do geoscience faculty have in helping students become quantitatively literate? Is it our role, or that of the mathematicians? How does quantitative literacy vary between different scientific and engineering fields? Or between science and nonscience fields? We will argue that successful quantitative literacy curricula must be an across-the-curriculum responsibility. We will share examples of how quantitative literacy can be developed within a geoscience curriculum, beginning with introductory classes for nonmajors (using the Mauna Loa CO2 data set) through graduate courses in inverse theory (using singular value decomposition). We will highlight six approaches to across-the curriculum efforts from national models: collaboration between mathematics and other faculty; gateway testing; intensive instructional support; workshops for nonmathematics faculty; quantitative reasoning requirement; and individual initiative by nonmathematics faculty.

  4. Quantitative analyses for elucidating mechanisms of cell fate commitment in the mouse blastocyst

    NASA Astrophysics Data System (ADS)

    Saiz, Néstor; Kang, Minjung; Puliafito, Alberto; Schrode, Nadine; Xenopoulos, Panagiotis; Lou, Xinghua; Di Talia, Stefano; Hadjantonakis, Anna-Katerina

    2015-03-01

    In recent years we have witnessed a shift from qualitative image analysis towards higher resolution, quantitative analyses of imaging data in developmental biology. This shift has been fueled by technological advances in both imaging and analysis software. We have recently developed a tool for accurate, semi-automated nuclear segmentation of imaging data from early mouse embryos and embryonic stem cells. We have applied this software to the study of the first lineage decisions that take place during mouse development and established analysis pipelines for both static and time-lapse imaging experiments. In this paper we summarize the conclusions from these studies to illustrate how quantitative, single-cell level analysis of imaging data can unveil biological processes that cannot be revealed by traditional qualitative studies.

  5. Quantitative magneto-optical investigation of superconductor/ferromagnet hybrid structures

    NASA Astrophysics Data System (ADS)

    Shaw, G.; Brisbois, J.; Pinheiro, L. B. G. L.; Müller, J.; Blanco Alvarez, S.; Devillers, T.; Dempsey, N. M.; Scheerder, J. E.; Van de Vondel, J.; Melinte, S.; Vanderbemden, P.; Motta, M.; Ortiz, W. A.; Hasselbach, K.; Kramer, R. B. G.; Silhanek, A. V.

    2018-02-01

    We present a detailed quantitative magneto-optical imaging study of several superconductor/ferromagnet hybrid structures, including Nb deposited on top of thermomagnetically patterned NdFeB and permalloy/niobium with erasable and tailored magnetic landscapes imprinted in the permalloy layer. The magneto-optical imaging data are complemented with and compared to scanning Hall probe microscopy measurements. Comprehensive protocols have been developed for calibrating, testing, and converting Faraday rotation data to magnetic field maps. Applied to the acquired data, they reveal the comparatively weaker magnetic response of the superconductor from the background of larger fields and field gradients generated by the magnetic layer.

  6. Dynamically monitoring the gene expression of dual fluorophore in the cell cycle with quantitative spectrum analysis

    NASA Astrophysics Data System (ADS)

    Lee, Ja-Yun; Wu, Tzong-Yuan; Hsu, I.-Jen

    2008-04-01

    The cloning and transcription techniques on gene cloned fluorescent proteins have been widely used in many applications. They have been used as reporters of some conditions in a series of reactions. However, it is usually difficult to monitor the specific target with the exactly number of proteins during the process in turbid media, especially at micrometer scales. We successfully revealed an alternative way to monitor the cell cycle behavior and quantitatively analyzed the target cells with green and red fluorescent proteins (GFP and RFP) during different phases of the cell cycle by quantitatively analyzing its behavior and also monitoring its spatial distribution.

  7. Quantitative evaluation of orbital hybridization in carbon nanotubes under radial deformation using π-orbital axis vector

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohnishi, Masato, E-mail: masato.ohnishi@rift.mech.tohoku.ac.jp; Suzuki, Ken; Miura, Hideo, E-mail: hmiura@rift.mech.tohoku.ac.jp

    2015-04-15

    When a radial strain is applied to a carbon nanotube (CNT), the increase in local curvature induces orbital hybridization. The effect of the curvature-induced orbital hybridization on the electronic properties of CNTs, however, has not been evaluated quantitatively. In this study, the strength of orbital hybridization in CNTs under homogeneous radial strain was evaluated quantitatively. Our analyses revealed the detailed procedure of the change in electronic structure of CNTs. In addition, the dihedral angle, the angle between π-orbital axis vectors of adjacent atoms, was found to effectively predict the strength of local orbital hybridization in deformed CNTs.

  8. Quantitative nucleolar proteomics reveals nuclear re-organization during stress- induced senescence in mouse fibroblast

    PubMed Central

    2011-01-01

    Background Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence. Results In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice. Conclusion Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence. PMID:21835027

  9. Quantitative Glycomics Strategies*

    PubMed Central

    Mechref, Yehia; Hu, Yunli; Desantos-Garcia, Janie L.; Hussein, Ahmed; Tang, Haixu

    2013-01-01

    The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. PMID:23325767

  10. Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells

    NASA Astrophysics Data System (ADS)

    Dvir, Liron; Nissim, Ronen; Alvarez-Elizondo, Martha B.; Weihs, Daphne

    2015-04-01

    Metastasis formation is a major cause of mortality in cancer patients and includes tumor cell relocation to distant organs. A metastatic cell invades through other cells and extracellular matrix by biochemical attachment and mechanical force application. Force is used to move on or through a 2- or 3-dimensional (3D) environment, respectively, or to penetrate a 2D substrate. We have previously shown that even when a gel substrate is impenetrable, metastatic breast cancer cells can still indent it by applying force. Cells typically apply force through the acto-myosin network, which is mechanically connected to the nucleus. We develop a 3D image-analysis to reveal relative locations of the cell elements, and show that as cells apply force to the gel, a coordinated process occurs that involves cytoskeletal remodeling and repositioning of the nucleus. Our approach shows that the actin and microtubules reorganize in the cell, bringing the actin to the leading edge of the cell. In parallel, the nucleus is transported behind the actin, likely by the cytoskeleton, into the indentation dimple formed in the gel. The nucleus volume below the gel surface correlates with indentation depth, when metastatic breast cancer cells indent gels deeply. However, the nucleus always remains above the gel in benign cells, even when small indentations are observed. Determining mechanical processes during metastatic cell invasion can reveal how cells disseminate in the body and can uncover targets for diagnosis and treatment.

  11. Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

    PubMed

    Radic-Sarikas, Branka; Tsafou, Kalliopi P; Emdal, Kristina B; Papamarkou, Theodore; Huber, Kilian V M; Mutz, Cornelia; Toretsky, Jeffrey A; Bennett, Keiryn L; Olsen, Jesper V; Brunak, Søren; Kovar, Heinrich; Superti-Furga, Giulio

    2017-01-01

    Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Quantitative tests for plate tectonics on Venus

    NASA Technical Reports Server (NTRS)

    Kaula, W. M.; Phillips, R. J.

    1981-01-01

    Quantitative comparisons are made between the characteristics of plate tectonics on the earth and those which are possible on Venus. Considerations of the factors influencing rise height and relating the decrease in rise height to plate velocity indicate that the rate of topographic dropoff from spreading centers should be about half that on earth due to greater rock-fluid density contrast and lower temperature differential between the surface and interior. Statistical analyses of Pioneer Venus radar altimetry data and global earth elevation data is used to identify 21,000 km of ridge on Venus and 33,000 km on earth, and reveal Venus ridges to have a less well-defined mode in crest heights and a greater concavity than earth ridges. Comparison of the Venus results with the spreading rates and associated heat flow on earth reveals plate creation rates on Venus to be 0.7 sq km/year or less and indicates that not more than 15% of Venus's energy is delivered to the surface by plate tectonics, in contrast to values of 2.9 sq km a year and 70% for earth.

  13. Affinity for Quantitative Tools: Undergraduate Marketing Students Moving beyond Quantitative Anxiety

    ERIC Educational Resources Information Center

    Tarasi, Crina O.; Wilson, J. Holton; Puri, Cheenu; Divine, Richard L.

    2013-01-01

    Marketing students are known as less likely to have an affinity for the quantitative aspects of the marketing discipline. In this article, we study the reasons why this might be true and develop a parsimonious 20-item scale for measuring quantitative affinity in undergraduate marketing students. The scale was administered to a sample of business…

  14. A dynamic regression analysis tool for quantitative assessment of bacterial growth written in Python.

    PubMed

    Hoeflinger, Jennifer L; Hoeflinger, Daniel E; Miller, Michael J

    2017-01-01

    Herein, an open-source method to generate quantitative bacterial growth data from high-throughput microplate assays is described. The bacterial lag time, maximum specific growth rate, doubling time and delta OD are reported. Our method was validated by carbohydrate utilization of lactobacilli, and visual inspection revealed 94% of regressions were deemed excellent. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Bigger Is Fitter? Quantitative Genetic Decomposition of Selection Reveals an Adaptive Evolutionary Decline of Body Mass in a Wild Rodent Population.

    PubMed

    Bonnet, Timothée; Wandeler, Peter; Camenisch, Glauco; Postma, Erik

    2017-01-01

    In natural populations, quantitative trait dynamics often do not appear to follow evolutionary predictions. Despite abundant examples of natural selection acting on heritable traits, conclusive evidence for contemporary adaptive evolution remains rare for wild vertebrate populations, and phenotypic stasis seems to be the norm. This so-called "stasis paradox" highlights our inability to predict evolutionary change, which is especially concerning within the context of rapid anthropogenic environmental change. While the causes underlying the stasis paradox are hotly debated, comprehensive attempts aiming at a resolution are lacking. Here, we apply a quantitative genetic framework to individual-based long-term data for a wild rodent population and show that despite a positive association between body mass and fitness, there has been a genetic change towards lower body mass. The latter represents an adaptive response to viability selection favouring juveniles growing up to become relatively small adults, i.e., with a low potential adult mass, which presumably complete their development earlier. This selection is particularly strong towards the end of the snow-free season, and it has intensified in recent years, coinciding which a change in snowfall patterns. Importantly, neither the negative evolutionary change, nor the selective pressures that drive it, are apparent on the phenotypic level, where they are masked by phenotypic plasticity and a non causal (i.e., non genetic) positive association between body mass and fitness, respectively. Estimating selection at the genetic level enabled us to uncover adaptive evolution in action and to identify the corresponding phenotypic selective pressure. We thereby demonstrate that natural populations can show a rapid and adaptive evolutionary response to a novel selective pressure, and that explicitly (quantitative) genetic models are able to provide us with an understanding of the causes and consequences of selection that is

  16. Bigger Is Fitter? Quantitative Genetic Decomposition of Selection Reveals an Adaptive Evolutionary Decline of Body Mass in a Wild Rodent Population

    PubMed Central

    Wandeler, Peter; Camenisch, Glauco

    2017-01-01

    In natural populations, quantitative trait dynamics often do not appear to follow evolutionary predictions. Despite abundant examples of natural selection acting on heritable traits, conclusive evidence for contemporary adaptive evolution remains rare for wild vertebrate populations, and phenotypic stasis seems to be the norm. This so-called “stasis paradox” highlights our inability to predict evolutionary change, which is especially concerning within the context of rapid anthropogenic environmental change. While the causes underlying the stasis paradox are hotly debated, comprehensive attempts aiming at a resolution are lacking. Here, we apply a quantitative genetic framework to individual-based long-term data for a wild rodent population and show that despite a positive association between body mass and fitness, there has been a genetic change towards lower body mass. The latter represents an adaptive response to viability selection favouring juveniles growing up to become relatively small adults, i.e., with a low potential adult mass, which presumably complete their development earlier. This selection is particularly strong towards the end of the snow-free season, and it has intensified in recent years, coinciding which a change in snowfall patterns. Importantly, neither the negative evolutionary change, nor the selective pressures that drive it, are apparent on the phenotypic level, where they are masked by phenotypic plasticity and a non causal (i.e., non genetic) positive association between body mass and fitness, respectively. Estimating selection at the genetic level enabled us to uncover adaptive evolution in action and to identify the corresponding phenotypic selective pressure. We thereby demonstrate that natural populations can show a rapid and adaptive evolutionary response to a novel selective pressure, and that explicitly (quantitative) genetic models are able to provide us with an understanding of the causes and consequences of selection that is

  17. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

    PubMed

    Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

    2015-10-30

    Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. The Quantitative Analysis of Chennai Automotive Industry Cluster

    NASA Astrophysics Data System (ADS)

    Bhaskaran, Ethirajan

    2016-07-01

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

  19. Towards quantitative condition assessment of biodiversity outcomes: Insights from Australian marine protected areas.

    PubMed

    Addison, Prue F E; Flander, Louisa B; Cook, Carly N

    2017-08-01

    Protected area management effectiveness (PAME) evaluation is increasingly undertaken to evaluate governance, assess conservation outcomes and inform evidence-based management of protected areas (PAs). Within PAME, quantitative approaches to assess biodiversity outcomes are now emerging, where biological monitoring data are directly assessed against quantitative (numerically defined) condition categories (termed quantitative condition assessments). However, more commonly qualitative condition assessments are employed in PAME, which use descriptive condition categories and are evaluated largely with expert judgement that can be subject to a range of biases, such as linguistic uncertainty and overconfidence. Despite the benefits of increased transparency and repeatability of evaluations, quantitative condition assessments are rarely used in PAME. To understand why, we interviewed practitioners from all Australian marine protected area (MPA) networks, which have access to long-term biological monitoring data and are developing or conducting PAME evaluations. Our research revealed that there is a desire within management agencies to implement quantitative condition assessment of biodiversity outcomes in Australian MPAs. However, practitioners report many challenges in transitioning from undertaking qualitative to quantitative condition assessments of biodiversity outcomes, which are hampering progress. Challenges include a lack of agency capacity (staff numbers and money), knowledge gaps, and diminishing public and political support for PAs. We point to opportunities to target strategies that will assist agencies overcome these challenges, including new decision support tools, approaches to better finance conservation efforts, and to promote more management relevant science. While a single solution is unlikely to achieve full evidence-based conservation, we suggest ways for agencies to target strategies and advance PAME evaluations toward best practice. Copyright

  20. Quantitative reduction of the TCR adapter protein SLP-76 unbalances immunity and immune regulation.

    PubMed

    Siggs, Owen M; Miosge, Lisa A; Daley, Stephen R; Asquith, Kelly; Foster, Paul S; Liston, Adrian; Goodnow, Christopher C

    2015-03-15

    Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies. Copyright © 2015 by The American Association of Immunologists, Inc.

  1. [Quantitative analysis of the structure of neuronal dendritic spines in the striatum using the Leitz-ASM system].

    PubMed

    Leontovich, T A; Zvegintseva, E G

    1985-10-01

    Two principal classes of striatum long axonal neurons (sparsely ramified reticular cells and densely ramified dendritic cells) were analyzed quantitatively in four animal species: hedgehog, rabbit, dog and monkey. The cross section area, total dendritic length and the area of dendritic field were measured using "LEITZ-ASM" system. Classes of neurons studied were significantly different in dogs and monkeys, while no differences were noted between hedgehog and rabbit. Reticular neurons of different species varied much more than dendritic ones. Quantitative analysis has revealed the progressive increase in the complexity of dendritic tree in mammals from rabbit to monkey.

  2. Allometric Trajectories and "Stress": A Quantitative Approach.

    PubMed

    Anfodillo, Tommaso; Petit, Giai; Sterck, Frank; Lechthaler, Silvia; Olson, Mark E

    2016-01-01

    The term "stress" is an important but vague term in plant biology. We show situations in which thinking in terms of "stress" is profitably replaced by quantifying distance from functionally optimal scaling relationships between plant parts. These relationships include, for example, the often-cited one between leaf area and sapwood area, which presumably reflects mutual dependence between sources and sink tissues and which scales positively within individuals and across species. These relationships seem to be so basic to plant functioning that they are favored by selection across nearly all plant lineages. Within a species or population, individuals that are far from the common scaling patterns are thus expected to perform negatively. For instance, "too little" leaf area (e.g., due to herbivory or disease) per unit of active stem mass would be expected to incur to low carbon income per respiratory cost and thus lead to lower growth. We present a framework that allows quantitative study of phenomena traditionally assigned to "stress," without need for recourse to this term. Our approach contrasts with traditional approaches for studying "stress," e.g., revealing that small "stressed" plants likely are in fact well suited to local conditions. We thus offer a quantitative perspective to the study of phenomena often referred to under such terms as "stress," plasticity, adaptation, and acclimation.

  3. Precocious quantitative cognition in monkeys.

    PubMed

    Ferrigno, Stephen; Hughes, Kelly D; Cantlon, Jessica F

    2016-02-01

    Basic quantitative abilities are thought to have an innate basis in humans partly because the ability to discriminate quantities emerges early in child development. If humans and nonhuman primates share this developmentally primitive foundation of quantitative reasoning, then this ability should be present early in development across species and should emerge earlier in monkeys than in humans because monkeys mature faster than humans. We report that monkeys spontaneously make accurate quantity choices by 1 year of age in a task that human children begin to perform only at 2.5 to 3 years of age. Additionally, we report that the quantitative sensitivity of infant monkeys is equal to that of the adult animals in their group and that rates of learning do not differ between infant and adult animals. This novel evidence of precocious quantitative reasoning in infant monkeys suggests that human quantitative reasoning shares its early developing foundation with other primates. The data further suggest that early developing components of primate quantitative reasoning are constrained by maturational factors related to genetic development as opposed to learning experience alone.

  4. ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.

    PubMed

    Chen, Xingqi; Shen, Ying; Draper, Will; Buenrostro, Jason D; Litzenburger, Ulrike; Cho, Seung Woo; Satpathy, Ansuman T; Carter, Ava C; Ghosh, Rajarshi P; East-Seletsky, Alexandra; Doudna, Jennifer A; Greenleaf, William J; Liphardt, Jan T; Chang, Howard Y

    2016-12-01

    Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

  5. Quantitative analysis and feature recognition in 3-D microstructural data sets

    NASA Astrophysics Data System (ADS)

    Lewis, A. C.; Suh, C.; Stukowski, M.; Geltmacher, A. B.; Spanos, G.; Rajan, K.

    2006-12-01

    A three-dimensional (3-D) reconstruction of an austenitic stainless-steel microstructure was used as input for an image-based finite-element model to simulate the anisotropic elastic mechanical response of the microstructure. The quantitative data-mining and data-warehousing techniques used to correlate regions of high stress with critical microstructural features are discussed. Initial analysis of elastic stresses near grain boundaries due to mechanical loading revealed low overall correlation with their location in the microstructure. However, the use of data-mining and feature-tracking techniques to identify high-stress outliers revealed that many of these high-stress points are generated near grain boundaries and grain edges (triple junctions). These techniques also allowed for the differentiation between high stresses due to boundary conditions of the finite volume reconstructed, and those due to 3-D microstructural features.

  6. Focus Groups in Elderly Ophthalmologic Patients: Setting the Stage for Quantitative Preference Elicitation.

    PubMed

    Danner, Marion; Vennedey, Vera; Hiligsmann, Mickaël; Fauser, Sascha; Stock, Stephanie

    2016-02-01

    Patients suffering from age-related macular degeneration (AMD) are rarely actively involved in decision-making, despite facing preference-sensitive treatment decisions. This paper presents a qualitative study to prepare quantitative preference elicitation in AMD patients. The aims of this study were (1) to gain familiarity with and learn about the special requirements of the AMD patient population for quantitative data collection; and (2) to select/refine patient-relevant treatment attributes and levels, and gain insights into preference structures. Semi-structured focus group interviews were performed. An interview guide including preselected categories in the form of seven potentially patient-relevant treatment attributes was followed. To identify the most patient-relevant treatment attributes, a ranking exercise was performed. Deductive content analyses were done by two independent reviewers for each attribute to derive subcategories (potential levels of attributes) and depict preference trends. The focus group interviews included 21 patients. The interviews revealed that quantitative preference surveys in this population will have to be interviewer assisted to make the survey feasible for patients. The five most patient-relevant attributes were the effect on visual function [ranking score (RS): 139], injection frequency (RS: 101), approval status (RS: 83), side effects (RS: 79), and monitoring frequency (RS: 76). Attribute and level refinement was based on patients' statements. Preference trends and dependencies between attributes informed the quantitative instrument design. This study suggests that qualitative research is a very helpful step to prepare the design and administration of quantitative preference elicitation instruments. It especially facilitated familiarization with the target population and its preferences, and it supported attribute/level refinement.

  7. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6

    PubMed Central

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A.; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5′ untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5′ terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase. PMID:27877176

  8. Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

    PubMed

    Post, Harm; Penning, Renske; Fitzpatrick, Martin A; Garrigues, Luc B; Wu, W; MacGillavry, Harold D; Hoogenraad, Casper C; Heck, Albert J R; Altelaar, A F Maarten

    2017-02-03

    Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO 2 -based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 μg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 μg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

  9. Qualitative and quantitative processing of side-scan sonar data

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dwan, F.S.; Anderson, A.L.; Hilde, T.W.C.

    1990-06-01

    Modern side-scan sonar systems allow vast areas of seafloor to be rapidly imaged and quantitatively mapped in detail. The application of remote sensing image processing techniques can be used to correct for various distortions inherent in raw sonography. Corrections are possible for water column, slant-range, aspect ratio, speckle and striping noise, multiple returns, power drop-off, and for georeferencing. The final products reveal seafloor features and patterns that are geometrically correct, georeferenced, and have improved signal/noise ratio. These products can be merged with other georeferenced data bases for further database management and information extraction. In order to compare data collected bymore » different systems from a common area and to ground truth measurements and geoacoustic models, quantitative correction must be made for calibrated sonar system and bathymetry effects. Such data inversion must account for system source level, beam pattern, time-varying gain, processing gain, transmission loss, absorption, insonified area, and grazing angle effects. Seafloor classification can then be performed on the calculated back-scattering strength using Lambert's Law and regression analysis. Examples are given using both approaches: image analysis and inversion of data based on the sonar equation.« less

  10. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    PubMed

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

  11. Brain electromagnetic activity and lightning: potentially congruent scale-invariant quantitative properties

    PubMed Central

    Persinger, Michael A.

    2012-01-01

    The space-time characteristics of the axonal action potential are remarkably similar to the scaled equivalents of lightning. The energy and current densities from these transients within their respective volumes or cross-sectional areas are the same order of magnitude. Length–velocity ratios and temporal durations are nearly identical. There are similar chemical consequences such as the production of nitric oxide. Careful, quantitative examination of the characteristics of lightning may reveal analogous features of the action potential that could lead to a more accurate understanding of these powerful correlates of neurocognitive processes. PMID:22615688

  12. Using metabarcoding to reveal and quantify plant-pollinator interactions

    PubMed Central

    Pornon, André; Escaravage, Nathalie; Burrus, Monique; Holota, Hélène; Khimoun, Aurélie; Mariette, Jérome; Pellizzari, Charlène; Iribar, Amaia; Etienne, Roselyne; Taberlet, Pierre; Vidal, Marie; Winterton, Peter; Zinger, Lucie; Andalo, Christophe

    2016-01-01

    Given the ongoing decline of both pollinators and plants, it is crucial to implement effective methods to describe complex pollination networks across time and space in a comprehensive and high-throughput way. Here we tested if metabarcoding may circumvent the limits of conventional methodologies in detecting and quantifying plant-pollinator interactions. Metabarcoding experiments on pollen DNA mixtures described a positive relationship between the amounts of DNA from focal species and the number of trnL and ITS1 sequences yielded. The study of pollen loads of insects captured in plant communities revealed that as compared to the observation of visits, metabarcoding revealed 2.5 times more plant species involved in plant-pollinator interactions. We further observed a tight positive relationship between the pollen-carrying capacities of insect taxa and the number of trnL and ITS1 sequences. The number of visits received per plant species also positively correlated to the number of their ITS1 and trnL sequences in insect pollen loads. By revealing interactions hard to observe otherwise, metabarcoding significantly enlarges the spatiotemporal observation window of pollination interactions. By providing new qualitative and quantitative information, metabarcoding holds great promise for investigating diverse facets of interactions and will provide a new perception of pollination networks as a whole. PMID:27255732

  13. Localization and quantitative co-localization of enamelin with amelogenin.

    PubMed

    Gallon, Victoria; Chen, Lisha; Yang, Xiudong; Moradian-Oldak, Janet

    2013-08-01

    Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1-8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

    PubMed

    Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J

    2013-03-01

    Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

  15. Clients' reasons for terminating psychotherapy: a quantitative and qualitative inquiry.

    PubMed

    Roe, David; Dekel, Rachel; Harel, Galit; Fennig, Shmuel

    2006-12-01

    To study private-practice clients' perspective on reasons for psychotherapy termination and how these are related to demographic and treatment variables and to satisfaction with therapy. Eighty-four persons who had been in extended private-practice psychotherapy which ended in the preceding three years participated in the study. Mean number of months in treatment was 27.70 (SD = 18.70). Assessment included rating scales and open-ended questions assessing demographic variables, reasons for terminating therapy, and satisfaction with therapy. Quantitative results revealed that the most frequent reasons for termination were accomplishment of goals, circumstantial constraints and dissatisfaction with therapy, and that client satisfaction was positively related to positive reasons for termination. Qualitative results revealed two additional frequently mentioned reasons for termination: the client's need for independence and the client's involvement in new meaningful relationships. Findings suggest that psychotherapy termination may at times be required to facilitate the pursuit of personally meaningful goals.

  16. Quantitative Finance

    NASA Astrophysics Data System (ADS)

    James, Jessica

    2017-01-01

    Quantitative finance is a field that has risen to prominence over the last few decades. It encompasses the complex models and calculations that value financial contracts, particularly those which reference events in the future, and apply probabilities to these events. While adding greatly to the flexibility of the market available to corporations and investors, it has also been blamed for worsening the impact of financial crises. But what exactly does quantitative finance encompass, and where did these ideas and models originate? We show that the mathematics behind finance and behind games of chance have tracked each other closely over the centuries and that many well-known physicists and mathematicians have contributed to the field.

  17. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

    PubMed

    Ziv, Omer; Zaritsky, Assaf; Yaffe, Yakey; Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-10-01

    Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  18. Nonparametric modeling of longitudinal covariance structure in functional mapping of quantitative trait loci.

    PubMed

    Yap, John Stephen; Fan, Jianqing; Wu, Rongling

    2009-12-01

    Estimation of the covariance structure of longitudinal processes is a fundamental prerequisite for the practical deployment of functional mapping designed to study the genetic regulation and network of quantitative variation in dynamic complex traits. We present a nonparametric approach for estimating the covariance structure of a quantitative trait measured repeatedly at a series of time points. Specifically, we adopt Huang et al.'s (2006, Biometrika 93, 85-98) approach of invoking the modified Cholesky decomposition and converting the problem into modeling a sequence of regressions of responses. A regularized covariance estimator is obtained using a normal penalized likelihood with an L(2) penalty. This approach, embedded within a mixture likelihood framework, leads to enhanced accuracy, precision, and flexibility of functional mapping while preserving its biological relevance. Simulation studies are performed to reveal the statistical properties and advantages of the proposed method. A real example from a mouse genome project is analyzed to illustrate the utilization of the methodology. The new method will provide a useful tool for genome-wide scanning for the existence and distribution of quantitative trait loci underlying a dynamic trait important to agriculture, biology, and health sciences.

  19. An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes

    PubMed Central

    Shafqat-Abbasi, Hamdah; Kowalewski, Jacob M; Kiss, Alexa; Gong, Xiaowei; Hernandez-Varas, Pablo; Berge, Ulrich; Jafari-Mamaghani, Mehrdad; Lock, John G; Strömblad, Staffan

    2016-01-01

    Mesenchymal (lamellipodial) migration is heterogeneous, although whether this reflects progressive variability or discrete, 'switchable' migration modalities, remains unclear. We present an analytical toolbox, based on quantitative single-cell imaging data, to interrogate this heterogeneity. Integrating supervised behavioral classification with multivariate analyses of cell motion, membrane dynamics, cell-matrix adhesion status and F-actin organization, this toolbox here enables the detection and characterization of two quantitatively distinct mesenchymal migration modes, termed 'Continuous' and 'Discontinuous'. Quantitative mode comparisons reveal differences in cell motion, spatiotemporal coordination of membrane protrusion/retraction, and how cells within each mode reorganize with changed cell speed. These modes thus represent distinctive migratory strategies. Additional analyses illuminate the macromolecular- and cellular-scale effects of molecular targeting (fibronectin, talin, ROCK), including 'adaptive switching' between Continuous (favored at high adhesion/full contraction) and Discontinuous (low adhesion/inhibited contraction) modes. Overall, this analytical toolbox now facilitates the exploration of both spontaneous and adaptive heterogeneity in mesenchymal migration. DOI: http://dx.doi.org/10.7554/eLife.11384.001 PMID:26821527

  20. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.

    2014-12-15

    Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each ofmore » which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes« less

  1. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks.

    PubMed

    Que, Emily L; Bleher, Reiner; Duncan, Francesca E; Kong, Betty Y; Gleber, Sophie C; Vogt, Stefan; Chen, Si; Garwin, Seth A; Bayer, Amanda R; Dravid, Vinayak P; Woodruff, Teresa K; O'Halloran, Thomas V

    2015-02-01

    Fertilization of a mammalian egg initiates a series of 'zinc sparks' that are necessary to induce the egg-to-embryo transition. Despite the importance of these zinc-efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches that resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy-dispersive spectroscopy, X-ray fluorescence microscopy and three-dimensional elemental tomography for high-resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 10(6) zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.

  2. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

    DOE PAGES

    Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.; ...

    2014-12-15

    Fertilization of a mammalian egg induces a series of ‘zinc sparks’ that are necessary for inducing the egg-to-embryo transition. Despite the importance of these zinc efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches to resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy dispersive spectroscopy, X-ray fluorescence microscopy, and 3D elemental tomography for high resolution elemental mapping. Here we show that the zinc spark arises from a system of thousands ofmore » zinc-loaded vesicles, each of which contains, on average, 106 zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. We conclude that the discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes.« less

  3. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks

    PubMed Central

    Que, Emily L.; Bleher, Reiner; Duncan, Francesca E.; Kong, Betty Y.; Gleber, Sophie C.; Vogt, Stefan; Chen, Si; Garwin, Seth A.; Bayer, Amanda R.; Dravid, Vinayak; Woodruff, Teresa K.; O’Halloran, Thomas V.

    2015-01-01

    Fertilization of a mammalian egg induces a series of ‘zinc sparks’ that are necessary for inducing the egg-to-embryo transition. Despite the importance of these zinc efflux events little is known about their origin. To understand the molecular mechanism of the zinc spark we combined four physical approaches to resolve zinc distributions in single cells: a chemical probe for dynamic live-cell fluorescence imaging and a combination of scanning transmission electron microscopy with energy dispersive spectroscopy, X-ray fluorescence microscopy, and 3D elemental tomography for high resolution elemental mapping. We show that the zinc spark arises from a system of thousands of zinc-loaded vesicles, each of which contains, on average, 106 zinc atoms. These vesicles undergo dynamic movement during oocyte maturation and exocytosis at the time of fertilization. The discovery of these vesicles and the demonstration that zinc sparks originate from them provides a quantitative framework for understanding how zinc fluxes regulate cellular processes. PMID:25615666

  4. QDMR: a quantitative method for identification of differentially methylated regions by entropy

    PubMed Central

    Zhang, Yan; Liu, Hongbo; Lv, Jie; Xiao, Xue; Zhu, Jiang; Liu, Xiaojuan; Su, Jianzhong; Li, Xia; Wu, Qiong; Wang, Fang; Cui, Ying

    2011-01-01

    DNA methylation plays critical roles in transcriptional regulation and chromatin remodeling. Differentially methylated regions (DMRs) have important implications for development, aging and diseases. Therefore, genome-wide mapping of DMRs across various temporal and spatial methylomes is important in revealing the impact of epigenetic modifications on heritable phenotypic variation. We present a quantitative approach, quantitative differentially methylated regions (QDMRs), to quantify methylation difference and identify DMRs from genome-wide methylation profiles by adapting Shannon entropy. QDMR was applied to synthetic methylation patterns and methylation profiles detected by methylated DNA immunoprecipitation microarray (MeDIP-chip) in human tissues/cells. This approach can give a reasonable quantitative measure of methylation difference across multiple samples. Then DMR threshold was determined from methylation probability model. Using this threshold, QDMR identified 10 651 tissue DMRs which are related to the genes enriched for cell differentiation, including 4740 DMRs not identified by the method developed by Rakyan et al. QDMR can also measure the sample specificity of each DMR. Finally, the application to methylation profiles detected by reduced representation bisulphite sequencing (RRBS) in mouse showed the platform-free and species-free nature of QDMR. This approach provides an effective tool for the high-throughput identification of potential functional regions involved in epigenetic regulation. PMID:21306990

  5. Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

    PubMed Central

    Lucas, James E; Siegel, Justin B

    2015-01-01

    Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

  6. Quantitative analysis of virgin coconut oil in cream cosmetics preparations using fourier transform infrared (FTIR) spectroscopy.

    PubMed

    Rohman, A; Man, Yb Che; Sismindari

    2009-10-01

    Today, virgin coconut oil (VCO) is becoming valuable oil and is receiving an attractive topic for researchers because of its several biological activities. In cosmetics industry, VCO is excellent material which functions as a skin moisturizer and softener. Therefore, it is important to develop a quantitative analytical method offering a fast and reliable technique. Fourier transform infrared (FTIR) spectroscopy with sample handling technique of attenuated total reflectance (ATR) can be successfully used to analyze VCO quantitatively in cream cosmetic preparations. A multivariate analysis using calibration of partial least square (PLS) model revealed the good relationship between actual value and FTIR-predicted value of VCO with coefficient of determination (R2) of 0.998.

  7. Teaching quantitative biology: goals, assessments, and resources

    PubMed Central

    Aikens, Melissa L.; Dolan, Erin L.

    2014-01-01

    More than a decade has passed since the publication of BIO2010, calling for an increased emphasis on quantitative skills in the undergraduate biology curriculum. In that time, relatively few papers have been published that describe educational innovations in quantitative biology or provide evidence of their effects on students. Using a “backward design” framework, we lay out quantitative skill and attitude goals, assessment strategies, and teaching resources to help biologists teach more quantitatively. Collaborations between quantitative biologists and education researchers are necessary to develop a broader and more appropriate suite of assessment tools, and to provide much-needed evidence on how particular teaching strategies affect biology students' quantitative skill development and attitudes toward quantitative work. PMID:25368425

  8. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    PubMed

    Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2013-01-01

    ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  9. Quantitative Proteomic Analysis of Human Lung Tumor Xenografts Treated with the Ectopic ATP Synthase Inhibitor Citreoviridin

    PubMed Central

    Wu, Yi-Hsuan; Hu, Chia-Wei; Chien, Chih-Wei; Chen, Yu-Ju; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2013-01-01

    ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy. PMID:23990911

  10. Developing Geoscience Students' Quantitative Skills

    NASA Astrophysics Data System (ADS)

    Manduca, C. A.; Hancock, G. S.

    2005-12-01

    Sophisticated quantitative skills are an essential tool for the professional geoscientist. While students learn many of these sophisticated skills in graduate school, it is increasingly important that they have a strong grounding in quantitative geoscience as undergraduates. Faculty have developed many strong approaches to teaching these skills in a wide variety of geoscience courses. A workshop in June 2005 brought together eight faculty teaching surface processes and climate change to discuss and refine activities they use and to publish them on the Teaching Quantitative Skills in the Geosciences website (serc.Carleton.edu/quantskills) for broader use. Workshop participants in consultation with two mathematics faculty who have expertise in math education developed six review criteria to guide discussion: 1) Are the quantitative and geologic goals central and important? (e.g. problem solving, mastery of important skill, modeling, relating theory to observation); 2) Does the activity lead to better problem solving? 3) Are the quantitative skills integrated with geoscience concepts in a way that makes sense for the learning environment and supports learning both quantitative skills and geoscience? 4) Does the methodology support learning? (e.g. motivate and engage students; use multiple representations, incorporate reflection, discussion and synthesis) 5) Are the materials complete and helpful to students? 6) How well has the activity worked when used? Workshop participants found that reviewing each others activities was very productive because they thought about new ways to teach and the experience of reviewing helped them think about their own activity from a different point of view. The review criteria focused their thinking about the activity and would be equally helpful in the design of a new activity. We invite a broad international discussion of the criteria(serc.Carleton.edu/quantskills/workshop05/review.html).The Teaching activities can be found on the

  11. TECHNOLOGICAL INNOVATION IN NEUROSURGERY: A QUANTITATIVE STUDY

    PubMed Central

    Marcus, Hani J; Hughes-Hallett, Archie; Kwasnicki, Richard M; Darzi, Ara; Yang, Guang-Zhong; Nandi, Dipankar

    2015-01-01

    Object Technological innovation within healthcare may be defined as the introduction of a new technology that initiates a change in clinical practice. Neurosurgery is a particularly technologically intensive surgical discipline, and new technologies have preceded many of the major advances in operative neurosurgical technique. The aim of the present study was to quantitatively evaluate technological innovation in neurosurgery using patents and peer-reviewed publications as metrics of technology development and clinical translation respectively. Methods A patent database was searched between 1960 and 2010 using the search terms “neurosurgeon” OR “neurosurgical” OR “neurosurgery”. The top 50 performing patent codes were then grouped into technology clusters. Patent and publication growth curves were then generated for these technology clusters. A top performing technology cluster was then selected as an exemplar for more detailed analysis of individual patents. Results In all, 11,672 patents and 208,203 publications relating to neurosurgery were identified. The top performing technology clusters over the 50 years were: image guidance devices, clinical neurophysiology devices, neuromodulation devices, operating microscopes and endoscopes. Image guidance and neuromodulation devices demonstrated a highly correlated rapid rise in patents and publications, suggesting they are areas of technology expansion. In-depth analysis of neuromodulation patents revealed that the majority of high performing patents were related to Deep Brain Stimulation (DBS). Conclusions Patent and publication data may be used to quantitatively evaluate technological innovation in neurosurgery. PMID:25699414

  12. Technological innovation in neurosurgery: a quantitative study.

    PubMed

    Marcus, Hani J; Hughes-Hallett, Archie; Kwasnicki, Richard M; Darzi, Ara; Yang, Guang-Zhong; Nandi, Dipankar

    2015-07-01

    Technological innovation within health care may be defined as the introduction of a new technology that initiates a change in clinical practice. Neurosurgery is a particularly technology-intensive surgical discipline, and new technologies have preceded many of the major advances in operative neurosurgical techniques. The aim of the present study was to quantitatively evaluate technological innovation in neurosurgery using patents and peer-reviewed publications as metrics of technology development and clinical translation, respectively. The authors searched a patent database for articles published between 1960 and 2010 using the Boolean search term "neurosurgeon OR neurosurgical OR neurosurgery." The top 50 performing patent codes were then grouped into technology clusters. Patent and publication growth curves were then generated for these technology clusters. A top-performing technology cluster was then selected as an exemplar for a more detailed analysis of individual patents. In all, 11,672 patents and 208,203 publications related to neurosurgery were identified. The top-performing technology clusters during these 50 years were image-guidance devices, clinical neurophysiology devices, neuromodulation devices, operating microscopes, and endoscopes. In relation to image-guidance and neuromodulation devices, the authors found a highly correlated rapid rise in the numbers of patents and publications, which suggests that these are areas of technology expansion. An in-depth analysis of neuromodulation-device patents revealed that the majority of well-performing patents were related to deep brain stimulation. Patent and publication data may be used to quantitatively evaluate technological innovation in neurosurgery.

  13. Label-free imaging of intracellular motility by low-coherent quantitative phase microscope in reflection geometry

    NASA Astrophysics Data System (ADS)

    Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

    2011-11-01

    We demonstrate tomographic imaging of intracellular activity of living cells by a low-coherent quantitative phase microscope. The intracellular organelles, such as the nucleus, nucleolus, and mitochondria, are moving around inside living cells, driven by the cellular physiological activity. In order to visualize the intracellular motility in a label-free manner we have developed a reflection-type quantitative phase microscope which employs the phase shifting interferometric technique with a low-coherent light source. The phase shifting interferometry enables us to quantitatively measure the intensity and phase of the optical field, and the low-coherence interferometry makes it possible to selectively probe a specific sectioning plane in the cell volume. The results quantitatively revealed the depth-resolved fluctuations of intracellular surfaces so that the plasma membrane and the membranes of intracellular organelles were independently measured. The transversal and the vertical spatial resolutions were 0.56 μm and 0.93 μm, respectively, and the mechanical sensitivity of the phase measurement was 1.2 nanometers. The mean-squared displacement was applied as a statistical tool to analyze the temporal fluctuation of the intracellular organelles. To the best of our knowledge, our system visualized depth-resolved intracellular organelles motion for the first time in sub-micrometer resolution without contrast agents.

  14. Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mu, Jun; Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing; Chongqing Key Laboratory of Neurobiology, Chongqing

    Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology andmore » proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of Apo

  15. Quantitation of Fine Displacement in Echography

    NASA Astrophysics Data System (ADS)

    Masuda, Kohji; Ishihara, Ken; Yoshii, Ken; Furukawa, Toshiyuki; Kumagai, Sadatoshi; Maeda, Hajime; Kodama, Shinzo

    1993-05-01

    A High-speed Digital Subtraction Echography was developed to visualize the fine displacement of human internal organs. This method indicates differences in position through time series images of high-frame-rate echography. Fine displacement less than ultrasonic wavelength can be observed. This method, however, lacks the ability to quantitatively measure displacement length. The subtraction between two successive images was affected by displacement direction in spite of the displacement length being the same. To solve this problem, convolution of an echogram with Gaussian distribution was used. To express displacement length as brightness quantitatively, normalization using a brightness gradient was applied. The quantitation algorithm was applied to successive B-mode images. Compared to the simply subtracted images, quantitated images express more precisely the motion of organs. Expansion of the carotid artery and fine motion of ventricular walls can be visualized more easily. Displacement length can be quantitated with wavelength. Under more static conditions, this system quantitates displacement length that is much less than wavelength.

  16. Clusters of Insomnia Disorder: An Exploratory Cluster Analysis of Objective Sleep Parameters Reveals Differences in Neurocognitive Functioning, Quantitative EEG, and Heart Rate Variability.

    PubMed

    Miller, Christopher B; Bartlett, Delwyn J; Mullins, Anna E; Dodds, Kirsty L; Gordon, Christopher J; Kyle, Simon D; Kim, Jong Won; D'Rozario, Angela L; Lee, Rico S C; Comas, Maria; Marshall, Nathaniel S; Yee, Brendon J; Espie, Colin A; Grunstein, Ronald R

    2016-11-01

    To empirically derive and evaluate potential clusters of Insomnia Disorder through cluster analysis from polysomnography (PSG). We hypothesized that clusters would differ on neurocognitive performance, sleep-onset measures of quantitative ( q )-EEG and heart rate variability (HRV). Research volunteers with Insomnia Disorder (DSM-5) completed a neurocognitive assessment and overnight PSG measures of total sleep time (TST), wake time after sleep onset (WASO), and sleep onset latency (SOL) were used to determine clusters. From 96 volunteers with Insomnia Disorder, cluster analysis derived at least two clusters from objective sleep parameters: Insomnia with normal objective sleep duration (I-NSD: n = 53) and Insomnia with short sleep duration (I-SSD: n = 43). At sleep onset, differences in HRV between I-NSD and I-SSD clusters suggest attenuated parasympathetic activity in I-SSD (P < 0.05). Preliminary work suggested three clusters by retaining the I-NSD and splitting the I-SSD cluster into two: I-SSD A (n = 29): defined by high WASO and I-SSD B (n = 14): a second I-SSD cluster with high SOL and medium WASO. The I-SSD B cluster performed worse than I-SSD A and I-NSD for sustained attention (P ≤ 0.05). In an exploratory analysis, q -EEG revealed reduced spectral power also in I-SSD B before (Delta, Alpha, Beta-1) and after sleep-onset (Beta-2) compared to I-SSD A and I-NSD (P ≤ 0.05). Two insomnia clusters derived from cluster analysis differ in sleep onset HRV. Preliminary data suggest evidence for three clusters in insomnia with differences for sustained attention and sleep-onset q -EEG. Insomnia 100 sleep study: Australia New Zealand Clinical Trials Registry (ANZCTR) identification number 12612000049875. URL: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=347742. © 2016 Associated Professional Sleep Societies, LLC.

  17. Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

    PubMed

    Okahashi, Yumiko; Iwamoto, Takaaki; Suzuki, Naomi; Shibutani, Shinya; Sugiura, Shigeki; Itoh, Shinji; Nishiwaki, Tomohisa; Ueno, Satoshi; Mori, Toshio

    2010-07-01

    Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

  18. Applications of Microfluidics in Quantitative Biology.

    PubMed

    Bai, Yang; Gao, Meng; Wen, Lingling; He, Caiyun; Chen, Yuan; Liu, Chenli; Fu, Xiongfei; Huang, Shuqiang

    2018-05-01

    Quantitative biology is dedicated to taking advantage of quantitative reasoning and advanced engineering technologies to make biology more predictable. Microfluidics, as an emerging technique, provides new approaches to precisely control fluidic conditions on small scales and collect data in high-throughput and quantitative manners. In this review, the authors present the relevant applications of microfluidics to quantitative biology based on two major categories (channel-based microfluidics and droplet-based microfluidics), and their typical features. We also envision some other microfluidic techniques that may not be employed in quantitative biology right now, but have great potential in the near future. © 2017 Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  19. Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies.

    PubMed

    Sun, Wanxin; Chang, Shi; Tai, Dean C S; Tan, Nancy; Xiao, Guangfa; Tang, Huihuan; Yu, Hanry

    2008-01-01

    Liver fibrosis is associated with an abnormal increase in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semiquantitative or computerized image analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second-harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

  20. Quantitative refractive index distribution of single cell by combining phase-shifting interferometry and AFM imaging.

    PubMed

    Zhang, Qinnan; Zhong, Liyun; Tang, Ping; Yuan, Yingjie; Liu, Shengde; Tian, Jindong; Lu, Xiaoxu

    2017-05-31

    Cell refractive index, an intrinsic optical parameter, is closely correlated with the intracellular mass and concentration. By combining optical phase-shifting interferometry (PSI) and atomic force microscope (AFM) imaging, we constructed a label free, non-invasive and quantitative refractive index of single cell measurement system, in which the accurate phase map of single cell was retrieved with PSI technique and the cell morphology with nanoscale resolution was achieved with AFM imaging. Based on the proposed AFM/PSI system, we achieved quantitative refractive index distributions of single red blood cell and Jurkat cell, respectively. Further, the quantitative change of refractive index distribution during Daunorubicin (DNR)-induced Jurkat cell apoptosis was presented, and then the content changes of intracellular biochemical components were achieved. Importantly, these results were consistent with Raman spectral analysis, indicating that the proposed PSI/AFM based refractive index system is likely to become a useful tool for intracellular biochemical components analysis measurement, and this will facilitate its application for revealing cell structure and pathological state from a new perspective.

  1. Systems-level identification of PKA-dependent signaling in epithelial cells.

    PubMed

    Isobe, Kiyoshi; Jung, Hyun Jun; Yang, Chin-Rang; Claxton, J'Neka; Sandoval, Pablo; Burg, Maurice B; Raghuram, Viswanathan; Knepper, Mark A

    2017-10-17

    G protein stimulatory α-subunit (G αs )-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the G αs -coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

  2. Factors influencing students' perceptions of their quantitative skills

    NASA Astrophysics Data System (ADS)

    Matthews, Kelly E.; Hodgson, Yvonne; Varsavsky, Cristina

    2013-09-01

    There is international agreement that quantitative skills (QS) are an essential graduate competence in science. QS refer to the application of mathematical and statistical thinking and reasoning in science. This study reports on the use of the Science Students Skills Inventory to capture final year science students' perceptions of their QS across multiple indicators, at two Australian research-intensive universities. Statistical analysis reveals several variables predicting higher levels of self-rated competence in QS: students' grade point average, students' perceptions of inclusion of QS in the science degree programme, their confidence in QS, and their belief that QS will be useful in the future. The findings are discussed in terms of implications for designing science curricula more effectively to build students' QS throughout science degree programmes. Suggestions for further research are offered.

  3. Isotopic Zonation Within Sulfate Evaporite Mineral Crystals Reveal Quantitative Paleoenvironment Details

    NASA Astrophysics Data System (ADS)

    Coleman, M.; Rhorssen, M.; Mielke, R. E.

    2008-12-01

    developed a new analytical method [2]. We use a modification of the standard TC/EA continuous-flow protocol to measure both hydrogen and oxygen of water of hydration from the same small sample. We have proved the concept of this new approach by analyzing zones within crystals and individual grains, growing epsomite (magnesium sulfate heptahydrate) in the laboratory and by analysis of natural gypsum evaporites. We are now exploring the effects of varying the controlling parameters. Eventual application to Martian sulfates will reveal amount of water involved in sulfate formation, its isotopic composition(s) and details of the paleo-atmospheric humidity. [1] Gat JR and Gonfiantini R, (Eds) (1981) IAEA Technical Report Series. [2] Rohrssen MK, Brunner B Mielke RE and Coleman M (2008) Analyt. Chem. (in press).

  4. Differential signaling through p190 and p210 BCR-ABL fusion proteins revealed by interactome and phosphoproteome analysis.

    PubMed

    Cutler, J A; Tahir, R; Sreenivasamurthy, S K; Mitchell, C; Renuse, S; Nirujogi, R S; Patil, A H; Heydarian, M; Wong, X; Wu, X; Huang, T-C; Kim, M-S; Reddy, K L; Pandey, A

    2017-07-01

    Two major types of leukemogenic BCR-ABL fusion proteins are p190 BCR-ABL and p210 BCR-ABL . Although the two fusion proteins are closely related, they can lead to different clinical outcomes. A thorough understanding of the signaling programs employed by these two fusion proteins is necessary to explain these clinical differences. We took an integrated approach by coupling protein-protein interaction analysis using biotinylation identification with global phosphorylation analysis to investigate the differences in signaling between these two fusion proteins. Our findings suggest that p190 BCR-ABL and p210 BCR-ABL differentially activate important signaling pathways, such as JAK-STAT, and engage with molecules that indicate interaction with different subcellular compartments. In the case of p210 BCR-ABL , we observed an increased engagement of molecules active proximal to the membrane and in the case of p190 BCR-ABL , an engagement of molecules of the cytoskeleton. These differences in signaling could underlie the distinct leukemogenic process induced by these two protein variants.

  5. Quantitative mass spectrometry: an overview

    NASA Astrophysics Data System (ADS)

    Urban, Pawel L.

    2016-10-01

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

  6. Quantitative imaging methods in osteoporosis.

    PubMed

    Oei, Ling; Koromani, Fjorda; Rivadeneira, Fernando; Zillikens, M Carola; Oei, Edwin H G

    2016-12-01

    Osteoporosis is characterized by a decreased bone mass and quality resulting in an increased fracture risk. Quantitative imaging methods are critical in the diagnosis and follow-up of treatment effects in osteoporosis. Prior radiographic vertebral fractures and bone mineral density (BMD) as a quantitative parameter derived from dual-energy X-ray absorptiometry (DXA) are among the strongest known predictors of future osteoporotic fractures. Therefore, current clinical decision making relies heavily on accurate assessment of these imaging features. Further, novel quantitative techniques are being developed to appraise additional characteristics of osteoporosis including three-dimensional bone architecture with quantitative computed tomography (QCT). Dedicated high-resolution (HR) CT equipment is available to enhance image quality. At the other end of the spectrum, by utilizing post-processing techniques such as the trabecular bone score (TBS) information on three-dimensional architecture can be derived from DXA images. Further developments in magnetic resonance imaging (MRI) seem promising to not only capture bone micro-architecture but also characterize processes at the molecular level. This review provides an overview of various quantitative imaging techniques based on different radiological modalities utilized in clinical osteoporosis care and research.

  7. Development of a Postcolumn Infused-Internal Standard Liquid Chromatography Mass Spectrometry Method for Quantitative Metabolomics Studies.

    PubMed

    Liao, Hsiao-Wei; Chen, Guan-Yuan; Wu, Ming-Shiang; Liao, Wei-Chih; Lin, Ching-Hung; Kuo, Ching-Hua

    2017-02-03

    Quantitative metabolomics has become much more important in clinical research in recent years. Individual differences in matrix effects (MEs) and the injection order effect are two major factors that reduce the quantification accuracy in liquid chromatography-electrospray ionization-mass spectrometry-based (LC-ESI-MS) metabolomics studies. This study proposed a postcolumn infused-internal standard (PCI-IS) combined with a matrix normalization factor (MNF) strategy to improve the analytical accuracy of quantitative metabolomics. The PCI-IS combined with the MNF method was applied for a targeted metabolomics study of amino acids (AAs). D8-Phenylalanine was used as the PCI-IS, and it was postcolumn-infused into the ESI interface for calibration purposes. The MNF was used to bridge the AA response in a standard solution with the plasma samples. The MEs caused signal changes that were corrected by dividing the AA signal intensities by the PCI-IS intensities after adjustment with the MNF. After the method validation, we evaluated the method applicability for breast cancer research using 100 plasma samples. The quantification results revealed that the 11 tested AAs exhibit an accuracy between 88.2 and 110.7%. The principal component analysis score plot revealed that the injection order effect can be successfully removed, and most of the within-group variation of the tested AAs decreased after the PCI-IS correction. Finally, targeted metabolomics studies on the AAs showed that tryptophan was expressed more in malignant patients than in the benign group. We anticipate that a similar approach can be applied to other endogenous metabolites to facilitate quantitative metabolomics studies.

  8. Understanding quantitative research: part 1.

    PubMed

    Hoe, Juanita; Hoare, Zoë

    This article, which is the first in a two-part series, provides an introduction to understanding quantitative research, basic statistics and terminology used in research articles. Critical appraisal of research articles is essential to ensure that nurses remain up to date with evidence-based practice to provide consistent and high-quality nursing care. This article focuses on developing critical appraisal skills and understanding the use and implications of different quantitative approaches to research. Part two of this article will focus on explaining common statistical terms and the presentation of statistical data in quantitative research.

  9. Hypothalamus proteomics from mouse models with obesity and anorexia reveals therapeutic targets of appetite regulation

    PubMed Central

    Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D

    2016-01-01

    Objective: This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Methods: Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Results: Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (q<0.05). Of these, 7718 protein groups were profiled with a minimum of two unique peptides for each. Hierachical clustering of the differentiated proteome revealed distinct proteomic signatures for the hypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. Conclusions: High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the

  10. Hypothalamus proteomics from mouse models with obesity and anorexia reveals therapeutic targets of appetite regulation.

    PubMed

    Manousopoulou, A; Koutmani, Y; Karaliota, S; Woelk, C H; Manolakos, E S; Karalis, K; Garbis, S D

    2016-04-25

    This study examined the proteomic profile of the hypothalamus in mice exposed to a high-fat diet (HFD) or with the anorexia of acute illness. This comparison could provide insight on the effects of these two opposite states of energy balance on appetite regulation. Four to six-week-old male C56BL/6J mice were fed a normal (control 1 group; n=7) or a HFD (HFD group; n=10) for 8 weeks. The control 2 (n=7) and lipopolysaccharide (LPS) groups (n=10) were fed a normal diet for 8 weeks before receiving an injection of saline and LPS, respectively. Hypothalamic regions were analysed using a quantitative proteomics method based on a combination of techniques including iTRAQ stable isotope labeling, orthogonal two-dimensional liquid chromatography hyphenated with nanospray ionization and high-resolution mass spectrometry. Key proteins were validated with quantitative PCR. Quantitative proteomics of the hypothalamous regions profiled a total of 9249 protein groups (q<0.05). Of these, 7718 protein groups were profiled with a minimum of two unique peptides for each. Hierachical clustering of the differentiated proteome revealed distinct proteomic signatures for the hypothalamus under the HFD and LPS nutritional conditions. Literature research with in silico bioinformatics interpretation of the differentiated proteome identified key biological relevant proteins and implicated pathways. Furthermore, the study identified potential pharmacologic targets. In the LPS groups, the anorexigen pro-opiomelanocortin was downregulated. In mice with obesity, nuclear factor-κB, glycine receptor subunit alpha-4 (GlyR) and neuropeptide Y levels were elevated, whereas serotonin receptor 1B levels decreased. High-precision quantitative proteomics revealed that under acute systemic inflammation in the hypothalamus as a response to LPS, homeostatic mechanisms mediating loss of appetite take effect. Conversely, under chronic inflammation in the hypothalamus as a response to HFD, mechanisms

  11. New features of global climatology revealed by satellite-derived oceanic rainfall maps

    NASA Technical Reports Server (NTRS)

    Rao, M. S. V.; Theon, J. S.

    1977-01-01

    Quantitative rainfall maps over the oceanic areas of the globe were derived from the Nimbus 5 Electrically Scanning Microwave Radiometer (ESMR) data. Analysis of satellite derived oceanic rainfall maps reveal certain distinctive characteristics of global patterns for the years 1973-74. The main ones are (1) the forking of the Intertropical Convergence Zone in the Pacific, (2) a previously unrecognized rain area in the South Atlantic, (3) the bimodal behavior of rainbelts in the Indian Ocean and (4) the large interannual variability in oceanic rainfall. These features are discussed.

  12. How to quantitatively evaluate safety of driver behavior upon accident? A biomechanical methodology

    PubMed Central

    Zhang, Wen; Cao, Jieer

    2017-01-01

    How to evaluate driver spontaneous reactions in various collision patterns in a quantitative way is one of the most important topics in vehicle safety. Firstly, this paper constructs representative numerical crash scenarios described by impact velocity, impact angle and contact position based on finite element (FE) computation platform. Secondly, a driver cabin model is extracted and described in the well validated multi-rigid body (MB) model to compute the value of weighted injury criterion to quantitatively assess drivers’ overall injury under certain circumstances. Furthermore, based on the coupling of FE and MB, parametric studies on various crash scenarios are conducted. It is revealed that the WIC (Weighted Injury Criteria) value variation law under high impact velocities is quite distinct comparing with the one in low impact velocities. In addition, the coupling effect can be elucidated by the fact that the difference of WIC value among three impact velocities under smaller impact angles tends to be distinctly higher than that under larger impact angles. Meanwhile, high impact velocity also increases the sensitivity of WIC under different collision positions and impact angles. Results may provide a new methodology to quantitatively evaluate driving behaviors and serve as a significant guiding step towards collision avoidance for autonomous driving vehicles. PMID:29240789

  13. How to quantitatively evaluate safety of driver behavior upon accident? A biomechanical methodology.

    PubMed

    Zhang, Wen; Cao, Jieer; Xu, Jun

    2017-01-01

    How to evaluate driver spontaneous reactions in various collision patterns in a quantitative way is one of the most important topics in vehicle safety. Firstly, this paper constructs representative numerical crash scenarios described by impact velocity, impact angle and contact position based on finite element (FE) computation platform. Secondly, a driver cabin model is extracted and described in the well validated multi-rigid body (MB) model to compute the value of weighted injury criterion to quantitatively assess drivers' overall injury under certain circumstances. Furthermore, based on the coupling of FE and MB, parametric studies on various crash scenarios are conducted. It is revealed that the WIC (Weighted Injury Criteria) value variation law under high impact velocities is quite distinct comparing with the one in low impact velocities. In addition, the coupling effect can be elucidated by the fact that the difference of WIC value among three impact velocities under smaller impact angles tends to be distinctly higher than that under larger impact angles. Meanwhile, high impact velocity also increases the sensitivity of WIC under different collision positions and impact angles. Results may provide a new methodology to quantitatively evaluate driving behaviors and serve as a significant guiding step towards collision avoidance for autonomous driving vehicles.

  14. T1, diffusion tensor, and quantitative magnetization transfer imaging of the hippocampus in an Alzheimer's disease mouse model.

    PubMed

    Whittaker, Heather T; Zhu, Shenghua; Di Curzio, Domenico L; Buist, Richard; Li, Xin-Min; Noy, Suzanna; Wiseman, Frances K; Thiessen, Jonathan D; Martin, Melanie

    2018-07-01

    Alzheimer's disease (AD) pathology causes microstructural changes in the brain. These changes, if quantified with magnetic resonance imaging (MRI), could be studied for use as an early biomarker for AD. The aim of our study was to determine if T 1 relaxation, diffusion tensor imaging (DTI), and quantitative magnetization transfer imaging (qMTI) metrics could reveal changes within the hippocampus and surrounding white matter structures in ex vivo transgenic mouse brains overexpressing human amyloid precursor protein with the Swedish mutation. Delineation of hippocampal cell layers using DTI color maps allows more detailed analysis of T 1 -weighted imaging, DTI, and qMTI metrics, compared with segmentation of gross anatomy based on relaxation images, and with analysis of DTI or qMTI metrics alone. These alterations are observed in the absence of robust intracellular Aβ accumulation or plaque deposition as revealed by histology. This work demonstrates that multiparametric quantitative MRI methods are useful for characterizing changes within the hippocampal substructures and surrounding white matter tracts of mouse models of AD. Copyright © 2018. Published by Elsevier Inc.

  15. Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

    PubMed

    Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

    2017-06-15

    Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate

  16. Diagnostic performance of semi-quantitative and quantitative stress CMR perfusion analysis: a meta-analysis.

    PubMed

    van Dijk, R; van Assen, M; Vliegenthart, R; de Bock, G H; van der Harst, P; Oudkerk, M

    2017-11-27

    Stress cardiovascular magnetic resonance (CMR) perfusion imaging is a promising modality for the evaluation of coronary artery disease (CAD) due to high spatial resolution and absence of radiation. Semi-quantitative and quantitative analysis of CMR perfusion are based on signal-intensity curves produced during the first-pass of gadolinium contrast. Multiple semi-quantitative and quantitative parameters have been introduced. Diagnostic performance of these parameters varies extensively among studies and standardized protocols are lacking. This study aims to determine the diagnostic accuracy of semi- quantitative and quantitative CMR perfusion parameters, compared to multiple reference standards. Pubmed, WebOfScience, and Embase were systematically searched using predefined criteria (3272 articles). A check for duplicates was performed (1967 articles). Eligibility and relevance of the articles was determined by two reviewers using pre-defined criteria. The primary data extraction was performed independently by two researchers with the use of a predefined template. Differences in extracted data were resolved by discussion between the two researchers. The quality of the included studies was assessed using the 'Quality Assessment of Diagnostic Accuracy Studies Tool' (QUADAS-2). True positives, false positives, true negatives, and false negatives were subtracted/calculated from the articles. The principal summary measures used to assess diagnostic accuracy were sensitivity, specificity, andarea under the receiver operating curve (AUC). Data was pooled according to analysis territory, reference standard and perfusion parameter. Twenty-two articles were eligible based on the predefined study eligibility criteria. The pooled diagnostic accuracy for segment-, territory- and patient-based analyses showed good diagnostic performance with sensitivity of 0.88, 0.82, and 0.83, specificity of 0.72, 0.83, and 0.76 and AUC of 0.90, 0.84, and 0.87, respectively. In per territory

  17. Macroscopic X-ray Powder Diffraction Scanning: Possibilities for Quantitative and Depth-Selective Parchment Analysis.

    PubMed

    Vanmeert, Frederik; De Nolf, Wout; Dik, Joris; Janssens, Koen

    2018-06-05

    At or below the surface of painted works of art, valuable information is present that provides insights into an object's past, such as the artist's technique and the creative process that was followed or its conservation history but also on its current state of preservation. Various noninvasive techniques have been developed over the past 2 decades that can probe this information either locally (via point analysis) or on a macroscopic scale (e.g., full-field imaging and raster scanning). Recently macroscopic X-ray powder diffraction (MA-XRPD) mapping using laboratory X-ray sources was developed. This method can visualize highly specific chemical distributions at the macroscale (dm 2 ). In this work we demonstrate the synergy between the quantitative aspects of powder diffraction and the noninvasive scanning capability of MA-XRPD highlighting the potential of the method to reveal new types of information. Quantitative data derived from a 15th/16th century illuminated sheet of parchment revealed three lead white pigments with different hydrocerussite-cerussite compositions in specific pictorial elements, while quantification analysis of impurities in the blue azurite pigment revealed two distinct azurite types: one rich in barite and one in quartz. Furthermore, on the same artifact, the depth-selective possibilities of the method that stem from an exploitation of the shift of the measured diffraction peaks with respect to reference data are highlighted. The influence of different experimental parameters on the depth-selective analysis results is briefly discussed. Promising stratigraphic information could be obtained, even though the analysis is hampered by not completely understood variations in the unit cell dimensions of the crystalline pigment phases.

  18. Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony.

    PubMed

    Ganter, Markus; Goldberg, Jonathan M; Dvorin, Jeffrey D; Paulo, Joao A; King, Jonas G; Tripathi, Abhai K; Paul, Aditya S; Yang, Jing; Coppens, Isabelle; Jiang, Rays H Y; Elsworth, Brendan; Baker, David A; Dinglasan, Rhoel R; Gygi, Steven P; Duraisingh, Manoj T

    2017-02-17

    Plasmodium parasites, the causative agents of malaria, have evolved a unique cell division cycle in the clinically relevant asexual blood stage of infection 1 . DNA replication commences approximately halfway through the intracellular development following invasion and parasite growth. The schizont stage is associated with multiple rounds of DNA replication and nuclear division without cytokinesis, resulting in a multinucleated cell. Nuclei divide asynchronously through schizogony, with only the final round of DNA replication and segregation being synchronous and coordinated with daughter cell assembly 2,3 . However, the control mechanisms for this divergent mode of replication are unknown. Here, we show that the Plasmodium-specific kinase PfCRK4 is a key cell-cycle regulator that orchestrates multiple rounds of DNA replication throughout schizogony in Plasmodium falciparum. PfCRK4 depletion led to a complete block in nuclear division and profoundly inhibited DNA replication. Quantitative phosphoproteomic profiling identified a set of PfCRK4-regulated phosphoproteins with greatest functional similarity to CDK2 substrates, particularly proteins involved in the origin of replication firing. PfCRK4 was required for initial and subsequent rounds of DNA replication during schizogony and, in addition, was essential for development in the mosquito vector. Our results identified an essential S-phase promoting factor of the unconventional P. falciparum cell cycle. PfCRK4 is required for both a prolonged period of the intraerythrocytic stage of Plasmodium infection, as well as for transmission, revealing a broad window for PfCRK4-targeted chemotherapeutics.

  19. Quantitative Analysis of Color Differences within High Contrast, Low Power Reversible Electrophoretic Displays

    DOE PAGES

    Giera, Brian; Bukosky, Scott; Lee, Elaine; ...

    2018-01-23

    Here, quantitative color analysis is performed on videos of high contrast, low power reversible electrophoretic deposition (EPD)-based displays operated under different applied voltages. This analysis is coded in an open-source software, relies on a color differentiation metric, ΔE * 00, derived from digital video, and provides an intuitive relationship between the operating conditions of the devices and their performance. Time-dependent ΔE * 00 color analysis reveals color relaxation behavior, recoverability for different voltage sequences, and operating conditions that can lead to optimal performance.

  20. Quantitative Analysis of Color Differences within High Contrast, Low Power Reversible Electrophoretic Displays

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Giera, Brian; Bukosky, Scott; Lee, Elaine

    Here, quantitative color analysis is performed on videos of high contrast, low power reversible electrophoretic deposition (EPD)-based displays operated under different applied voltages. This analysis is coded in an open-source software, relies on a color differentiation metric, ΔE * 00, derived from digital video, and provides an intuitive relationship between the operating conditions of the devices and their performance. Time-dependent ΔE * 00 color analysis reveals color relaxation behavior, recoverability for different voltage sequences, and operating conditions that can lead to optimal performance.