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

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

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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

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.

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

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.

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.

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

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.

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

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

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

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.

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

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

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.

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.

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

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

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

In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118-124, 2012).Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen ((15)N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of (15)N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369-380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins

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.

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

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.

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.

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

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

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.

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

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.

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

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

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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

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].

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

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.

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

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

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

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

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.

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

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

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

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

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.

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.

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

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.

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

An overview of quantitative approaches in Gestalt perception.

PubMed

Jäkel, Frank; Singh, Manish; Wichmann, Felix A; Herzog, Michael H

2016-09-01

Gestalt psychology is often criticized as lacking quantitative measurements and precise mathematical models. While this is true of the early Gestalt school, today there are many quantitative approaches in Gestalt perception and the special issue of Vision Research "Quantitative Approaches in Gestalt Perception" showcases the current state-of-the-art. In this article we give an overview of these current approaches. For example, ideal observer models are one of the standard quantitative tools in vision research and there is a clear trend to try and apply this tool to Gestalt perception and thereby integrate Gestalt perception into mainstream vision research. More generally, Bayesian models, long popular in other areas of vision research, are increasingly being employed to model perceptual grouping as well. Thus, although experimental and theoretical approaches to Gestalt perception remain quite diverse, we are hopeful that these quantitative trends will pave the way for a unified theory. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

A framework for organizing and selecting quantitative approaches for benefit-harm assessment.

PubMed

Puhan, Milo A; Singh, Sonal; Weiss, Carlos O; Varadhan, Ravi; Boyd, Cynthia M

2012-11-19

Several quantitative approaches for benefit-harm assessment of health care interventions exist but it is unclear how the approaches differ. Our aim was to review existing quantitative approaches for benefit-harm assessment and to develop an organizing framework that clarifies differences and aids selection of quantitative approaches for a particular benefit-harm assessment. We performed a review of the literature to identify quantitative approaches for benefit-harm assessment. Our team, consisting of clinicians, epidemiologists, and statisticians, discussed the approaches and identified their key characteristics. We developed a framework that helps investigators select quantitative approaches for benefit-harm assessment that are appropriate for a particular decisionmaking context. Our framework for selecting quantitative approaches requires a concise definition of the treatment comparison and population of interest, identification of key benefit and harm outcomes, and determination of the need for a measure that puts all outcomes on a single scale (which we call a benefit and harm comparison metric). We identified 16 quantitative approaches for benefit-harm assessment. These approaches can be categorized into those that consider single or multiple key benefit and harm outcomes, and those that use a benefit-harm comparison metric or not. Most approaches use aggregate data and can be used in the context of single studies or systematic reviews. Although the majority of approaches provides a benefit and harm comparison metric, only four approaches provide measures of uncertainty around the benefit and harm comparison metric (such as a 95 percent confidence interval). None of the approaches considers the actual joint distribution of benefit and harm outcomes, but one approach considers competing risks when calculating profile-specific event rates. Nine approaches explicitly allow incorporating patient preferences. The choice of quantitative approaches depends on the

A framework for organizing and selecting quantitative approaches for benefit-harm assessment

PubMed Central

2012-01-01

Background Several quantitative approaches for benefit-harm assessment of health care interventions exist but it is unclear how the approaches differ. Our aim was to review existing quantitative approaches for benefit-harm assessment and to develop an organizing framework that clarifies differences and aids selection of quantitative approaches for a particular benefit-harm assessment. Methods We performed a review of the literature to identify quantitative approaches for benefit-harm assessment. Our team, consisting of clinicians, epidemiologists, and statisticians, discussed the approaches and identified their key characteristics. We developed a framework that helps investigators select quantitative approaches for benefit-harm assessment that are appropriate for a particular decisionmaking context. Results Our framework for selecting quantitative approaches requires a concise definition of the treatment comparison and population of interest, identification of key benefit and harm outcomes, and determination of the need for a measure that puts all outcomes on a single scale (which we call a benefit and harm comparison metric). We identified 16 quantitative approaches for benefit-harm assessment. These approaches can be categorized into those that consider single or multiple key benefit and harm outcomes, and those that use a benefit-harm comparison metric or not. Most approaches use aggregate data and can be used in the context of single studies or systematic reviews. Although the majority of approaches provides a benefit and harm comparison metric, only four approaches provide measures of uncertainty around the benefit and harm comparison metric (such as a 95 percent confidence interval). None of the approaches considers the actual joint distribution of benefit and harm outcomes, but one approach considers competing risks when calculating profile-specific event rates. Nine approaches explicitly allow incorporating patient preferences. Conclusion The choice of

Less label, more free: approaches in label-free quantitative mass spectrometry.

PubMed

Neilson, Karlie A; Ali, Naveid A; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Gariné; Lee, Albert; van Sluyter, Steven C; Haynes, Paul A

2011-02-01

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

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

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

PubMed

Calderón-Celis, Francisco; Encinar, Jorge Ruiz; Sanz-Medel, Alfredo

2017-07-31

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and

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.

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.

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.

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.

Sexual Harassment Prevention Initiatives: Quantitative and Qualitative Approaches

DTIC Science & Technology

2010-10-28

design , and the time series with nonequivalent control group design . The experimental research approach will randomly assign participants...Leedy & Ormrod, 2005). According to Fife- Schaw (2006) there are three quasi-experimental designs : the nonequivalent control group design , the time...that have controlled and isolated variables. A specific quantitative approach available to the researcher is the use of surveys. Surveys, in

Quantitative approaches in climate change ecology

PubMed Central

Brown, Christopher J; Schoeman, David S; Sydeman, William J; Brander, Keith; Buckley, Lauren B; Burrows, Michael; Duarte, Carlos M; Moore, Pippa J; Pandolfi, John M; Poloczanska, Elvira; Venables, William; Richardson, Anthony J

2011-01-01

Contemporary impacts of anthropogenic climate change on ecosystems are increasingly being recognized. Documenting the extent of these impacts requires quantitative tools for analyses of ecological observations to distinguish climate impacts in noisy data and to understand interactions between climate variability and other drivers of change. To assist the development of reliable statistical approaches, we review the marine climate change literature and provide suggestions for quantitative approaches in climate change ecology. We compiled 267 peer-reviewed articles that examined relationships between climate change and marine ecological variables. Of the articles with time series data (n = 186), 75% used statistics to test for a dependency of ecological variables on climate variables. We identified several common weaknesses in statistical approaches, including marginalizing other important non-climate drivers of change, ignoring temporal and spatial autocorrelation, averaging across spatial patterns and not reporting key metrics. We provide a list of issues that need to be addressed to make inferences more defensible, including the consideration of (i) data limitations and the comparability of data sets; (ii) alternative mechanisms for change; (iii) appropriate response variables; (iv) a suitable model for the process under study; (v) temporal autocorrelation; (vi) spatial autocorrelation and patterns; and (vii) the reporting of rates of change. While the focus of our review was marine studies, these suggestions are equally applicable to terrestrial studies. Consideration of these suggestions will help advance global knowledge of climate impacts and understanding of the processes driving ecological change.

A Quantitative Approach to the Formal Verification of Real-Time Systems.

DTIC Science & Technology

1996-09-01

Computer Science A Quantitative Approach to the Formal Verification of Real - Time Systems Sergio Vale Aguiar Campos September 1996 CMU-CS-96-199...ptisiic raieaiSI v Diambimos Lboiamtad _^ A Quantitative Approach to the Formal Verification of Real - Time Systems Sergio Vale Aguiar Campos...implied, of NSF, the Semiconduc- tor Research Corporation, ARPA or the U.S. government. Keywords: real - time systems , formal verification, symbolic

Enhancing quantitative approaches for assessing community resilience

USGS Publications Warehouse

Chuang, W. C.; Garmestani, A.S.; Eason, T. N.; Spanbauer, T. L.; Fried-Peterson, H. B.; Roberts, C.P.; Sundstrom, Shana M.; Burnett, J.L.; Angeler, David G.; Chaffin, Brian C.; Gunderson, L.; Twidwell, Dirac; Allen, Craig R.

2018-01-01

Scholars from many different intellectual disciplines have attempted to measure, estimate, or quantify resilience. However, there is growing concern that lack of clarity on the operationalization of the concept will limit its application. In this paper, we discuss the theory, research development and quantitative approaches in ecological and community resilience. Upon noting the lack of methods that quantify the complexities of the linked human and natural aspects of community resilience, we identify several promising approaches within the ecological resilience tradition that may be useful in filling these gaps. Further, we discuss the challenges for consolidating these approaches into a more integrated perspective for managing social-ecological systems.

Enhancing quantitative approaches for assessing community resilience.

PubMed

Chuang, W C; Garmestani, A; Eason, T N; Spanbauer, T L; Fried-Petersen, H B; Roberts, C P; Sundstrom, S M; Burnett, J L; Angeler, D G; Chaffin, B C; Gunderson, L; Twidwell, D; Allen, C R

2018-05-01

Scholars from many different intellectual disciplines have attempted to measure, estimate, or quantify resilience. However, there is growing concern that lack of clarity on the operationalization of the concept will limit its application. In this paper, we discuss the theory, research development and quantitative approaches in ecological and community resilience. Upon noting the lack of methods that quantify the complexities of the linked human and natural aspects of community resilience, we identify several promising approaches within the ecological resilience tradition that may be useful in filling these gaps. Further, we discuss the challenges for consolidating these approaches into a more integrated perspective for managing social-ecological systems. Published by Elsevier Ltd.

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

Using the Blended Learning Approach in a Quantitative Literacy Course

ERIC Educational Resources Information Center

Botts, Ryan T.; Carter, Lori; Crockett, Catherine

2018-01-01

The efforts to improve the quantitative reasoning (quantitative literacy) skills of college students in the United States have been gaining momentum in recent years. At the same time, the blended learning approach to course delivery has gained in popularity, promising better learning with flexible modalities and pace. This paper presents the…

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

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.

Toward a quantitative approach to migrants integration

NASA Astrophysics Data System (ADS)

Barra, A.; Contucci, P.

2010-03-01

Migration phenomena and all the related issues, like integration of different social groups, are intrinsically complex problems since they strongly depend on several competitive mechanisms as economic factors, cultural differences and many others. By identifying a few essential assumptions, and using the statistical mechanics of complex systems, we propose a novel quantitative approach that provides a minimal theory for those phenomena. We show that the competitive interactions in decision making between a population of N host citizens and P immigrants, a bi-partite spin-glass, give rise to a social consciousness inside the host community in the sense of the associative memory of neural networks. The theory leads to a natural quantitative definition of migrant's "integration" inside the community. From the technical point of view this minimal picture assumes, as control parameters, only general notions like the strength of the random interactions, the ratio between the sizes of the two parties and the cultural influence. Few steps forward, toward more refined models, which include a digression on the kind of the felt experiences and some structure on the random interaction topology (as dilution to avoid the plain mean-field approach) and correlations of experiences felt between the two parties (biasing the distribution of the coupling) are discussed at the end, where we show the robustness of our approach.

IWGT report on quantitative approaches to genotoxicity risk ...

EPA Pesticide Factsheets

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the need for quantitative dose–response analysis of genetic toxicology data, the existence and appropriate evaluation of threshold responses, and methods to analyze exposure-response relationships and derive points of departure (PoDs) from which acceptable exposure levels could be determined. This report summarizes the QWG discussions and recommendations regarding appropriate approaches to evaluate exposure-related risks of genotoxic damage, including extrapolation below identified PoDs and across test systems and species. Recommendations include the selection of appropriate genetic endpoints and target tissues, uncertainty factors and extrapolation methods to be considered, the importance and use of information on mode of action, toxicokinetics, metabolism, and exposure biomarkers when using quantitative exposure-response data to determine acceptable exposure levels in human populations or to assess the risk associated with known or anticipated exposures. The empirical relationship between genetic damage (mutation and chromosomal aberration) and cancer in animal models was also examined. It was concluded that there is a general correlation between cancer induction and mutagenic and/or clast

A Quantitative Approach to Scar Analysis

PubMed Central

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

2011-01-01

Analysis of collagen architecture is essential to wound healing research. However, to date no consistent methodologies exist for quantitatively assessing dermal collagen architecture in scars. In this study, we developed a standardized approach for quantitative analysis of scar collagen morphology by confocal microscopy using fractal dimension and lacunarity analysis. Full-thickness wounds were created on adult mice, closed by primary intention, and harvested at 14 days after wounding for morphometrics and standard Fourier transform-based scar analysis as well as fractal dimension and lacunarity analysis. In addition, transmission electron microscopy was used to evaluate collagen ultrastructure. We demonstrated that fractal dimension and lacunarity analysis were superior to Fourier transform analysis in discriminating scar versus unwounded tissue in a wild-type mouse model. To fully test the robustness of this scar analysis approach, a fibromodulin-null mouse model that heals with increased scar was also used. Fractal dimension and lacunarity analysis effectively discriminated unwounded fibromodulin-null versus wild-type skin as well as healing fibromodulin-null versus wild-type wounds, whereas Fourier transform analysis failed to do so. Furthermore, fractal dimension and lacunarity data also correlated well with transmission electron microscopy collagen ultrastructure analysis, adding to their validity. These results demonstrate that fractal dimension and lacunarity are more sensitive than Fourier transform analysis for quantification of scar morphology. PMID:21281794

Infusing Quantitative Approaches throughout the Biological Sciences Curriculum

ERIC Educational Resources Information Center

Thompson, Katerina V.; Cooke, Todd J.; Fagan, William F.; Gulick, Denny; Levy, Doron; Nelson, Kären C.; Redish, Edward F.; Smith, Robert F.; Presson, Joelle

2013-01-01

A major curriculum redesign effort at the University of Maryland is infusing all levels of our undergraduate biological sciences curriculum with increased emphasis on interdisciplinary connections and quantitative approaches. The curriculum development efforts have largely been guided by recommendations in the National Research Council's "Bio…

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.

Developing a Research Program Using Qualitative and Quantitative Approaches.

ERIC Educational Resources Information Center

Beck, Cheryl Tatano

1997-01-01

A research program on postpartum depression is used to illustrate the use of both qualitative and quantitative approaches. The direction of a research program is thus not limited by the type of methods in which a researcher has expertise. (SK)

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

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

A quantitative approach to evolution of music and philosophy

NASA Astrophysics Data System (ADS)

Vieira, Vilson; Fabbri, Renato; Travieso, Gonzalo; Oliveira, Osvaldo N., Jr.; da Fontoura Costa, Luciano

2012-08-01

The development of new statistical and computational methods is increasingly making it possible to bridge the gap between hard sciences and humanities. In this study, we propose an approach based on a quantitative evaluation of attributes of objects in fields of humanities, from which concepts such as dialectics and opposition are formally defined mathematically. As case studies, we analyzed the temporal evolution of classical music and philosophy by obtaining data for 8 features characterizing the corresponding fields for 7 well-known composers and philosophers, which were treated with multivariate statistics and pattern recognition methods. A bootstrap method was applied to avoid statistical bias caused by the small sample data set, with which hundreds of artificial composers and philosophers were generated, influenced by the 7 names originally chosen. Upon defining indices for opposition, skewness and counter-dialectics, we confirmed the intuitive analysis of historians in that classical music evolved according to a master-apprentice tradition, while in philosophy changes were driven by opposition. Though these case studies were meant only to show the possibility of treating phenomena in humanities quantitatively, including a quantitative measure of concepts such as dialectics and opposition, the results are encouraging for further application of the approach presented here to many other areas, since it is entirely generic.

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

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

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.

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

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.

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

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.

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.

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

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

Quantitative and qualitative approaches in the study of poverty and adolescent development: separation or integration?

PubMed

Leung, Janet T Y; Shek, Daniel T L

2011-01-01

This paper examines the use of quantitative and qualitative approaches to study the impact of economic disadvantage on family processes and adolescent development. Quantitative research has the merits of objectivity, good predictive and explanatory power, parsimony, precision and sophistication of analysis. Qualitative research, in contrast, provides a detailed, holistic, in-depth understanding of social reality and allows illumination of new insights. With the pragmatic considerations of methodological appropriateness, design flexibility, and situational responsiveness in responding to the research inquiry, a mixed methods approach could be a possibility of integrating quantitative and qualitative approaches and offers an alternative strategy to study the impact of economic disadvantage on family processes and adolescent development.

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.

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.

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.

A Systematic Approach for Quantitative Analysis of Multidisciplinary Design Optimization Framework

NASA Astrophysics Data System (ADS)

Kim, Sangho; Park, Jungkeun; Lee, Jeong-Oog; Lee, Jae-Woo

An efficient Multidisciplinary Design and Optimization (MDO) framework for an aerospace engineering system should use and integrate distributed resources such as various analysis codes, optimization codes, Computer Aided Design (CAD) tools, Data Base Management Systems (DBMS), etc. in a heterogeneous environment, and need to provide user-friendly graphical user interfaces. In this paper, we propose a systematic approach for determining a reference MDO framework and for evaluating MDO frameworks. The proposed approach incorporates two well-known methods, Analytic Hierarchy Process (AHP) and Quality Function Deployment (QFD), in order to provide a quantitative analysis of the qualitative criteria of MDO frameworks. Identification and hierarchy of the framework requirements and the corresponding solutions for the reference MDO frameworks, the general one and the aircraft oriented one were carefully investigated. The reference frameworks were also quantitatively identified using AHP and QFD. An assessment of three in-house frameworks was then performed. The results produced clear and useful guidelines for improvement of the in-house MDO frameworks and showed the feasibility of the proposed approach for evaluating an MDO framework without a human interference.

Research design: qualitative, quantitative and mixed methods approaches Research design: qualitative, quantitative and mixed methods approaches Creswell John W Sage 320 £29 0761924426 0761924426 [Formula: see text].

PubMed

2004-09-01

The second edition of Creswell's book has been significantly revised and updated. The author clearly sets out three approaches to research: quantitative, qualitative and mixed methods. As someone who has used mixed methods in my research, it is refreshing to read a textbook that addresses this. The differences between the approaches are clearly identified and a rationale for using each methodological stance provided.

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

High resolution quantitative phase imaging of live cells with constrained optimization approach

NASA Astrophysics Data System (ADS)

Pandiyan, Vimal Prabhu; Khare, Kedar; John, Renu

2016-03-01

Quantitative phase imaging (QPI) aims at studying weakly scattering and absorbing biological specimens with subwavelength accuracy without any external staining mechanisms. Use of a reference beam at an angle is one of the necessary criteria for recording of high resolution holograms in most of the interferometric methods used for quantitative phase imaging. The spatial separation of the dc and twin images is decided by the reference beam angle and Fourier-filtered reconstructed image will have a very poor resolution if hologram is recorded below a minimum reference angle condition. However, it is always inconvenient to have a large reference beam angle while performing high resolution microscopy of live cells and biological specimens with nanometric features. In this paper, we treat reconstruction of digital holographic microscopy images as a constrained optimization problem with smoothness constraint in order to recover only complex object field in hologram plane even with overlapping dc and twin image terms. We solve this optimization problem by gradient descent approach iteratively and the smoothness constraint is implemented by spatial averaging with appropriate size. This approach will give excellent high resolution image recovery compared to Fourier filtering while keeping a very small reference angle. We demonstrate this approach on digital holographic microscopy of live cells by recovering the quantitative phase of live cells from a hologram recorded with nearly zero reference angle.

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.

Quantitative Outcomes of a One Health approach to Study Global Health Challenges.

PubMed

Falzon, Laura C; Lechner, Isabel; Chantziaras, Ilias; Collineau, Lucie; Courcoul, Aurélie; Filippitzi, Maria-Eleni; Laukkanen-Ninios, Riikka; Peroz, Carole; Pinto Ferreira, Jorge; Postma, Merel; Prestmo, Pia G; Phythian, Clare J; Sarno, Eleonora; Vanantwerpen, Gerty; Vergne, Timothée; Grindlay, Douglas J C; Brennan, Marnie L

2018-03-01

Having gained momentum in the last decade, the One Health initiative promotes a holistic approach to address complex global health issues. Before recommending its adoption to stakeholders, however, it is paramount to first compile quantitative evidence of the benefit of such an approach. The aim of this scoping review was to identify and summarize primary research that describes monetary and non-monetary outcomes following adoption of a One Health approach. An extensive literature search yielded a total of 42,167 references, of which 85 were included in the final analysis. The top two biotic health issues addressed in these studies were rabies and malaria; the top abiotic health issue was air pollution. Most studies described collaborations between human and animal (n = 42), or human and environmental disciplines (n = 41); commonly reported interventions included vector control and animal vaccination. Monetary outcomes were commonly expressed as cost-benefit or cost-utility ratios; non-monetary outcomes were described using disease frequency or disease burden measurements. The majority of the studies reported positive or partially positive outcomes. This paper illustrates the variety of health challenges that can be addressed using a One Health approach, and provides tangible quantitative measures that can be used to evaluate future implementations of the One Health approach.

A simple approach to quantitative analysis using three-dimensional spectra based on selected Zernike moments.

PubMed

Zhai, Hong Lin; Zhai, Yue Yuan; Li, Pei Zhen; Tian, Yue Li

2013-01-21

A very simple approach to quantitative analysis is proposed based on the technology of digital image processing using three-dimensional (3D) spectra obtained by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). As the region-based shape features of a grayscale image, Zernike moments with inherently invariance property were employed to establish the linear quantitative models. This approach was applied to the quantitative analysis of three compounds in mixed samples using 3D HPLC-DAD spectra, and three linear models were obtained, respectively. The correlation coefficients (R(2)) for training and test sets were more than 0.999, and the statistical parameters and strict validation supported the reliability of established models. The analytical results suggest that the Zernike moment selected by stepwise regression can be used in the quantitative analysis of target compounds. Our study provides a new idea for quantitative analysis using 3D spectra, which can be extended to the analysis of other 3D spectra obtained by different methods or instruments.

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

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.

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

A novel approach for quantitative harmonization in PET.

PubMed

Namías, M; Bradshaw, T; Menezes, V O; Machado, M A D; Jeraj, R

2018-05-04

Positron emission tomography (PET) imaging allows for measurement of activity concentrations of a given radiotracer in vivo. The quantitative capabilities of PET imaging are particularly important in the context of monitoring response to treatment, where quantitative changes in tracer uptake could be used as a biomarker of treatment response. Reconstruction algorithms and settings have a significant impact on PET quantification. In this work we introduce a novel harmonization methodology requiring only a simple cylindrical phantom and show that it can match the performance of more complex harmonization approaches based on phantoms with spherical inserts. Resolution and noise measurements from cylindrical phantoms are used to simulate the spherical inserts from NEMA image quality phantoms. An optimization algorithm was used to find the optimal smoothing filters for the simulated NEMA phantom images to identify those that best harmonized the PET scanners. Our methodology was tested on seven different PET models from two manufacturers installed at five institutions. Our methodology is able to predict contrast recovery coefficients (CRCs) from NEMA phantoms with errors within  ±5.2% for CRCmax and  ±3.7% for CRCmean (limits of agreement  =  95%). After applying the proposed harmonization protocol, all the CRC values were within the tolerances from EANM. Quantitative harmonization in compliance with the EARL FDG-PET/CT accreditation program is achieved in a simpler way, without the need of NEMA phantoms. This may lead to simplified scanner harmonization workflows more accessible to smaller institutions.

Tau-U: A Quantitative Approach for Analysis of Single-Case Experimental Data in Aphasia.

PubMed

Lee, Jaime B; Cherney, Leora R

2018-03-01

Tau-U is a quantitative approach for analyzing single-case experimental design (SCED) data. It combines nonoverlap between phases with intervention phase trend and can correct for a baseline trend (Parker, Vannest, & Davis, 2011). We demonstrate the utility of Tau-U by comparing it with the standardized mean difference approach (Busk & Serlin, 1992) that is widely reported within the aphasia SCED literature. Repeated writing measures from 3 participants with chronic aphasia who received computer-based writing treatment are analyzed visually and quantitatively using both Tau-U and the standardized mean difference approach. Visual analysis alone was insufficient for determining an effect between the intervention and writing improvement. The standardized mean difference yielded effect sizes ranging from 4.18 to 26.72 for trained items and 1.25 to 3.20 for untrained items. Tau-U yielded significant (p < .05) effect sizes for 2 of 3 participants for trained probes and 1 of 3 participants for untrained probes. A baseline trend correction was applied to data from 2 of 3 participants. Tau-U has the unique advantage of allowing for the correction of an undesirable baseline trend. Although further study is needed, Tau-U shows promise as a quantitative approach to augment visual analysis of SCED data in aphasia.

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

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

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

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.

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.

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples

PubMed Central

Licier, Rígel; Miranda, Eric; Serrano, Horacio

2016-01-01

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine. PMID:28248241

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.

PubMed

Licier, Rígel; Miranda, Eric; Serrano, Horacio

2016-10-17

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

Qualitative and quantitative approaches in the dose-response assessment of genotoxic carcinogens.

PubMed

Fukushima, Shoji; Gi, Min; Kakehashi, Anna; Wanibuchi, Hideki; Matsumoto, Michiharu

2016-05-01

Qualitative and quantitative approaches are important issues in field of carcinogenic risk assessment of the genotoxic carcinogens. Herein, we provide quantitative data on low-dose hepatocarcinogenicity studies for three genotoxic hepatocarcinogens: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodiethylamine (DEN). Hepatocarcinogenicity was examined by quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci, which are the preneoplastic lesions in rat hepatocarcinogenesis and the endpoint carcinogenic marker in the rat liver medium-term carcinogenicity bioassay. We also examined DNA damage and gene mutations which occurred through the initiation stage of carcinogenesis. For the establishment of points of departure (PoD) from which the cancer-related risk can be estimated, we analyzed the above events by quantitative no-observed-effect level and benchmark dose approaches. MeIQx at low doses induced formation of DNA-MeIQx adducts; somewhat higher doses caused elevation of 8-hydroxy-2'-deoxyquanosine levels; at still higher doses gene mutations occurred; and the highest dose induced formation of GST-P positive foci. These data indicate that early genotoxic events in the pathway to carcinogenesis showed the expected trend of lower PoDs for earlier events in the carcinogenic process. Similarly, only the highest dose of IQ caused an increase in the number of GST-P positive foci in the liver, while IQ-DNA adduct formation was observed with low doses. Moreover, treatment with DEN at low doses had no effect on development of GST-P positive foci in the liver. These data on PoDs for the markers contribute to understand whether genotoxic carcinogens have a threshold for their carcinogenicity. The most appropriate approach to use in low dose-response assessment must be approved on the basis of scientific judgment. © The Author 2015. Published by Oxford University Press on behalf of

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

'Stories' or 'snapshots'? A study directed at comparing qualitative and quantitative approaches to curriculum evaluation.

PubMed

Pateman, B; Jinks, A M

1999-01-01

The focus of this paper is a study designed to explore the validity of quantitative approaches of student evaluation in a pre-registration degree programme. As managers of the students' education we were concerned that the quantitative method, which used lecturer criteria, may not fully represent students' views. The approach taken is that of a process-type strategy for curriculum evaluation as described by Parlett and Hamilton (1972). The aim of the study is to produce illuminative data, or students' 'stories' of their educational experiences through use of semi-structured interviews. The results are then compared to the current quantitative measurement tools designed to obtain 'snapshots' of the educational effectiveness of the curriculum. The quantitative measurement tools use Likert scale measurements of teacher-devised criterion statements. The results of the study give a rich source of qualitative data which can be used to inform future curriculum development. However, complete validation of the current quantitative instruments used was not achieved in this study. Student and teacher agendas in respect of important issues pertaining to the course programme were found to differ. Limitations of the study are given. There is discussion of the options open to the management team with regard to future development of curriculum evaluation systems.

Application of Person-Centered Approaches to Critical Quantitative Research: Exploring Inequities in College Financing Strategies

ERIC Educational Resources Information Center

Malcom-Piqueux, Lindsey

2014-01-01

This chapter discusses the utility of person-centered approaches to critical quantitative researchers. These techniques, which identify groups of individuals who share similar attributes, experiences, or outcomes, are contrasted with more commonly used variable-centered approaches. An illustrative example of a latent class analysis of the college…

Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules.

PubMed

Jones, Barry R; Schultz, Gary A; Eckstein, James A; Ackermann, Bradley L

2012-10-01

Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

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

Quantitative Assessment of Arrhythmia Using Non-linear Approach: A Non-invasive Prognostic Tool

NASA Astrophysics Data System (ADS)

Chakraborty, Monisha; Ghosh, Dipak

2017-12-01

Accurate prognostic tool to identify severity of Arrhythmia is yet to be investigated, owing to the complexity of the ECG signal. In this paper, we have shown that quantitative assessment of Arrhythmia is possible using non-linear technique based on "Hurst Rescaled Range Analysis". Although the concept of applying "non-linearity" for studying various cardiac dysfunctions is not entirely new, the novel objective of this paper is to identify the severity of the disease, monitoring of different medicine and their dose, and also to assess the efficiency of different medicine. The approach presented in this work is simple which in turn will help doctors in efficient disease management. In this work, Arrhythmia ECG time series are collected from MIT-BIH database. Normal ECG time series are acquired using POLYPARA system. Both time series are analyzed in thelight of non-linear approach following the method "Rescaled Range Analysis". The quantitative parameter, "Fractal Dimension" (D) is obtained from both types of time series. The major finding is that Arrhythmia ECG poses lower values of D as compared to normal. Further, this information can be used to access the severity of Arrhythmia quantitatively, which is a new direction of prognosis as well as adequate software may be developed for the use of medical practice.

Quantitative Assessment of Arrhythmia Using Non-linear Approach: A Non-invasive Prognostic Tool

NASA Astrophysics Data System (ADS)

Chakraborty, Monisha; Ghosh, Dipak

2018-04-01

Accurate prognostic tool to identify severity of Arrhythmia is yet to be investigated, owing to the complexity of the ECG signal. In this paper, we have shown that quantitative assessment of Arrhythmia is possible using non-linear technique based on "Hurst Rescaled Range Analysis". Although the concept of applying "non-linearity" for studying various cardiac dysfunctions is not entirely new, the novel objective of this paper is to identify the severity of the disease, monitoring of different medicine and their dose, and also to assess the efficiency of different medicine. The approach presented in this work is simple which in turn will help doctors in efficient disease management. In this work, Arrhythmia ECG time series are collected from MIT-BIH database. Normal ECG time series are acquired using POLYPARA system. Both time series are analyzed in thelight of non-linear approach following the method "Rescaled Range Analysis". The quantitative parameter, "Fractal Dimension" (D) is obtained from both types of time series. The major finding is that Arrhythmia ECG poses lower values of D as compared to normal. Further, this information can be used to access the severity of Arrhythmia quantitatively, which is a new direction of prognosis as well as adequate software may be developed for the use of medical practice.

Predictive value of EEG in postanoxic encephalopathy: A quantitative model-based approach.

PubMed

Efthymiou, Evdokia; Renzel, Roland; Baumann, Christian R; Poryazova, Rositsa; Imbach, Lukas L

2017-10-01

The majority of comatose patients after cardiac arrest do not regain consciousness due to severe postanoxic encephalopathy. Early and accurate outcome prediction is therefore essential in determining further therapeutic interventions. The electroencephalogram is a standardized and commonly available tool used to estimate prognosis in postanoxic patients. The identification of pathological EEG patterns with poor prognosis relies however primarily on visual EEG scoring by experts. We introduced a model-based approach of EEG analysis (state space model) that allows for an objective and quantitative description of spectral EEG variability. We retrospectively analyzed standard EEG recordings in 83 comatose patients after cardiac arrest between 2005 and 2013 in the intensive care unit of the University Hospital Zürich. Neurological outcome was assessed one month after cardiac arrest using the Cerebral Performance Category. For a dynamic and quantitative EEG analysis, we implemented a model-based approach (state space analysis) to quantify EEG background variability independent from visual scoring of EEG epochs. Spectral variability was compared between groups and correlated with clinical outcome parameters and visual EEG patterns. Quantitative assessment of spectral EEG variability (state space velocity) revealed significant differences between patients with poor and good outcome after cardiac arrest: Lower mean velocity in temporal electrodes (T4 and T5) was significantly associated with poor prognostic outcome (p<0.005) and correlated with independently identified visual EEG patterns such as generalized periodic discharges (p<0.02). Receiver operating characteristic (ROC) analysis confirmed the predictive value of lower state space velocity for poor clinical outcome after cardiac arrest (AUC 80.8, 70% sensitivity, 15% false positive rate). Model-based quantitative EEG analysis (state space analysis) provides a novel, complementary marker for prognosis in postanoxic

Conspicuous Strategies in Teaching Expressive Writing: A Quantitative Study Comparing Two Approaches to Process Writing

ERIC Educational Resources Information Center

Fontenot, Jennifer; Carney, Karen J.; Hansen, Kay

2015-01-01

A process-writing approach (BW) with novel concepts was developed by the authors to teach writing to elementary-level students. They believed the BW approach was effective but was particularly effective for special-needs students. Consequently, they decided to quantitatively test these assertions. Instead of testing students taught using the BW…

A novel logic-based approach for quantitative toxicology prediction.

PubMed

Amini, Ata; Muggleton, Stephen H; Lodhi, Huma; Sternberg, Michael J E

2007-01-01

There is a pressing need for accurate in silico methods to predict the toxicity of molecules that are being introduced into the environment or are being developed into new pharmaceuticals. Predictive toxicology is in the realm of structure activity relationships (SAR), and many approaches have been used to derive such SAR. Previous work has shown that inductive logic programming (ILP) is a powerful approach that circumvents several major difficulties, such as molecular superposition, faced by some other SAR methods. The ILP approach reasons with chemical substructures within a relational framework and yields chemically understandable rules. Here, we report a general new approach, support vector inductive logic programming (SVILP), which extends the essentially qualitative ILP-based SAR to quantitative modeling. First, ILP is used to learn rules, the predictions of which are then used within a novel kernel to derive a support-vector generalization model. For a highly heterogeneous dataset of 576 molecules with known fathead minnow fish toxicity, the cross-validated correlation coefficients (R2CV) from a chemical descriptor method (CHEM) and SVILP are 0.52 and 0.66, respectively. The ILP, CHEM, and SVILP approaches correctly predict 55, 58, and 73%, respectively, of toxic molecules. In a set of 165 unseen molecules, the R2 values from the commercial software TOPKAT and SVILP are 0.26 and 0.57, respectively. In all calculations, SVILP showed significant improvements in comparison with the other methods. The SVILP approach has a major advantage in that it uses ILP automatically and consistently to derive rules, mostly novel, describing fragments that are toxicity alerts. The SVILP is a general machine-learning approach and has the potential of tackling many problems relevant to chemoinformatics including in silico drug design.

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.

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.

A Quantitative Approach to Assessing System Evolvability

NASA Technical Reports Server (NTRS)

Christian, John A., III

2004-01-01

When selecting a system from multiple candidates, the customer seeks the one that best meets his or her needs. Recently the desire for evolvable systems has become more important and engineers are striving to develop systems that accommodate this need. In response to this search for evolvability, we present a historical perspective on evolvability, propose a refined definition of evolvability, and develop a quantitative method for measuring this property. We address this quantitative methodology from both a theoretical and practical perspective. This quantitative model is then applied to the problem of evolving a lunar mission to a Mars mission as a case study.

Two approaches to improving mental health care: positivist/quantitative versus skill-based/qualitative.

PubMed

Luchins, Daniel

2012-01-01

The quality improvement model currently used in medicine and mental health was adopted from industry, where it developed out of early 20th-century efforts to apply a positivist/quantitative agenda to improving manufacturing. This article questions the application of this model to mental health care. It argues that (1) developing "operational definitions" for something as value-laden as "quality" risks conflating two realms, what we measure with what we value; (2) when measurements that are tied to individuals are aggregated to establish benchmarks and goals, unwarranted mathematical assumptions are made; (3) choosing clinical outcomes is problematic; (4) there is little relationship between process measures and clinical outcomes; and (5) since changes in quality indices do not relate to improved clinical care, management's reliance on such indices provides an illusory sense of control. An alternative model is the older, skill-based/qualitative approach to knowing, which relies on "implicit/ expert" knowledge. These two approaches offer a series of contrasts: quality versus excellence, competence versus expertise, management versus leadership, extrinsic versus intrinsic rewards. The article concludes that we need not totally dispense with the current quality improvement model, but rather should balance quantitative efforts with the older qualitative approach in a mixed methods model.

Quantitative evaluation of phase processing approaches in susceptibility weighted imaging

NASA Astrophysics Data System (ADS)

Li, Ningzhi; Wang, Wen-Tung; Sati, Pascal; Pham, Dzung L.; Butman, John A.

2012-03-01

Susceptibility weighted imaging (SWI) takes advantage of the local variation in susceptibility between different tissues to enable highly detailed visualization of the cerebral venous system and sensitive detection of intracranial hemorrhages. Thus, it has been increasingly used in magnetic resonance imaging studies of traumatic brain injury as well as other intracranial pathologies. In SWI, magnitude information is combined with phase information to enhance the susceptibility induced image contrast. Because of global susceptibility variations across the image, the rate of phase accumulation varies widely across the image resulting in phase wrapping artifacts that interfere with the local assessment of phase variation. Homodyne filtering is a common approach to eliminate this global phase variation. However, filter size requires careful selection in order to preserve image contrast and avoid errors resulting from residual phase wraps. An alternative approach is to apply phase unwrapping prior to high pass filtering. A suitable phase unwrapping algorithm guarantees no residual phase wraps but additional computational steps are required. In this work, we quantitatively evaluate these two phase processing approaches on both simulated and real data using different filters and cutoff frequencies. Our analysis leads to an improved understanding of the relationship between phase wraps, susceptibility effects, and acquisition parameters. Although homodyne filtering approaches are faster and more straightforward, phase unwrapping approaches perform more accurately in a wider variety of acquisition scenarios.

Benefit-risk analysis : a brief review and proposed quantitative approaches.

PubMed

Holden, William L

2003-01-01

Given the current status of benefit-risk analysis as a largely qualitative method, two techniques for a quantitative synthesis of a drug's benefit and risk are proposed to allow a more objective approach. The recommended methods, relative-value adjusted number-needed-to-treat (RV-NNT) and its extension, minimum clinical efficacy (MCE) analysis, rely upon efficacy or effectiveness data, adverse event data and utility data from patients, describing their preferences for an outcome given potential risks. These methods, using hypothetical data for rheumatoid arthritis drugs, demonstrate that quantitative distinctions can be made between drugs which would better inform clinicians, drug regulators and patients about a drug's benefit-risk profile. If the number of patients needed to treat is less than the relative-value adjusted number-needed-to-harm in an RV-NNT analysis, patients are willing to undergo treatment with the experimental drug to derive a certain benefit knowing that they may be at risk for any of a series of potential adverse events. Similarly, the results of an MCE analysis allow for determining the worth of a new treatment relative to an older one, given not only the potential risks of adverse events and benefits that may be gained, but also by taking into account the risk of disease without any treatment. Quantitative methods of benefit-risk analysis have a place in the evaluative armamentarium of pharmacovigilance, especially those that incorporate patients' perspectives.

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.

High throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence

PubMed Central

Shultzaberger, Ryan K.; Paddock, Mark L.; Katsuki, Takeo; Greenspan, Ralph J.; Golden, Susan S.

2016-01-01

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise, and generate meaningful quantitative measurements of clock output levels for advanced analysis. PMID:25662451

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

A novel approach to teach the generation of bioelectrical potentials from a descriptive and quantitative perspective.

PubMed

Rodriguez-Falces, Javier

2013-12-01

In electrophysiology studies, it is becoming increasingly common to explain experimental observations using both descriptive methods and quantitative approaches. However, some electrophysiological phenomena, such as the generation of extracellular potentials that results from the propagation of the excitation source along the muscle fiber, are difficult to describe and conceptualize. In addition, most traditional approaches aimed at describing extracellular potentials consist of complex mathematical machinery that gives no chance for physical interpretation. The aim of the present study is to present a new method to teach the formation of extracellular potentials around a muscle fiber from both a descriptive and quantitative perspective. The implementation of this method was tested through a written exam and a satisfaction survey. The new method enhanced the ability of students to visualize the generation of bioelectrical potentials. In addition, the new approach improved students' understanding of how changes in the fiber-to-electrode distance and in the shape of the excitation source are translated into changes in the extracellular potential. The survey results show that combining general principles of electrical fields with accurate graphic imagery gives students an intuitive, yet quantitative, feel for electrophysiological signals and enhances their motivation to continue their studies in the biomedical engineering field.

Using the realist perspective to link theory from qualitative evidence synthesis to quantitative studies: Broadening the matrix approach.

PubMed

van Grootel, Leonie; van Wesel, Floryt; O'Mara-Eves, Alison; Thomas, James; Hox, Joop; Boeije, Hennie

2017-09-01

This study describes an approach for the use of a specific type of qualitative evidence synthesis in the matrix approach, a mixed studies reviewing method. The matrix approach compares quantitative and qualitative data on the review level by juxtaposing concrete recommendations from the qualitative evidence synthesis against interventions in primary quantitative studies. However, types of qualitative evidence syntheses that are associated with theory building generate theoretical models instead of recommendations. Therefore, the output from these types of qualitative evidence syntheses cannot directly be used for the matrix approach but requires transformation. This approach allows for the transformation of these types of output. The approach enables the inference of moderation effects instead of direct effects from the theoretical model developed in a qualitative evidence synthesis. Recommendations for practice are formulated on the basis of interactional relations inferred from the qualitative evidence synthesis. In doing so, we apply the realist perspective to model variables from the qualitative evidence synthesis according to the context-mechanism-outcome configuration. A worked example shows that it is possible to identify recommendations from a theory-building qualitative evidence synthesis using the realist perspective. We created subsets of the interventions from primary quantitative studies based on whether they matched the recommendations or not and compared the weighted mean effect sizes of the subsets. The comparison shows a slight difference in effect sizes between the groups of studies. The study concludes that the approach enhances the applicability of the matrix approach. Copyright © 2017 John Wiley & Sons, Ltd.

Quantitative meta-analytic approaches for the analysis of animal toxicology and epidemiologic data in human health risk assessments

EPA Science Inventory

Often, human health risk assessments have relied on qualitative approaches for hazard identification to integrate evidence across multiple studies to conclude whether particular hazards exist. However, quantitative approaches for evidence integration, including the application o...

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.

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

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.

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

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

Standardized Approach to Quantitatively Measure Residual Limb Skin Health in Individuals with Lower Limb Amputation.

PubMed

Rink, Cameron L; Wernke, Matthew M; Powell, Heather M; Tornero, Mark; Gnyawali, Surya C; Schroeder, Ryan M; Kim, Jayne Y; Denune, Jeffrey A; Albury, Alexander W; Gordillo, Gayle M; Colvin, James M; Sen, Chandan K

2017-07-01

Objective: (1) Develop a standardized approach to quantitatively measure residual limb skin health. (2) Report reference residual limb skin health values in people with transtibial and transfemoral amputation. Approach: Residual limb health outcomes in individuals with transtibial ( n  = 5) and transfemoral ( n  = 5) amputation were compared to able-limb controls ( n  = 4) using noninvasive imaging (hyperspectral imaging and laser speckle flowmetry) and probe-based approaches (laser doppler flowmetry, transcutaneous oxygen, transepidermal water loss, surface electrical capacitance). Results: A standardized methodology that employs noninvasive imaging and probe-based approaches to measure residual limb skin health are described. Compared to able-limb controls, individuals with transtibial and transfemoral amputation have significantly lower transcutaneous oxygen tension, higher transepidermal water loss, and higher surface electrical capacitance in the residual limb. Innovation: Residual limb health as a critical component of prosthesis rehabilitation for individuals with lower limb amputation is understudied in part due to a lack of clinical measures. Here, we present a standardized approach to measure residual limb health in people with transtibial and transfemoral amputation. Conclusion: Technology advances in noninvasive imaging and probe-based measures are leveraged to develop a standardized approach to quantitatively measure residual limb health in individuals with lower limb loss. Compared to able-limb controls, resting residual limb physiology in people that have had transfemoral or transtibial amputation is characterized by lower transcutaneous oxygen tension and poorer skin barrier function.

LuciPHOr: Algorithm for Phosphorylation Site Localization with False Localization Rate Estimation Using Modified Target-Decoy Approach*

PubMed Central

Fermin, Damian; Walmsley, Scott J.; Gingras, Anne-Claude; Choi, Hyungwon; Nesvizhskii, Alexey I.

2013-01-01

The localization of phosphorylation sites in peptide sequences is a challenging problem in large-scale phosphoproteomics analysis. The intense neutral loss peaks and the coexistence of multiple serine/threonine and/or tyrosine residues are limiting factors for objectively scoring site patterns across thousands of peptides. Various computational approaches for phosphorylation site localization have been proposed, including Ascore, Mascot Delta score, and ProteinProspector, yet few address direct estimation of the false localization rate (FLR) in each experiment. Here we propose LuciPHOr, a modified target-decoy-based approach that uses mass accuracy and peak intensities for site localization scoring and FLR estimation. Accurate estimation of the FLR is a difficult task at the individual-site level because the degree of uncertainty in localization varies significantly across different peptides. LuciPHOr carries out simultaneous localization on all candidate sites in each peptide and estimates the FLR based on the target-decoy framework, where decoy phosphopeptides generated by placing artificial phosphorylation(s) on non-candidate residues compete with the non-decoy phosphopeptides. LuciPHOr also reports approximate site-level confidence scores for all candidate sites as a means to localize additional sites from multiphosphorylated peptides in which localization can be partially achieved. Unlike the existing tools, LuciPHOr is compatible with any search engine output processed through the Trans-Proteomic Pipeline. We evaluated the performance of LuciPHOr in terms of the sensitivity and accuracy of FLR estimates using two synthetic phosphopeptide libraries and a phosphoproteomic dataset generated from complex mouse brain samples. PMID:23918812

A quantitative risk analysis approach to port hydrocarbon logistics.

PubMed

Ronza, A; Carol, S; Espejo, V; Vílchez, J A; Arnaldos, J

2006-01-16

A method is presented that allows quantitative risk analysis to be performed on marine hydrocarbon terminals sited in ports. A significant gap was identified in the technical literature on QRA for the handling of hazardous materials in harbours published prior to this work. The analysis is extended to tanker navigation through port waters and loading and unloading facilities. The steps of the method are discussed, beginning with data collecting. As to accident scenario identification, an approach is proposed that takes into account minor and massive spills due to loading arm failures and tank rupture. Frequency estimation is thoroughly reviewed and a shortcut approach is proposed for frequency calculation. This allows for the two-fold possibility of a tanker colliding/grounding at/near the berth or while navigating to/from the berth. A number of probability data defining the possibility of a cargo spill after an external impact on a tanker are discussed. As to consequence and vulnerability estimates, a scheme is proposed for the use of ratios between the numbers of fatal victims, injured and evacuated people. Finally, an example application is given, based on a pilot study conducted in the Port of Barcelona, where the method was tested.

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.

Rock Slide Risk Assessment: A Semi-Quantitative Approach

NASA Astrophysics Data System (ADS)

Duzgun, H. S. B.

2009-04-01

Rock slides can be better managed by systematic risk assessments. Any risk assessment methodology for rock slides involves identification of rock slide risk components, which are hazard, elements at risk and vulnerability. For a quantitative/semi-quantitative risk assessment for rock slides, a mathematical value the risk has to be computed and evaluated. The quantitative evaluation of risk for rock slides enables comparison of the computed risk with the risk of other natural and/or human-made hazards and providing better decision support and easier communication for the decision makers. A quantitative/semi-quantitative risk assessment procedure involves: Danger Identification, Hazard Assessment, Elements at Risk Identification, Vulnerability Assessment, Risk computation, Risk Evaluation. On the other hand, the steps of this procedure require adaptation of existing or development of new implementation methods depending on the type of landslide, data availability, investigation scale and nature of consequences. In study, a generic semi-quantitative risk assessment (SQRA) procedure for rock slides is proposed. The procedure has five consecutive stages: Data collection and analyses, hazard assessment, analyses of elements at risk and vulnerability and risk assessment. The implementation of the procedure for a single rock slide case is illustrated for a rock slope in Norway. Rock slides from mountain Ramnefjell to lake Loen are considered to be one of the major geohazards in Norway. Lake Loen is located in the inner part of Nordfjord in Western Norway. Ramnefjell Mountain is heavily jointed leading to formation of vertical rock slices with height between 400-450 m and width between 7-10 m. These slices threaten the settlements around Loen Valley and tourists visiting the fjord during summer season, as the released slides have potential of creating tsunami. In the past, several rock slides had been recorded from the Mountain Ramnefjell between 1905 and 1950. Among them

Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics.

PubMed

Marchand, Jérémy; Martineau, Estelle; Guitton, Yann; Dervilly-Pinel, Gaud; Giraudeau, Patrick

2017-02-01

Multi-dimensional NMR is an appealing approach for dealing with the challenging complexity of biological samples in metabolomics. This article describes how spectroscopists have recently challenged their imagination in order to make 2D NMR a powerful tool for quantitative metabolomics, based on innovative pulse sequences combined with meticulous analytical chemistry approaches. Clever time-saving strategies have also been explored to make 2D NMR a high-throughput tool for metabolomics, relying on alternative data acquisition schemes such as ultrafast NMR. Currently, much work is aimed at drastically boosting the NMR sensitivity thanks to hyperpolarisation techniques, which have been used in combination with fast acquisition methods and could greatly expand the application potential of NMR metabolomics. Copyright © 2016 Elsevier Ltd. All rights reserved.

The craniocaudal extension of posterolateral approaches and their combination: a quantitative anatomic and clinical analysis.

PubMed

Safavi-Abbasi, Sam; de Oliveira, Jean G; Deshmukh, Pushpa; Reis, Cassius V; Brasiliense, Leonardo B C; Crawford, Neil R; Feiz-Erfan, Iman; Spetzler, Robert F; Preul, Mark C

2010-03-01

The aim of this study was to describe quantitatively the properties of the posterolateral approaches and their combination. Six silicone-injected cadaveric heads were dissected bilaterally. Quantitative data were generated with the Optotrak 3020 system (Northern Digital, Waterloo, Canada) and Surgiscope (Elekta Instruments, Inc., Atlanta, GA), including key anatomic points on the skull base and brainstem. All parameters were measured after the basic retrosigmoid craniectomy and then after combination with a basic far-lateral extension. The clinical results of 20 patients who underwent a combined retrosigmoid and far-lateral approach were reviewed. The change in accessibility to the lower clivus was greatest after the far-lateral extension (mean change, 43.62 +/- 10.98 mm2; P = .001). Accessibility to the constant landmarks, Meckel's cave, internal auditory meatus, and jugular foramen did not change significantly between the 2 approaches (P > .05). The greatest change in accessibility to soft tissue between the 2 approaches was to the lower brainstem (mean change, 33.88 +/- 5.25 mm2; P = .0001). Total removal was achieved in 75% of the cases. The average postoperative Glasgow Outcome Scale score of patients who underwent the combined retrosigmoid and far-lateral approach improved significantly, compared with the preoperative scores. The combination of the far-lateral and simple retrosigmoid approaches significantly increases the petroclival working area and access to the cranial nerves. However, risk of injury to neurovascular structures and time needed to extend the craniotomy must be weighed against the increased working area and angles of attack.

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.

Pro-Social Behavior Amongst Students of Tertiary Institutions: An Explorative and a Quantitative Approach

ERIC Educational Resources Information Center

Quain, Samuel; Yidana, Xiaaba Dantallah; Ambotumah, Bernard Baba; Mensah-Livivnstone, Ike Joe Nii Annang

2016-01-01

The purpose of this paper was to explore antecedents of pro-social behavior amongst university students, using a private university as a case study. Following an explorative research, the study was guided by some theories relating to the phenomenon, focusing on gender and location factors. A quantitative approach was used in the follow up to the…

Decoding brain cancer dynamics: a quantitative histogram-based approach using temporal MRI

NASA Astrophysics Data System (ADS)

Zhou, Mu; Hall, Lawrence O.; Goldgof, Dmitry B.; Russo, Robin; Gillies, Robert J.; Gatenby, Robert A.

2015-03-01

Brain tumor heterogeneity remains a challenge for probing brain cancer evolutionary dynamics. In light of evolution, it is a priority to inspect the cancer system from a time-domain perspective since it explicitly tracks the dynamics of cancer variations. In this paper, we study the problem of exploring brain tumor heterogeneity from temporal clinical magnetic resonance imaging (MRI) data. Our goal is to discover evidence-based knowledge from such temporal imaging data, where multiple clinical MRI scans from Glioblastoma multiforme (GBM) patients are generated during therapy. In particular, we propose a quantitative histogram-based approach that builds a prediction model to measure the difference in histograms obtained from pre- and post-treatment. The study could significantly assist radiologists by providing a metric to identify distinctive patterns within each tumor, which is crucial for the goal of providing patient-specific treatments. We examine the proposed approach for a practical application - clinical survival group prediction. Experimental results show that our approach achieved 90.91% accuracy.

A quantitative approach to neuropsychiatry: The why and the how.

PubMed

Kas, Martien J; Penninx, Brenda; Sommer, Bernd; Serretti, Alessandro; Arango, Celso; Marston, Hugh

2017-12-12

The current nosology of neuropsychiatric disorders allows for a pragmatic approach to treatment choice, regulation and clinical research. However, without a biological rationale for these disorders, drug development has stagnated. The recently EU-funded PRISM project aims to develop a quantitative biological approach to the understanding and classification of neuropsychiatric diseases to accelerate the discovery and development of better treatments. By combining clinical data sets from major worldwide disease cohorts and by applying innovative technologies to deeply phenotype stratified patient groups, we will define a set of quantifiable biological parameters for social withdrawal and cognitive deficits common to Schizophrenia (SZ), Major Depression (MD), and Alzheimer's Disease (AD). These studies aim to provide new classification and assessment tools for social and cognitive performance across neuropsychiatric disorders, clinically relevant substrates for treatment development, and predictive, preclinical animal systems. With patients and regulatory agencies, we seek to provide clear routes for the future translation and regulatory approval for new treatments and provide solutions to the growing public health challenges of psychiatry and neurology. Copyright © 2017. Published by Elsevier Ltd.

CRAFT (complete reduction to amplitude frequency table)--robust and time-efficient Bayesian approach for quantitative mixture analysis by NMR.

PubMed

Krishnamurthy, Krish

2013-12-01

The intrinsic quantitative nature of NMR is increasingly exploited in areas ranging from complex mixture analysis (as in metabolomics and reaction monitoring) to quality assurance/control. Complex NMR spectra are more common than not, and therefore, extraction of quantitative information generally involves significant prior knowledge and/or operator interaction to characterize resonances of interest. Moreover, in most NMR-based metabolomic experiments, the signals from metabolites are normally present as a mixture of overlapping resonances, making quantification difficult. Time-domain Bayesian approaches have been reported to be better than conventional frequency-domain analysis at identifying subtle changes in signal amplitude. We discuss an approach that exploits Bayesian analysis to achieve a complete reduction to amplitude frequency table (CRAFT) in an automated and time-efficient fashion - thus converting the time-domain FID to a frequency-amplitude table. CRAFT uses a two-step approach to FID analysis. First, the FID is digitally filtered and downsampled to several sub FIDs, and secondly, these sub FIDs are then modeled as sums of decaying sinusoids using the Bayesian approach. CRAFT tables can be used for further data mining of quantitative information using fingerprint chemical shifts of compounds of interest and/or statistical analysis of modulation of chemical quantity in a biological study (metabolomics) or process study (reaction monitoring) or quality assurance/control. The basic principles behind this approach as well as results to evaluate the effectiveness of this approach in mixture analysis are presented. Copyright © 2013 John Wiley & Sons, Ltd.

Poem Generator: A Comparative Quantitative Evaluation of a Microworlds-Based Learning Approach for Teaching English

ERIC Educational Resources Information Center

Jenkins, Craig

2015-01-01

This paper is a comparative quantitative evaluation of an approach to teaching poetry in the subject domain of English that employs a "guided discovery" pedagogy using computer-based microworlds. It uses a quasi-experimental design in order to measure performance gains in computational thinking and poetic thinking following a…

An Exploration of a Quantitative Reasoning Instructional Approach to Linear Equations in Two Variables with Community College Students

ERIC Educational Resources Information Center

Belue, Paul T.; Cavey, Laurie Overman; Kinzel, Margaret T.

2017-01-01

In this exploratory study, we examined the effects of a quantitative reasoning instructional approach to linear equations in two variables on community college students' conceptual understanding, procedural fluency, and reasoning ability. This was done in comparison to the use of a traditional procedural approach for instruction on the same topic.…

Quantitative and qualitative approaches in educational research — problems and examples of controlled understanding through interpretive methods

NASA Astrophysics Data System (ADS)

Neumann, Karl

1987-06-01

In the methodological discussion of recent years it has become apparent that many research problems, including problems relating to the theory of educational science, cannot be solved by using quantitative methods. The multifaceted aspects of human behaviour and all its environment-bound subtle nuances, especially the process of education or the development of identity, cannot fully be taken into account within a rigid neopositivist approach. In employing the paradigm of symbolic interactionism as a suitable model for the analysis of processes of education and formation, the research has generally to start out from complex reciprocal social interactions instead of unambigious connections of causes. In analysing several particular methodological problems, the article demonstrates some weaknesses of quantitative approaches and then shows the advantages in and the necessity for using qualitative research tools.

Computational modeling approaches to quantitative structure-binding kinetics relationships in drug discovery.

PubMed

De Benedetti, Pier G; Fanelli, Francesca

2018-03-21

Simple comparative correlation analyses and quantitative structure-kinetics relationship (QSKR) models highlight the interplay of kinetic rates and binding affinity as an essential feature in drug design and discovery. The choice of the molecular series, and their structural variations, used in QSKR modeling is fundamental to understanding the mechanistic implications of ligand and/or drug-target binding and/or unbinding processes. Here, we discuss the implications of linear correlations between kinetic rates and binding affinity constants and the relevance of the computational approaches to QSKR modeling. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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.

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

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

A Novel Approach to Teach the Generation of Bioelectrical Potentials from a Descriptive and Quantitative Perspective

ERIC Educational Resources Information Center

Rodriguez-Falces, Javier

2013-01-01

In electrophysiology studies, it is becoming increasingly common to explain experimental observations using both descriptive methods and quantitative approaches. However, some electrophysiological phenomena, such as the generation of extracellular potentials that results from the propagation of the excitation source along the muscle fiber, are…

A Quantitative Corpus-Based Approach to English Spatial Particles: Conceptual Symmetry and Its Pedagogical Implications

ERIC Educational Resources Information Center

Chen, Alvin Cheng-Hsien

2014-01-01

The present study aims to investigate how conceptual symmetry plays a role in the use of spatial particles in English and to further examine its pedagogical implications via a corpus-based evaluation of the course books in senior high schools in Taiwan. More specifically, we adopt a quantitative corpus-based approach to investigate whether bipolar…

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

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches

PubMed Central

Preuner, Sandra; Barna, Agnes; Frommlet, Florian; Czurda, Stefan; Konstantin, Byrgazov; Alikian, Mary; Machova Polakova, Katerina; Sacha, Tomasz; Richter, Johan; Lion, Thomas; Gabriel, Christian

2016-01-01

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points. PMID:27136541

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

PubMed

Preuner, Sandra; Barna, Agnes; Frommlet, Florian; Czurda, Stefan; Konstantin, Byrgazov; Alikian, Mary; Machova Polakova, Katerina; Sacha, Tomasz; Richter, Johan; Lion, Thomas; Gabriel, Christian

2016-04-29

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

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

[A novel quantitative approach to study dynamic anaerobic process at micro scale].

PubMed

Zhang, Zhong-Liang; Wu, Jing; Jiang, Jian-Kai; Jiang, Jie; Li, Huai-Zhi

2012-11-01

Anaerobic digestion is attracting more and more interests because of its advantages such as low cost and recovery of clean energy etc. In order to overcome the drawbacks of the existed methods to study the dynamic anaerobic process, a novel microscopical quantitative approach at the granule level was developed combining both the microdevice and the quantitative image analysis techniques. This experiment displayed the process and characteristics of the gas production at static state for the first time and the results indicated that the method was of satisfactory repeatability. The gas production process at static state could be divided into three stages including rapid linear increasing stage, decelerated increasing stage and slow linear increasing stage. The rapid linear increasing stage was long and the biogas rate was high under high initial organic loading rate. The results showed that it was feasible to make the anaerobic process to be carried out in the microdevice; furthermore this novel method was reliable and could clearly display the dynamic process of the anaerobic reaction at the micro scale. The results are helpful to understand the anaerobic process.

New approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Pohl, L.; Kaiser, M.; Ketelhut, S.; Pereira, S.; Goycoolea, F.; Kemper, Björn

2016-03-01

Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

Evaluating Attitudes, Skill, and Performance in a Learning-Enhanced Quantitative Methods Course: A Structural Modeling Approach.

ERIC Educational Resources Information Center

Harlow, Lisa L.; Burkholder, Gary J.; Morrow, Jennifer A.

2002-01-01

Used a structural modeling approach to evaluate relations among attitudes, initial skills, and performance in a Quantitative Methods course that involved students in active learning. Results largely confirmed hypotheses offering support for educational reform efforts that propose actively involving students in the learning process, especially in…

Integrated genomics and molecular breeding approaches for dissecting the complex quantitative traits in crop plants.

PubMed

Kujur, Alice; Saxena, Maneesha S; Bajaj, Deepak; Laxmi; Parida, Swarup K

2013-12-01

The enormous population growth, climate change and global warming are now considered major threats to agriculture and world's food security. To improve the productivity and sustainability of agriculture, the development of highyielding and durable abiotic and biotic stress-tolerant cultivars and/climate resilient crops is essential. Henceforth, understanding the molecular mechanism and dissection of complex quantitative yield and stress tolerance traits is the prime objective in current agricultural biotechnology research. In recent years, tremendous progress has been made in plant genomics and molecular breeding research pertaining to conventional and next-generation whole genome, transcriptome and epigenome sequencing efforts, generation of huge genomic, transcriptomic and epigenomic resources and development of modern genomics-assisted breeding approaches in diverse crop genotypes with contrasting yield and abiotic stress tolerance traits. Unfortunately, the detailed molecular mechanism and gene regulatory networks controlling such complex quantitative traits is not yet well understood in crop plants. Therefore, we propose an integrated strategies involving available enormous and diverse traditional and modern -omics (structural, functional, comparative and epigenomics) approaches/resources and genomics-assisted breeding methods which agricultural biotechnologist can adopt/utilize to dissect and decode the molecular and gene regulatory networks involved in the complex quantitative yield and stress tolerance traits in crop plants. This would provide clues and much needed inputs for rapid selection of novel functionally relevant molecular tags regulating such complex traits to expedite traditional and modern marker-assisted genetic enhancement studies in target crop species for developing high-yielding stress-tolerant varieties.

Modular Classification of Endoscopic Endonasal Transsphenoidal Approaches to Sellar Region: Anatomic Quantitative Study.

PubMed

Belotti, Francesco; Doglietto, Francesco; Schreiber, Alberto; Ravanelli, Marco; Ferrari, Marco; Lancini, Davide; Rampinelli, Vittorio; Hirtler, Lena; Buffoli, Barbara; Bolzoni Villaret, Andrea; Maroldi, Roberto; Rodella, Luigi Fabrizio; Nicolai, Piero; Fontanella, Marco Maria

2018-01-01

Endoscopic visualization does not necessarily correspond to an adequate working space. The need for balancing invasiveness and adequacy of sellar tumor exposure has recently led to the description of multiple endoscopic endonasal transsphenoidal approaches. Comparative anatomic data on these variants are lacking. We sought to quantitatively compare endoscopic endonasal transsphenoidal approaches to the sella and parasellar region, using the concept of "surgical pyramid." Four endoscopic transsphenoidal approaches were performed in 10 injected specimens: 1) hemisphenoidotomy; 2) transrostral; 3) extended transrostral (with superior turbinectomy); and 4) extended transrostral with posterior ethmoidectomy. ApproachViewer software (part of GTx-Eyes II, University Health Network, Toronto, Canada) with a dedicated navigation system was used to quantify the surgical pyramid volume, as well as exposure of sellar and parasellar areas. Statistical analyses were performed with Friedman's tests and Nemenyi's procedure. Hemisphenoidotomy provided limited exposure of the sellar area and a small working volume. A transrostral approach was necessary to expose the entire sella. Exposure of lateral parasellar areas required superior turbinectomy or posterior ethmoidectomy. The differences between each of the modules was statistically significant. The present study validates, from an anatomic point of view, a modular classification of endoscopic endonasal transsphenoidal approaches to the sellar region. Copyright © 2017 Elsevier Inc. All rights reserved.

The quantitation of buffering action II. Applications of the formal & general approach.

PubMed

Schmitt, Bernhard M

2005-03-16

The paradigm of "buffering" originated in acid-base physiology, but was subsequently extended to other fields and is now used for a wide and diverse set of phenomena. In the preceding article, we have presented a formal and general approach to the quantitation of buffering action. Here, we use that buffering concept for a systematic treatment of selected classical and other buffering phenomena. H+ buffering by weak acids and "self-buffering" in pure water represent "conservative buffered systems" whose analysis reveals buffering properties that contrast in important aspects from classical textbook descriptions. The buffering of organ perfusion in the face of variable perfusion pressure (also termed "autoregulation") can be treated in terms of "non-conservative buffered systems", the general form of the concept. For the analysis of cytoplasmic Ca++ concentration transients (also termed "muffling"), we develop a related unit that is able to faithfully reflect the time-dependent quantitative aspect of buffering during the pre-steady state period. Steady-state buffering is shown to represent the limiting case of time-dependent muffling, namely for infinitely long time intervals and infinitely small perturbations. Finally, our buffering concept provides a stringent definition of "buffering" on the level of systems and control theory, resulting in four absolute ratio scales for control performance that are suited to measure disturbance rejection and setpoint tracking, and both their static and dynamic aspects. Our concept of buffering provides a powerful mathematical tool for the quantitation of buffering action in all its appearances.

Study of O-Phosphorylation Sites in Proteins Involved in Photosynthesis-Related Processes in Synechocystis sp. Strain PCC 6803: Application of the SRM Approach.

PubMed

Angeleri, Martina; Muth-Pawlak, Dorota; Aro, Eva-Mari; Battchikova, Natalia

2016-12-02

O-Phosphorylation has been shown in photosynthesis-related proteins in a cyanobacterium Synechocystis sp. strain PCC 6803 (thereafter Synechocystis 6803), suggesting that phosphorylation of S, T, and Y residues might be important in photosynthesis-related processes. Investigation of biological roles of these phosphorylation events requires confident knowledge of the phosphorylated sites and prospects for their individual assessment. We performed phosphoproteomic analysis of Synechocystis 6803 using TiO 2 enrichment of the phosphopeptides, followed by LC-MS/MS, and discovered 367 phosphorylation sites in 190 proteins participating in various cellular functions. Furthermore, we focused on the large group of phosphoproteins that are involved in light harvesting, photosynthesis-driven electron flow, photoprotection, and CO 2 fixation. The SRM approach was applied to verify/improve assignments of phosphorylation sites in these proteins and to investigate possibilities for analysis of phosphopeptide isomers. The SRM assays were designed for peptides comprising 45 phosphorylation sites. The assays contain peptide iRT values and Q1/Q3 transitions comprising those discriminating between phosphopeptide isoforms. The majority of investigated phosphopeptides and phosphorylated isoforms could be individually assessed with the SRM technique. The assays could be potentially used in future quantitative studies to evaluate an extent of phosphorylation in photosynthesis-related proteins in Synechocystis 6803 cells challenged with various environmental stresses.

Qualitative and Quantitative Approaches to the Study of Poverty: Taming the Tensions and Appreciating the Complementarities

ERIC Educational Resources Information Center

Balarabe Kura, Sulaiman Y.

2012-01-01

There is a germane relationship between qualitative and quantitative approaches to social science research. The relationship is empirically and theoretically demonstrated by poverty researchers. The study of poverty, as argued in this article, is a study of both numbers and contextualities. This article provides a general overview of qualitative…

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems.

PubMed

Pereira, Rui P A; Peplies, Jörg; Brettar, Ingrid; Höfle, Manfred G

2017-03-31

Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples. Our results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella

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

Quantitative Surface Chirality Detection with Sum Frequency Generation Vibrational Spectroscopy: Twin Polarization Angle Approach

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

Wei, Feng; Xu, Yanyan; Guo, Yuan

2009-12-27

Here we report a novel twin polarization angle (TPA) approach in the quantitative chirality detection with the surface sum-frequency generation vibrational spectroscopy (SFG-VS). Generally, the achiral contribution dominates the surface SFG-VS signal, and the pure chiral signal is usually two or three orders of magnitude smaller. Therefore, it has been difficult to make quantitative detection and analysis of the chiral contributions to the surface SFG- VS signal. In the TPA method, by varying together the polarization angles of the incoming visible light and the sum frequency signal at fixed s or p polarization of the incoming infrared beam, the polarizationmore » dependent SFG signal can give not only direct signature of the chiral contribution in the total SFG-VS signal, but also the accurate measurement of the chiral and achiral components in the surface SFG signal. The general description of the TPA method is presented and the experiment test of the TPA approach is also presented for the SFG-VS from the S- and R-limonene chiral liquid surfaces. The most accurate degree of chiral excess values thus obtained for the 2878 cm⁻¹ spectral peak of the S- and R-limonene liquid surfaces are (23.7±0.4)% and ({25.4±1.3)%, respectively.« less

The Advantages and Disadvantages of Using Qualitative and Quantitative Approaches and Methods in Language "Testing and Assessment" Research: A Literature Review

ERIC Educational Resources Information Center

Rahman, Md Shidur

2017-01-01

The researchers of various disciplines often use qualitative and quantitative research methods and approaches for their studies. Some of these researchers like to be known as qualitative researchers; others like to be regarded as quantitative researchers. The researchers, thus, are sharply polarised; and they involve in a competition of pointing…

Testing process predictions of models of risky choice: a quantitative model comparison approach

PubMed Central

Pachur, Thorsten; Hertwig, Ralph; Gigerenzer, Gerd; Brandstätter, Eduard

2013-01-01

This article presents a quantitative model comparison contrasting the process predictions of two prominent views on risky choice. One view assumes a trade-off between probabilities and outcomes (or non-linear functions thereof) and the separate evaluation of risky options (expectation models). Another view assumes that risky choice is based on comparative evaluation, limited search, aspiration levels, and the forgoing of trade-offs (heuristic models). We derived quantitative process predictions for a generic expectation model and for a specific heuristic model, namely the priority heuristic (Brandstätter et al., 2006), and tested them in two experiments. The focus was on two key features of the cognitive process: acquisition frequencies (i.e., how frequently individual reasons are looked up) and direction of search (i.e., gamble-wise vs. reason-wise). In Experiment 1, the priority heuristic predicted direction of search better than the expectation model (although neither model predicted the acquisition process perfectly); acquisition frequencies, however, were inconsistent with both models. Additional analyses revealed that these frequencies were primarily a function of what Rubinstein (1988) called “similarity.” In Experiment 2, the quantitative model comparison approach showed that people seemed to rely more on the priority heuristic in difficult problems, but to make more trade-offs in easy problems. This finding suggests that risky choice may be based on a mental toolbox of strategies. PMID:24151472

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

Quantitative Approaches to Group Research: Suggestions for Best Practices

ERIC Educational Resources Information Center

McCarthy, Christopher J.; Whittaker, Tiffany A.; Boyle, Lauren H.; Eyal, Maytal

2017-01-01

Rigorous scholarship is essential to the continued growth of group work, yet the unique nature of this counseling specialty poses challenges for quantitative researchers. The purpose of this proposal is to overview unique challenges to quantitative research with groups in the counseling field, including difficulty in obtaining large sample sizes…

The FAQUIRE Approach: FAst, QUantitative, hIghly Resolved and sEnsitivity Enhanced 1H, 13C Data.

PubMed

Farjon, Jonathan; Milande, Clément; Martineau, Estelle; Akoka, Serge; Giraudeau, Patrick

2018-02-06

The targeted analysis of metabolites in complex mixtures is a challenging issue. NMR is one of the major tools in this field, but there is a strong need for more sensitive, better-resolved, and faster quantitative methods. In this framework, we introduce the concept of FAst, QUantitative, hIghly Resolved and sEnsitivity enhanced (FAQUIRE) NMR to push forward the limits of metabolite NMR analysis. 2D 1 H, 13 C 2D quantitative maps are promising alternatives for enhancing the spectral resolution but are highly time-consuming because of (i) the intrinsic nature of 2D, (ii) the longer recycling times required for quantitative conditions, and (iii) the higher number of scans needed to reduce the level of detection/quantification to access low concentrated metabolites. To reach this aim, speeding up the recently developed QUantItative Perfected and pUre shifted HSQC (QUIPU HSQC) is an interesting attempt to develop the FAQUIRE concept. Thanks to the combination of spectral aliasing, nonuniform sampling, and variable repetition time, the acquisition time of 2D quantitative maps is reduced by a factor 6 to 9, while conserving a high spectral resolution thanks to a pure shift approach. The analytical potential of the new Quick QUIPU HSQC (Q QUIPU HSQC) is evaluated on a model metabolite sample, and its potential is shown on breast-cell extracts embedding metabolites at millimolar to submillimolar concentrations.

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

An effective approach to quantitative analysis of ternary amino acids in foxtail millet substrate based on terahertz spectroscopy.

PubMed

Lu, Shao Hua; Li, Bao Qiong; Zhai, Hong Lin; Zhang, Xin; Zhang, Zhuo Yong

2018-04-25

Terahertz time-domain spectroscopy has been applied to many fields, however, it still encounters drawbacks in multicomponent mixtures analysis due to serious spectral overlapping. Here, an effective approach to quantitative analysis was proposed, and applied on the determination of the ternary amino acids in foxtail millet substrate. Utilizing three parameters derived from the THz-TDS, the images were constructed and the Tchebichef image moments were used to extract the information of target components. Then the quantitative models were obtained by stepwise regression. The correlation coefficients of leave-one-out cross-validation (R loo-cv 2 ) were more than 0.9595. As for external test set, the predictive correlation coefficients (R p 2 ) were more than 0.8026 and the root mean square error of prediction (RMSE p ) were less than 1.2601. Compared with the traditional methods (PLS and N-PLS methods), our approach is more accurate, robust and reliable, and can be a potential excellent approach to quantify multicomponent with THz-TDS spectroscopy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mixed qualitative and quantitative approach for validating an information booklet before total hip arthroplasty.

PubMed

Chabaud, Aurore; Eschalier, Bénédicte; Zullian, Myriam; Plan-Paquet, Anne; Aubreton, Sylvie; Saragaglia, Dominique; Descamps, Stéphane; Coudeyre, Emmanuel

2018-05-01

Providing patients with validated information before total hip arthroplasty may help lessen discrepancies between patients' expectations and the surgical result. This study sought to validate an information booklet for candidates for hip arthroplasty by using a mixed qualitative and quantitative approach based on a panel of patients and a sample of healthcare professionals. We developed a booklet in accordance with the standard methods and then conducted focus groups to collect the opinions of a sample of multidisciplinary experts involved in the care of patients with hip osteoarthritis. The number of focus groups and experts was determined according to the data saturation principle. A panel of patients awaiting hip arthroplasty or those in the immediate post-operative period assessed the booklet with self-reporting questionnaires (knowledge, beliefs, and expectations) and semi-structured interviews. All experts and both patient groups validated the booklet in terms of content and presentation. Semi-structured interviews were uninformative, especially for post-operative patients. Reading the booklet significantly (P<0.001) improved the knowledge scores in both groups, with no intergroup differences, but did not affect beliefs in either patient group. Only pre-operative patients significantly changed their expectations. Our mixed qualitative and quantitative approach allowed us to validate a booklet for patients awaiting hip arthroplasty, taking into account the opinions of both patients and healthcare professionals. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Comparative and Quantitative Global Proteomics Approaches: An Overview

PubMed Central

Deracinois, Barbara; Flahaut, Christophe; Duban-Deweer, Sophie; Karamanos, Yannis

2013-01-01

Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other. PMID:28250403

Quantitative and qualitative approaches to identifying migration chronology in a continental migrant

USGS Publications Warehouse

Beatty, William S.; Kesler, Dylan C.; Webb, Elisabeth B.; Raedeke, Andrew H.; Naylor, Luke W.; Humburg, Dale D.

2013-01-01

The degree to which extrinsic factors influence migration chronology in North American waterfowl has not been quantified, particularly for dabbling ducks. Previous studies have examined waterfowl migration using various methods, however, quantitative approaches to define avian migration chronology over broad spatio-temporal scales are limited, and the implications for using different approaches have not been assessed. We used movement data from 19 female adult mallards (Anas platyrhynchos) equipped with solar-powered global positioning system satellite transmitters to evaluate two individual level approaches for quantifying migration chronology. The first approach defined migration based on individual movements among geopolitical boundaries (state, provincial, international), whereas the second method modeled net displacement as a function of time using nonlinear models. Differences in migration chronologies identified by each of the approaches were examined with analysis of variance. The geopolitical method identified mean autumn migration midpoints at 15 November 2010 and 13 November 2011, whereas the net displacement method identified midpoints at 15 November 2010 and 14 November 2011. The mean midpoints for spring migration were 3 April 2011 and 20 March 2012 using the geopolitical method and 31 March 2011 and 22 March 2012 using the net displacement method. The duration, initiation date, midpoint, and termination date for both autumn and spring migration did not differ between the two individual level approaches. Although we did not detect differences in migration parameters between the different approaches, the net displacement metric offers broad potential to address questions in movement ecology for migrating species. Ultimately, an objective definition of migration chronology will allow researchers to obtain a comprehensive understanding of the extrinsic factors that drive migration at the individual and population levels. As a result, targeted

Quantitative and qualitative approaches to identifying migration chronology in a continental migrant.

PubMed

Beatty, William S; Kesler, Dylan C; Webb, Elisabeth B; Raedeke, Andrew H; Naylor, Luke W; Humburg, Dale D

2013-01-01

The degree to which extrinsic factors influence migration chronology in North American waterfowl has not been quantified, particularly for dabbling ducks. Previous studies have examined waterfowl migration using various methods, however, quantitative approaches to define avian migration chronology over broad spatio-temporal scales are limited, and the implications for using different approaches have not been assessed. We used movement data from 19 female adult mallards (Anas platyrhynchos) equipped with solar-powered global positioning system satellite transmitters to evaluate two individual level approaches for quantifying migration chronology. The first approach defined migration based on individual movements among geopolitical boundaries (state, provincial, international), whereas the second method modeled net displacement as a function of time using nonlinear models. Differences in migration chronologies identified by each of the approaches were examined with analysis of variance. The geopolitical method identified mean autumn migration midpoints at 15 November 2010 and 13 November 2011, whereas the net displacement method identified midpoints at 15 November 2010 and 14 November 2011. The mean midpoints for spring migration were 3 April 2011 and 20 March 2012 using the geopolitical method and 31 March 2011 and 22 March 2012 using the net displacement method. The duration, initiation date, midpoint, and termination date for both autumn and spring migration did not differ between the two individual level approaches. Although we did not detect differences in migration parameters between the different approaches, the net displacement metric offers broad potential to address questions in movement ecology for migrating species. Ultimately, an objective definition of migration chronology will allow researchers to obtain a comprehensive understanding of the extrinsic factors that drive migration at the individual and population levels. As a result, targeted

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.

A quantitative approach to developing Parkinsonian monkeys (Macaca fascicularis) with intracerebroventricular 1-methyl-4-phenylpyridinium injections.

PubMed

Li, Hao; Lei, Xiaoguang; Huang, Baihui; Rizak, Joshua D; Yang, Lichuan; Yang, Shangchuan; Wu, Jing; Lü, Longbao; Wang, Jianhong; Yan, Ting; Li, Hongwei; Wang, Zhengbo; Hu, Yingzhou; Le, Weidong; Deng, Xingli; Li, Jiali; Xu, Lin; Zhang, Baorong; Hu, Xintian

2015-08-15

Non-human primate Parkinson's disease (PD) models are essential for PD research. The most extensively used PD monkey models are induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the modeling processes of developing PD monkeys cannot be quantitatively controlled with MPTP. Therefore, a new approach to quantitatively develop chronic PD monkey models will help to advance the goals of "reduction, replacement and refinement" in animal experiments. A novel chronic PD monkey models was reported using the intracerebroventricular administration of 1-methyl-4-phenylpyridinium (MPP(+)) in Cynomolgus monkeys (Macaca fascicularis). This approach successfully produced stable and consistent PD monkeys with typical motor symptoms and pathological changes. More importantly, a sigmoidal relationship (Y=8.15801e(-0.245/x); R=0.73) was discovered between PD score (Y) and cumulative dose of MPP(+) (X). This relationship was then used to develop two additional PD monkeys under a specific time schedule (4 weeks), with planned PD scores (7) by controlling the dose and frequency of the MPP(+) administration as an independent validation of the formula. We developed Parkinsonian monkeys within controlled time frames by regulating the accumulated dose of MPP(+) intracerebroventricular administered, while limiting side effects often witnessed in models developed with the peripheral administration of MPTP, makes this model highly suitable for treatment development. This novel approach provides an edge in evaluating the mechanisms of PD pathology associated with environmental toxins and novel treatment approaches as the formula developed provides a "map" to control and predict the modeling processes. Copyright © 2015 Elsevier B.V. All rights reserved.

RGB color calibration for quantitative image analysis: the "3D thin-plate spline" warping approach.

PubMed

Menesatti, Paolo; Angelini, Claudio; Pallottino, Federico; Antonucci, Francesca; Aguzzi, Jacopo; Costa, Corrado

2012-01-01

In the last years the need to numerically define color by its coordinates in n-dimensional space has increased strongly. Colorimetric calibration is fundamental in food processing and other biological disciplines to quantitatively compare samples' color during workflow with many devices. Several software programmes are available to perform standardized colorimetric procedures, but they are often too imprecise for scientific purposes. In this study, we applied the Thin-Plate Spline interpolation algorithm to calibrate colours in sRGB space (the corresponding Matlab code is reported in the Appendix). This was compared with other two approaches. The first is based on a commercial calibration system (ProfileMaker) and the second on a Partial Least Square analysis. Moreover, to explore device variability and resolution two different cameras were adopted and for each sensor, three consecutive pictures were acquired under four different light conditions. According to our results, the Thin-Plate Spline approach reported a very high efficiency of calibration allowing the possibility to create a revolution in the in-field applicative context of colour quantification not only in food sciences, but also in other biological disciplines. These results are of great importance for scientific color evaluation when lighting conditions are not controlled. Moreover, it allows the use of low cost instruments while still returning scientifically sound quantitative data.

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.

Navigational Traffic Conflict Technique: A Proactive Approach to Quantitative Measurement of Collision Risks in Port Waters

NASA Astrophysics Data System (ADS)

Debnath, Ashim Kumar; Chin, Hoong Chor

Navigational safety analysis relying on collision statistics is often hampered because of the low number of observations. A promising alternative approach that overcomes this problem is proposed in this paper. By analyzing critical vessel interactions this approach proactively measures collision risk in port waters. The proposed method is illustrated for quantitative measurement of collision risks in Singapore port fairways, and validated by examining correlations between the measured risks with those perceived by pilots. This method is an ethically appealing alternative to the collision-based analysis for fast, reliable and effective safety assessment, thus possessing great potential for managing collision risks in port waters.

Quantitative analysis of breast cancer diagnosis using a probabilistic modelling approach.

PubMed

Liu, Shuo; Zeng, Jinshu; Gong, Huizhou; Yang, Hongqin; Zhai, Jia; Cao, Yi; Liu, Junxiu; Luo, Yuling; Li, Yuhua; Maguire, Liam; Ding, Xuemei

2018-01-01

Breast cancer is the most prevalent cancer in women in most countries of the world. Many computer-aided diagnostic methods have been proposed, but there are few studies on quantitative discovery of probabilistic dependencies among breast cancer data features and identification of the contribution of each feature to breast cancer diagnosis. This study aims to fill this void by utilizing a Bayesian network (BN) modelling approach. A K2 learning algorithm and statistical computation methods are used to construct BN structure and assess the obtained BN model. The data used in this study were collected from a clinical ultrasound dataset derived from a Chinese local hospital and a fine-needle aspiration cytology (FNAC) dataset from UCI machine learning repository. Our study suggested that, in terms of ultrasound data, cell shape is the most significant feature for breast cancer diagnosis, and the resistance index presents a strong probabilistic dependency on blood signals. With respect to FNAC data, bare nuclei are the most important discriminating feature of malignant and benign breast tumours, and uniformity of both cell size and cell shape are tightly interdependent. The BN modelling approach can support clinicians in making diagnostic decisions based on the significant features identified by the model, especially when some other features are missing for specific patients. The approach is also applicable to other healthcare data analytics and data modelling for disease diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protocol for Standardizing High-to-Moderate Abundance Protein Biomarker Assessments Through an MRM-with-Standard-Peptides Quantitative Approach.

PubMed

Percy, Andrew J; Yang, Juncong; Chambers, Andrew G; Mohammed, Yassene; Miliotis, Tasso; Borchers, Christoph H

2016-01-01

Quantitative mass spectrometry (MS)-based approaches are emerging as a core technology for addressing health-related queries in systems biology and in the biomedical and clinical fields. In several 'omics disciplines (proteomics included), an approach centered on selected or multiple reaction monitoring (SRM or MRM)-MS with stable isotope-labeled standards (SIS), at the protein or peptide level, has emerged as the most precise technique for quantifying and screening putative analytes in biological samples. To enable the widespread use of MRM-based protein quantitation for disease biomarker assessment studies and its ultimate acceptance for clinical analysis, the technique must be standardized to facilitate precise and accurate protein quantitation. To that end, we have developed a number of kits for assessing method/platform performance, as well as for screening proposed candidate protein biomarkers in various human biofluids. Collectively, these kits utilize a bottom-up LC-MS methodology with SIS peptides as internal standards and quantify proteins using regression analysis of standard curves. This chapter details the methodology used to quantify 192 plasma proteins of high-to-moderate abundance (covers a 6 order of magnitude range from 31 mg/mL for albumin to 18 ng/mL for peroxidredoxin-2), and a 21-protein subset thereof. We also describe the application of this method to patient samples for biomarker discovery and verification studies. Additionally, we introduce our recently developed Qualis-SIS software, which is used to expedite the analysis and assessment of protein quantitation data in control and patient samples.

Molecule kernels: a descriptor- and alignment-free quantitative structure-activity relationship approach.

PubMed

Mohr, Johannes A; Jain, Brijnesh J; Obermayer, Klaus

2008-09-01

Quantitative structure activity relationship (QSAR) analysis is traditionally based on extracting a set of molecular descriptors and using them to build a predictive model. In this work, we propose a QSAR approach based directly on the similarity between the 3D structures of a set of molecules measured by a so-called molecule kernel, which is independent of the spatial prealignment of the compounds. Predictors can be build using the molecule kernel in conjunction with the potential support vector machine (P-SVM), a recently proposed machine learning method for dyadic data. The resulting models make direct use of the structural similarities between the compounds in the test set and a subset of the training set and do not require an explicit descriptor construction. We evaluated the predictive performance of the proposed method on one classification and four regression QSAR datasets and compared its results to the results reported in the literature for several state-of-the-art descriptor-based and 3D QSAR approaches. In this comparison, the proposed molecule kernel method performed better than the other QSAR methods.

A quantitative approach to assessing the efficacy of occupant protection programs: A case study from Montana.

PubMed

Manlove, Kezia; Stanley, Laura; Peck, Alyssa

2015-10-01

Quantitative evaluation of vehicle occupant protection programs is critical for ensuring efficient government resource allocation, but few methods exist for conducting evaluation across multiple programs simultaneously. Here we present an analysis of occupant protection efficacy in the state of Montana. This approach relies on seat belt compliance rates as measured by the National Occupant Protection Usage Survey (NOPUS). A hierarchical logistic regression model is used to estimate the impacts of four Montana Department of Transportation (MDT)-funded occupant protection programs used in the state of Montana, following adjustment for a suite of potential confounders. Activity from two programs, Buckle Up coalitions and media campaigns, are associated with increased seat belt use in Montana, whereas the impact of another program, Selective Traffic Enforcement, is potentially masked by other program activity. A final program, Driver's Education, is not associated with any shift in seat belt use. This method allows for a preliminary quantitative estimation of program impacts without requiring states to obtain any new seat belt use data. This approach provides states a preliminary look at program impacts, and a means for carefully planning future program allocation and investigation. Copyright © 2015 Elsevier Ltd. All rights reserved.

The ACCE method: an approach for obtaining quantitative or qualitative estimates of residual confounding that includes unmeasured confounding

PubMed Central

Smith, Eric G.

2015-01-01

Background:  Nonrandomized studies typically cannot account for confounding from unmeasured factors.  Method:  A method is presented that exploits the recently-identified phenomenon of  “confounding amplification” to produce, in principle, a quantitative estimate of total residual confounding resulting from both measured and unmeasured factors.  Two nested propensity score models are constructed that differ only in the deliberate introduction of an additional variable(s) that substantially predicts treatment exposure.  Residual confounding is then estimated by dividing the change in treatment effect estimate between models by the degree of confounding amplification estimated to occur, adjusting for any association between the additional variable(s) and outcome. Results:  Several hypothetical examples are provided to illustrate how the method produces a quantitative estimate of residual confounding if the method’s requirements and assumptions are met.  Previously published data is used to illustrate that, whether or not the method routinely provides precise quantitative estimates of residual confounding, the method appears to produce a valuable qualitative estimate of the likely direction and general size of residual confounding. Limitations:  Uncertainties exist, including identifying the best approaches for: 1) predicting the amount of confounding amplification, 2) minimizing changes between the nested models unrelated to confounding amplification, 3) adjusting for the association of the introduced variable(s) with outcome, and 4) deriving confidence intervals for the method’s estimates (although bootstrapping is one plausible approach). Conclusions:  To this author’s knowledge, it has not been previously suggested that the phenomenon of confounding amplification, if such amplification is as predictable as suggested by a recent simulation, provides a logical basis for estimating total residual confounding. The method's basic approach is

A Quantitative and Novel Approach to the Prioritization of Zoonotic Diseases in North America: A Public Perspective

PubMed Central

Ng, Victoria; Sargeant, Jan M.

2012-01-01

Background Zoonoses account for over half of all communicable diseases causing illness in humans. As there are limited resources available for the control and prevention of zoonotic diseases, a framework for their prioritization is necessary to ensure resources are directed into those of highest importance. Although zoonotic outbreaks are a significant burden of disease in North America, the systematic prioritization of zoonoses in this region has not been previously evaluated. Methodology/Principal Findings This study describes the novel use of a well-established quantitative method, conjoint analysis (CA), to identify the relative importance of 21 key characteristics of zoonotic diseases that can be used for their prioritization in Canada and the US. Relative importance weights from the CA were used to develop a point-scoring system to derive a recommended list of zoonoses for prioritization in Canada and the US. Over 1,500 participants from the general public were recruited to complete the online survey (761 from Canada and 778 from the US). Hierarchical Bayes models were fitted to the survey data to derive CA-weighted scores. Scores were applied to 62 zoonotic diseases of public health importance in Canada and the US to rank diseases in order of priority. Conclusions/Significance This was the first study to describe a systematic and quantitative approach to the prioritization of zoonoses in North America involving public participants. We found individuals with no prior knowledge or experience in prioritizing zoonoses were capable of producing meaningful results using CA as a novel quantitative approach to prioritization. More similarities than differences were observed between countries suggesting general agreement in disease prioritization between Canadians and Americans. We demonstrate CA as a potential tool for the prioritization of zoonoses; other prioritization exercises may also consider this approach. PMID:23133639

An adaptive model approach for quantitative wrist rigidity evaluation during deep brain stimulation surgery.

PubMed

Assis, Sofia; Costa, Pedro; Rosas, Maria Jose; Vaz, Rui; Silva Cunha, Joao Paulo

2016-08-01

Intraoperative evaluation of the efficacy of Deep Brain Stimulation includes evaluation of the effect on rigidity. A subjective semi-quantitative scale is used, dependent on the examiner perception and experience. A system was proposed previously, aiming to tackle this subjectivity, using quantitative data and providing real-time feedback of the computed rigidity reduction, hence supporting the physician decision. This system comprised of a gyroscope-based motion sensor in a textile band, placed in the patients hand, which communicated its measurements to a laptop. The latter computed a signal descriptor from the angular velocity of the hand during wrist flexion in DBS surgery. The first approach relied on using a general rigidity reduction model, regardless of the initial severity of the symptom. Thus, to enhance the performance of the previously presented system, we aimed to develop models for high and low baseline rigidity, according to the examiner assessment before any stimulation. This would allow a more patient-oriented approach. Additionally, usability was improved by having in situ processing in a smartphone, instead of a computer. Such system has shown to be reliable, presenting an accuracy of 82.0% and a mean error of 3.4%. Relatively to previous results, the performance was similar, further supporting the importance of considering the cogwheel rigidity to better infer about the reduction in rigidity. Overall, we present a simple, wearable, mobile system, suitable for intra-operatory conditions during DBS, supporting a physician in decision-making when setting stimulation parameters.

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

PubMed

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

A Quantitative Systems Pharmacology Approach to Infer Pathways Involved in Complex Disease Phenotypes.

PubMed

Schurdak, Mark E; Pei, Fen; Lezon, Timothy R; Carlisle, Diane; Friedlander, Robert; Taylor, D Lansing; Stern, Andrew M

2018-01-01

Designing effective therapeutic strategies for complex diseases such as cancer and neurodegeneration that involve tissue context-specific interactions among multiple gene products presents a major challenge for precision medicine. Safe and selective pharmacological modulation of individual molecular entities associated with a disease often fails to provide efficacy in the clinic. Thus, development of optimized therapeutic strategies for individual patients with complex diseases requires a more comprehensive, systems-level understanding of disease progression. Quantitative systems pharmacology (QSP) is an approach to drug discovery that integrates computational and experimental methods to understand the molecular pathogenesis of a disease at the systems level more completely. Described here is the chemogenomic component of QSP for the inference of biological pathways involved in the modulation of the disease phenotype. The approach involves testing sets of compounds of diverse mechanisms of action in a disease-relevant phenotypic assay, and using the mechanistic information known for the active compounds, to infer pathways and networks associated with the phenotype. The example used here is for monogenic Huntington's disease (HD), which due to the pleiotropic nature of the mutant phenotype has a complex pathogenesis. The overall approach, however, is applicable to any complex disease.

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

Flexible automated approach for quantitative liquid handling of complex biological samples.

PubMed

Palandra, Joe; Weller, David; Hudson, Gary; Li, Jeff; Osgood, Sarah; Hudson, Emily; Zhong, Min; Buchholz, Lisa; Cohen, Lucinda H

2007-11-01

A fully automated protein precipitation technique for biological sample preparation has been developed for the quantitation of drugs in various biological matrixes. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and controls from a Watson laboratory information management system generated work list, shaking of 96-well plates, and vacuum application. Processing time is less than 30 s per sample or approximately 45 min per 96-well plate, which is then immediately ready for injection onto an LC-MS/MS system. An overview of the process workflow is discussed, including the software development. Validation data are also provided, including specific liquid class data as well as comparative data of automated vs manual preparation using both quality controls and actual sample data. The efficiencies gained from this automated approach are described.

Developing patient-centered treatment protocols in brain stimulation: a rationale for combining quantitative and qualitative approaches in persons with HIV.

PubMed

Rosedale, Mary; Malaspina, Dolores; Malamud, Daniel; Strauss, Shiela M; Horne, Jaclyn D; Abouzied, Salman; Cruciani, Ricardo A; Knotkova, Helena

2012-01-01

This article reports and discusses how quantitative (physiological and behavioral) and qualitative methods are being combined in an open-label pilot feasibility study. The study evaluates safety, tolerability, and acceptability of a protocol to treat depression in HIV-infected individuals, using a 2-week block of transcranial direct current stimulation (tDCS) over the dorsolateral prefrontal cortex. Major depressive disorder (MDD) is the second most prevalent psychiatric disorder after substance abuse among HIV-positive adults, and novel antidepressant treatments are needed for this vulnerable population. The authors describe the challenges and contributions derived from different research perspectives and methodological approaches and provide a philosophical framework for combining quantitative and qualitative measurements for a fuller examination of the disorder. Four methodological points are presented: (1) the value of combining quantitative and qualitative approaches; (2) the need for context-specific measures when studying patients with medical and psychiatric comorbidities; (3) the importance of research designs that integrate physiological, behavioral, and qualitative approaches when evaluating novel treatments; and (4) the need to explore the relationships between biomarkers, clinical symptom assessments, patient self-evaluations, and patient experiences when developing new, patient-centered protocols. The authors conclude that the complexity of studying novel treatments in complex and new patient populations requires complex research designs to capture the richness of data that inform translational research.

Quantitative DIC microscopy using an off-axis self-interference approach.

PubMed

Fu, Dan; Oh, Seungeun; Choi, Wonshik; Yamauchi, Toyohiko; Dorn, August; Yaqoob, Zahid; Dasari, Ramachandra R; Feld, Michael S

2010-07-15

Traditional Normarski differential interference contrast (DIC) microscopy is a very powerful method for imaging nonstained biological samples. However, one of its major limitations is the nonquantitative nature of the imaging. To overcome this problem, we developed a quantitative DIC microscopy method based on off-axis sample self-interference. The digital holography algorithm is applied to obtain quantitative phase gradients in orthogonal directions, which leads to a quantitative phase image through a spiral integration of the phase gradients. This method is practically simple to implement on any standard microscope without stringent requirements on polarization optics. Optical sectioning can be obtained through enlarged illumination NA.

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.

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.

An analytical approach based on ESI-MS, LC-MS and PCA for the quali-quantitative analysis of cycloartane derivatives in Astragalus spp.

PubMed

Napolitano, Assunta; Akay, Seref; Mari, Angela; Bedir, Erdal; Pizza, Cosimo; Piacente, Sonia

2013-11-01

Astragalus species are widely used as health foods and dietary supplements, as well as drugs in traditional medicine. To rapidly evaluate metabolite similarities and differences among the EtOH extracts of the roots of eight commercial Astragalus spp., an approach based on direct analyses by ESI-MS followed by PCA of ESI-MS data, was carried out. Successively, quali-quantitative analyses of cycloartane derivatives in the eight Astragalus spp. by LC-ESI-MS(n) and PCA of LC-ESI-MS data were performed. This approach allowed to promptly highlighting metabolite similarities and differences among the various Astragalus spp. PCA results from LC-ESI-MS data of Astragalus samples were in reasonable agreement with both PCA results of ESI-MS data and quantitative results. This study affords an analytical method for the quali-quantitative determination of cycloartane derivatives in herbal preparations used as health and food supplements. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantitative approach for optimizing e-beam condition of photoresist inspection and measurement

NASA Astrophysics Data System (ADS)

Lin, Chia-Jen; Teng, Chia-Hao; Cheng, Po-Chung; Sato, Yoshishige; Huang, Shang-Chieh; Chen, Chu-En; Maruyama, Kotaro; Yamazaki, Yuichiro

2018-03-01

Severe process margin in advanced technology node of semiconductor device is controlled by e-beam metrology system and e-beam inspection system with scanning electron microscopy (SEM) image. By using SEM, larger area image with higher image quality is required to collect massive amount of data for metrology and to detect defect in a large area for inspection. Although photoresist is the one of the critical process in semiconductor device manufacturing, observing photoresist pattern by SEM image is crucial and troublesome especially in the case of large image. The charging effect by e-beam irradiation on photoresist pattern causes deterioration of image quality, and it affect CD variation on metrology system and causes difficulties to continue defect inspection in a long time for a large area. In this study, we established a quantitative approach for optimizing e-beam condition with "Die to Database" algorithm of NGR3500 on photoresist pattern to minimize charging effect. And we enhanced the performance of measurement and inspection on photoresist pattern by using optimized e-beam condition. NGR3500 is the geometry verification system based on "Die to Database" algorithm which compares SEM image with design data [1]. By comparing SEM image and design data, key performance indicator (KPI) of SEM image such as "Sharpness", "S/N", "Gray level variation in FOV", "Image shift" can be retrieved. These KPIs were analyzed with different e-beam conditions which consist of "Landing Energy", "Probe Current", "Scanning Speed" and "Scanning Method", and the best e-beam condition could be achieved with maximum image quality, maximum scanning speed and minimum image shift. On this quantitative approach of optimizing e-beam condition, we could observe dependency of SEM condition on photoresist charging. By using optimized e-beam condition, measurement could be continued on photoresist pattern over 24 hours stably. KPIs of SEM image proved image quality during measurement and

Framework for quantitative evaluation of 3D vessel segmentation approaches using vascular phantoms in conjunction with 3D landmark localization and registration

NASA Astrophysics Data System (ADS)

Wörz, Stefan; Hoegen, Philipp; Liao, Wei; Müller-Eschner, Matthias; Kauczor, Hans-Ulrich; von Tengg-Kobligk, Hendrik; Rohr, Karl

2016-03-01

We introduce a framework for quantitative evaluation of 3D vessel segmentation approaches using vascular phantoms. Phantoms are designed using a CAD system and created with a 3D printer, and comprise realistic shapes including branches and pathologies such as abdominal aortic aneurysms (AAA). To transfer ground truth information to the 3D image coordinate system, we use a landmark-based registration scheme utilizing fiducial markers integrated in the phantom design. For accurate 3D localization of the markers we developed a novel 3D parametric intensity model that is directly fitted to the markers in the images. We also performed a quantitative evaluation of different vessel segmentation approaches for a phantom of an AAA.

A synchrotron-based local computed tomography combined with data-constrained modelling approach for quantitative analysis of anthracite coal microstructure

PubMed Central

Chen, Wen Hao; Yang, Sam Y. S.; Xiao, Ti Qiao; Mayo, Sherry C.; Wang, Yu Dan; Wang, Hai Peng

2014-01-01

Quantifying three-dimensional spatial distributions of pores and material compositions in samples is a key materials characterization challenge, particularly in samples where compositions are distributed across a range of length scales, and where such compositions have similar X-ray absorption properties, such as in coal. Consequently, obtaining detailed information within sub-regions of a multi-length-scale sample by conventional approaches may not provide the resolution and level of detail one might desire. Herein, an approach for quantitative high-definition determination of material compositions from X-ray local computed tomography combined with a data-constrained modelling method is proposed. The approach is capable of dramatically improving the spatial resolution and enabling finer details within a region of interest of a sample larger than the field of view to be revealed than by using conventional techniques. A coal sample containing distributions of porosity and several mineral compositions is employed to demonstrate the approach. The optimal experimental parameters are pre-analyzed. The quantitative results demonstrated that the approach can reveal significantly finer details of compositional distributions in the sample region of interest. The elevated spatial resolution is crucial for coal-bed methane reservoir evaluation and understanding the transformation of the minerals during coal processing. The method is generic and can be applied for three-dimensional compositional characterization of other materials. PMID:24763649

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.

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.

Quantitative glycomics.

PubMed

Orlando, Ron

2010-01-01

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

Olfaction preservation after removal of large tuberculum sellae meningiomas via a superior interhemispheric approach. A quantitative and qualitative study.

PubMed

Hannequin, P; Paviot, A; Chaussy, O; Gilard, V; Cébula, H; Marie, J-P; Proust, F

2015-10-01

We present a prospective series of tuberculum sellae meningioma (TSM) resected via a superior interhemispheric (IH) approach in 10 patients who preoperatively and postoperatively underwent extensive olfaction testing using a standardised test battery. This prospective longitudinal study evaluated the olfactory function after TSM resection. The resection was performed via a superior interhemispheric (IH) approach. The quantitative and qualitative analyses of the olfactory function were assessed with the Biolfa(®) olfactory test (at 6 months). Between November 2009 and April 2012, 10 consecutive patients with symptomatic TSM and preserved olfactory function were operated via a superior IH approach. For the self-evaluation criteria of the olfactory function, the mean preoperative visual analog scale score was 8.8. The mean preoperative total quantitative (/27) scored 18.2 ± 6.3 for this cohort of 10 patients. In the postoperative period, the mean total score decreased non-significantly to 15.8 ± 8.8 (Wilcoxon test, P = 0.085). The mean preoperative qualitative score (/8) was 5.5 ± 1.7 and in the postoperative period decreased, non-significantly, to 4.7 ± 2.6 (Wilcoxon test, P = 0.12). The olfactory function was quantitatively and qualitatively preserved in 6 patients (60%), but a postoperative deterioration occurred in 2 (20%) and an anosmia in 2 (20%). Size and invasive characteristics of the meningioma determined the post-surgical deterioration. Olfaction is an important factor of emotional and social life, which needs to be integrated into the challenge regarding the resection of TSM. The risks of nerve damage are reviewed. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Conventional liquid chromatography/triple quadrupole mass spectrometer-based metabolite identification and semi-quantitative estimation approach in the investigation of dabigatran etexilate in vitro metabolism

PubMed Central

Hu, Zhe-Yi; Parker, Robert B.; Herring, Vanessa L.; Laizure, S. Casey

2012-01-01

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometer (LC-MS/MS)-based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor-product ion pairs were predicted, to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects of tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using ‘metabolite standards’ generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 convert DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 (or M2) was further metabolized to DAB by CES2 (or CES1). The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples. PMID:23239178

The mathematics of cancer: integrating quantitative models.

PubMed

Altrock, Philipp M; Liu, Lin L; Michor, Franziska

2015-12-01

Mathematical modelling approaches have become increasingly abundant in cancer research. The complexity of cancer is well suited to quantitative approaches as it provides challenges and opportunities for new developments. In turn, mathematical modelling contributes to cancer research by helping to elucidate mechanisms and by providing quantitative predictions that can be validated. The recent expansion of quantitative models addresses many questions regarding tumour initiation, progression and metastases as well as intra-tumour heterogeneity, treatment responses and resistance. Mathematical models can complement experimental and clinical studies, but also challenge current paradigms, redefine our understanding of mechanisms driving tumorigenesis and shape future research in cancer biology.

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.

RGB Color Calibration for Quantitative Image Analysis: The “3D Thin-Plate Spline” Warping Approach

PubMed Central

Menesatti, Paolo; Angelini, Claudio; Pallottino, Federico; Antonucci, Francesca; Aguzzi, Jacopo; Costa, Corrado

2012-01-01

In the last years the need to numerically define color by its coordinates in n-dimensional space has increased strongly. Colorimetric calibration is fundamental in food processing and other biological disciplines to quantitatively compare samples' color during workflow with many devices. Several software programmes are available to perform standardized colorimetric procedures, but they are often too imprecise for scientific purposes. In this study, we applied the Thin-Plate Spline interpolation algorithm to calibrate colours in sRGB space (the corresponding Matlab code is reported in the Appendix). This was compared with other two approaches. The first is based on a commercial calibration system (ProfileMaker) and the second on a Partial Least Square analysis. Moreover, to explore device variability and resolution two different cameras were adopted and for each sensor, three consecutive pictures were acquired under four different light conditions. According to our results, the Thin-Plate Spline approach reported a very high efficiency of calibration allowing the possibility to create a revolution in the in-field applicative context of colour quantification not only in food sciences, but also in other biological disciplines. These results are of great importance for scientific color evaluation when lighting conditions are not controlled. Moreover, it allows the use of low cost instruments while still returning scientifically sound quantitative data. PMID:22969337

Quantitative traits and diversification.

PubMed

FitzJohn, Richard G

2010-12-01

Quantitative traits have long been hypothesized to affect speciation and extinction rates. For example, smaller body size or increased specialization may be associated with increased rates of diversification. Here, I present a phylogenetic likelihood-based method (quantitative state speciation and extinction [QuaSSE]) that can be used to test such hypotheses using extant character distributions. This approach assumes that diversification follows a birth-death process where speciation and extinction rates may vary with one or more traits that evolve under a diffusion model. Speciation and extinction rates may be arbitrary functions of the character state, allowing much flexibility in testing models of trait-dependent diversification. I test the approach using simulated phylogenies and show that a known relationship between speciation and a quantitative character could be recovered in up to 80% of the cases on large trees (500 species). Consistent with other approaches, detecting shifts in diversification due to differences in extinction rates was harder than when due to differences in speciation rates. Finally, I demonstrate the application of QuaSSE to investigate the correlation between body size and diversification in primates, concluding that clade-specific differences in diversification may be more important than size-dependent diversification in shaping the patterns of diversity within this group.

Approaches to advancing quantitative human health risk assessment of environmental chemicals in the post-genomic era.

PubMed

Chiu, Weihsueh A; Euling, Susan Y; Scott, Cheryl Siegel; Subramaniam, Ravi P

2013-09-15

The contribution of genomics and associated technologies to human health risk assessment for environmental chemicals has focused largely on elucidating mechanisms of toxicity, as discussed in other articles in this issue. However, there is interest in moving beyond hazard characterization to making more direct impacts on quantitative risk assessment (QRA)--i.e., the determination of toxicity values for setting exposure standards and cleanup values. We propose that the evolution of QRA of environmental chemicals in the post-genomic era will involve three, somewhat overlapping phases in which different types of approaches begin to mature. The initial focus (in Phase I) has been and continues to be on "augmentation" of weight of evidence--using genomic and related technologies qualitatively to increase the confidence in and scientific basis of the results of QRA. Efforts aimed towards "integration" of these data with traditional animal-based approaches, in particular quantitative predictors, or surrogates, for the in vivo toxicity data to which they have been anchored are just beginning to be explored now (in Phase II). In parallel, there is a recognized need for "expansion" of the use of established biomarkers of susceptibility or risk of human diseases and disorders for QRA, particularly for addressing the issues of cumulative assessment and population risk. Ultimately (in Phase III), substantial further advances could be realized by the development of novel molecular and pathway-based biomarkers and statistical and in silico models that build on anticipated progress in understanding the pathways of human diseases and disorders. Such efforts would facilitate a gradual "reorientation" of QRA towards approaches that more directly link environmental exposures to human outcomes. Published by Elsevier Inc.

A spectral approach for the quantitative description of cardiac collagen network from nonlinear optical imaging.

PubMed

Masè, Michela; Cristoforetti, Alessandro; Avogaro, Laura; Tessarolo, Francesco; Piccoli, Federico; Caola, Iole; Pederzolli, Carlo; Graffigna, Angelo; Ravelli, Flavia

2015-01-01

The assessment of collagen structure in cardiac pathology, such as atrial fibrillation (AF), is essential for a complete understanding of the disease. This paper introduces a novel methodology for the quantitative description of collagen network properties, based on the combination of nonlinear optical microscopy with a spectral approach of image processing and analysis. Second-harmonic generation (SHG) microscopy was applied to atrial tissue samples from cardiac surgery patients, providing label-free, selective visualization of the collagen structure. The spectral analysis framework, based on 2D-FFT, was applied to the SHG images, yielding a multiparametric description of collagen fiber orientation (angle and anisotropy indexes) and texture scale (dominant wavelength and peak dispersion indexes). The proof-of-concept application of the methodology showed the capability of our approach to detect and quantify differences in the structural properties of the collagen network in AF versus sinus rhythm patients. These results suggest the potential of our approach in the assessment of collagen properties in cardiac pathologies related to a fibrotic structural component.

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

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.

A Novel Quantitative Approach to Concept Analysis: The Internomological Network

PubMed Central

Cook, Paul F.; Larsen, Kai R.; Sakraida, Teresa J.; Pedro, Leli

2012-01-01

Background When a construct such as patients’ transition to self-management of chronic illness is studied by researchers across multiple disciplines, the meaning of key terms can become confused. This results from inherent problems in language where a term can have multiple meanings (polysemy) and different words can mean the same thing (synonymy). Objectives To test a novel quantitative method for clarifying the meaning of constructs by examining the similarity of published contexts in which they are used. Method Published terms related to the concept transition to self-management of chronic illness were analyzed using the internomological network (INN), a type of latent semantic analysis to calculate the mathematical relationships between constructs based on the contexts in which researchers use each term. This novel approach was tested by comparing results to those from concept analysis, a best-practice qualitative approach to clarifying meanings of terms. By comparing results of the two methods, the best synonyms of transition to self-management, as well as key antecedent, attribute, and consequence terms, were identified. Results Results from INN analysis were consistent with those from concept analysis. The potential synonyms self-management, transition, and adaptation had the greatest utility. Adaptation was the clearest overall synonym, but had lower cross-disciplinary use. The terms coping and readiness had more circumscribed meanings. The INN analysis confirmed key features of transition to self-management, and suggested related concepts not found by the previous review. Discussion The INN analysis is a promising novel methodology that allows researchers to quantify the semantic relationships between constructs. The method works across disciplinary boundaries, and may help to integrate the diverse literature on self-management of chronic illness. PMID:22592387

Multivariate calibration in Laser-Induced Breakdown Spectroscopy quantitative analysis: The dangers of a 'black box' approach and how to avoid them

NASA Astrophysics Data System (ADS)

Safi, A.; Campanella, B.; Grifoni, E.; Legnaioli, S.; Lorenzetti, G.; Pagnotta, S.; Poggialini, F.; Ripoll-Seguer, L.; Hidalgo, M.; Palleschi, V.

2018-06-01

The introduction of multivariate calibration curve approach in Laser-Induced Breakdown Spectroscopy (LIBS) quantitative analysis has led to a general improvement of the LIBS analytical performances, since a multivariate approach allows to exploit the redundancy of elemental information that are typically present in a LIBS spectrum. Software packages implementing multivariate methods are available in the most diffused commercial and open source analytical programs; in most of the cases, the multivariate algorithms are robust against noise and operate in unsupervised mode. The reverse of the coin of the availability and ease of use of such packages is the (perceived) difficulty in assessing the reliability of the results obtained which often leads to the consideration of the multivariate algorithms as 'black boxes' whose inner mechanism is supposed to remain hidden to the user. In this paper, we will discuss the dangers of a 'black box' approach in LIBS multivariate analysis, and will discuss how to overcome them using the chemical-physical knowledge that is at the base of any LIBS quantitative analysis.

A Semiparametric Approach for Composite Functional Mapping of Dynamic Quantitative Traits

PubMed Central

Yang, Runqing; Gao, Huijiang; Wang, Xin; Zhang, Ji; Zeng, Zhao-Bang; Wu, Rongling

2007-01-01

Functional mapping has emerged as a powerful tool for mapping quantitative trait loci (QTL) that control developmental patterns of complex dynamic traits. Original functional mapping has been constructed within the context of simple interval mapping, without consideration of separate multiple linked QTL for a dynamic trait. In this article, we present a statistical framework for mapping QTL that affect dynamic traits by capitalizing on the strengths of functional mapping and composite interval mapping. Within this so-called composite functional-mapping framework, functional mapping models the time-dependent genetic effects of a QTL tested within a marker interval using a biologically meaningful parametric function, whereas composite interval mapping models the time-dependent genetic effects of the markers outside the test interval to control the genome background using a flexible nonparametric approach based on Legendre polynomials. Such a semiparametric framework was formulated by a maximum-likelihood model and implemented with the EM algorithm, allowing for the estimation and the test of the mathematical parameters that define the QTL effects and the regression coefficients of the Legendre polynomials that describe the marker effects. Simulation studies were performed to investigate the statistical behavior of composite functional mapping and compare its advantage in separating multiple linked QTL as compared to functional mapping. We used the new mapping approach to analyze a genetic mapping example in rice, leading to the identification of multiple QTL, some of which are linked on the same chromosome, that control the developmental trajectory of leaf age. PMID:17947431

Climate change and dengue: a critical and systematic review of quantitative modelling approaches

PubMed Central

2014-01-01

Background Many studies have found associations between climatic conditions and dengue transmission. However, there is a debate about the future impacts of climate change on dengue transmission. This paper reviewed epidemiological evidence on the relationship between climate and dengue with a focus on quantitative methods for assessing the potential impacts of climate change on global dengue transmission. Methods A literature search was conducted in October 2012, using the electronic databases PubMed, Scopus, ScienceDirect, ProQuest, and Web of Science. The search focused on peer-reviewed journal articles published in English from January 1991 through October 2012. Results Sixteen studies met the inclusion criteria and most studies showed that the transmission of dengue is highly sensitive to climatic conditions, especially temperature, rainfall and relative humidity. Studies on the potential impacts of climate change on dengue indicate increased climatic suitability for transmission and an expansion of the geographic regions at risk during this century. A variety of quantitative modelling approaches were used in the studies. Several key methodological issues and current knowledge gaps were identified through this review. Conclusions It is important to assemble spatio-temporal patterns of dengue transmission compatible with long-term data on climate and other socio-ecological changes and this would advance projections of dengue risks associated with climate change. PMID:24669859

Applying Quantitative Approaches to the Formative Evaluation of Antismoking Campaign Messages

PubMed Central

Parvanta, Sarah; Gibson, Laura; Forquer, Heather; Shapiro-Luft, Dina; Dean, Lorraine; Freres, Derek; Lerman, Caryn; Mallya, Giridhar; Moldovan-Johnson, Mihaela; Tan, Andy; Cappella, Joseph; Hornik, Robert

2014-01-01

This article shares an in-depth summary of a formative evaluation that used quantitative data to inform the development and selection of promotional ads for the antismoking communication component of a social marketing campaign. A foundational survey provided cross-sectional data to identify beliefs about quitting smoking that campaign messages should target, as well as beliefs to avoid. Pretesting draft ads against quantitative indicators of message effectiveness further facilitated the selection and rejection of final campaign ads. Finally, we consider lessons learned from the process of balancing quantitative methods and judgment to make formative decisions about more and less promising persuasive messages for campaigns. PMID:24817829

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.

Theoretical foundations for a quantitative approach to paleogenetics. I, II.

NASA Technical Reports Server (NTRS)

Holmquist, R.

1972-01-01

It is shown that by neglecting the phenomena of multiple hits, back mutation, and chance coincidence errors larger than 100% can be introduced in the calculated value of the average number of nucleotide base differences to be expected between two homologous polynucleotides. Mathematical formulas are derived to correct quantitatively for these effects. It is pointed out that the effects change materially the quantitative aspects of phylogenics, such as the length of the legs of the trees. A number of problems are solved without approximation.-

A quantitative framework for assessing ecological resilience

EPA Science Inventory

Quantitative approaches to measure and assess resilience are needed to bridge gaps between science, policy, and management. In this paper, we suggest a quantitative framework for assessing ecological resilience. Ecological resilience as an emergent ecosystem phenomenon can be de...

Plasmodium knowlesi transmission: integrating quantitative approaches from epidemiology and ecology to understand malaria as a zoonosis.

PubMed

Brock, P M; Fornace, K M; Parmiter, M; Cox, J; Drakeley, C J; Ferguson, H M; Kao, R R

2016-04-01

The public health threat posed by zoonotic Plasmodium knowlesi appears to be growing: it is increasingly reported across South East Asia, and is the leading cause of malaria in Malaysian Borneo. Plasmodium knowlesi threatens progress towards malaria elimination as aspects of its transmission, such as spillover from wildlife reservoirs and reliance on outdoor-biting vectors, may limit the effectiveness of conventional methods of malaria control. The development of new quantitative approaches that address the ecological complexity of P. knowlesi, particularly through a focus on its primary reservoir hosts, will be required to control it. Here, we review what is known about P. knowlesi transmission, identify key knowledge gaps in the context of current approaches to transmission modelling, and discuss the integration of these approaches with clinical parasitology and geostatistical analysis. We highlight the need to incorporate the influences of fine-scale spatial variation, rapid changes to the landscape, and reservoir population and transmission dynamics. The proposed integrated approach would address the unique challenges posed by malaria as a zoonosis, aid the identification of transmission hotspots, provide insight into the mechanistic links between incidence and land use change and support the design of appropriate interventions.

Investigating the Pedagogical Approaches Related to Changes in Attitudes toward Statistics in a Quantitative Methods Course for Psychology Undergraduate Students

ERIC Educational Resources Information Center

Liau, Albert K.; Kiat, John E.; Nie, Youyan

2015-01-01

The purpose of this study was to examine the extent to which the pedagogical approaches used in the course were related to improvements in students' attitudes toward statistics in a Quantitative Methods course for psychology undergraduate students in a Malaysian University. The study examined whether increasing availability of the instructor and…

Fluvial drainage networks: the fractal approach as an improvement of quantitative geomorphic analyses

NASA Astrophysics Data System (ADS)

Melelli, Laura; Liucci, Luisa; Vergari, Francesca; Ciccacci, Sirio; Del Monte, Maurizio

2014-05-01

Drainage basins are primary landscape units for geomorphological investigations. Both hillslopes and river drainage system are fundamental components in drainage basins analysis. As other geomorphological systems, also the drainage basins aim to an equilibrium condition where the sequence of erosion, transport and sedimentation approach to a condition of minimum energy effort. This state is revealed by a typical geometry of landforms and of drainage net. Several morphometric indexes can measure how much a drainage basin is far from the theoretical equilibrium configuration, revealing possible external disarray. In active tectonic areas, the drainage basins have a primary importance in order to highlight style, amount and rate of tectonic impulses, and morphometric indexes allow to estimate the tectonic activity classes of different sectors in a study area. Moreover, drainage rivers are characterized by a self-similarity structure; this promotes the use of fractals theory to investigate the system. In this study, fractals techniques are employed together with quantitative geomorphological analysis to study the Upper Tiber Valley (UTV), a tectonic intermontane basin located in northern Apennines (Umbria, central Italy). The area is the result of different tectonic phases. From Late Pliocene until present time the UTV is strongly controlled by a regional uplift and by an extensional phase with different sets of normal faults playing a fundamental role in basin morphology. Thirty-four basins are taken into account for the quantitative analysis, twenty on the left side of the basin, the others on the right side. Using fractals dimension of drainage networks, Horton's laws results, concavity and steepness indexes, and hypsometric curves, this study aims to obtain an evolutionary model of the UTV, where the uplift is compared to local subsidence induced by normal fault activity. The results highlight a well defined difference between western and eastern tributary basins

A GIS-based Quantitative Approach for the Search of Clandestine Graves, Italy.

PubMed

Somma, Roberta; Cascio, Maria; Silvestro, Massimiliano; Torre, Eliana

2018-05-01

Previous research on the RAG color-coded prioritization systems for the discovery of clandestine graves has not considered all the factors influencing the burial site choice within a GIS project. The goal of this technical note was to discuss a GIS-based quantitative approach for the search of clandestine graves. The method is based on cross-referenced RAG maps with cumulative suitability factors to host a burial, leading to the editing of different search scenarios for ground searches showing high-(Red), medium-(Amber), and low-(Green) priority areas. The application of this procedure allowed several outcomes to be determined: If the concealment occurs at night, then the "search scenario without the visibility" will be the most effective one; if the concealment occurs in daylight, then the "search scenario with the DSM-based visibility" will be most appropriate; the different search scenarios may be cross-referenced with offender's confessions and eyewitnesses' testimonies to verify the veracity of their statements. © 2017 American Academy of Forensic Sciences.

AN APPROACH TO THE DEVELOPMENT OF MODELS TO QUANTITATIVELY ASSESS THE EFFECTS OF EXPOSURE TO ENVIRONMENTALLY RELEVANT LEVELS OF ENDOCRINE DISRUPTORS

EPA Science Inventory

An approach to the development of quantitative models to assess the effects of exposure to environmentally relevant levels of endocrine disruptors on homeostasis in adults.

Ben-Jonathan N, Cooper RL, Foster P, Hughes CL, Hoyer PB, Klotz D, Kohn M, Lamb DJ, Stancel GM.
<...

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.

QTest: Quantitative Testing of Theories of Binary Choice.

PubMed

Regenwetter, Michel; Davis-Stober, Clintin P; Lim, Shiau Hong; Guo, Ying; Popova, Anna; Zwilling, Chris; Cha, Yun-Shil; Messner, William

2014-01-01

The goal of this paper is to make modeling and quantitative testing accessible to behavioral decision researchers interested in substantive questions. We provide a novel, rigorous, yet very general, quantitative diagnostic framework for testing theories of binary choice. This permits the nontechnical scholar to proceed far beyond traditionally rather superficial methods of analysis, and it permits the quantitatively savvy scholar to triage theoretical proposals before investing effort into complex and specialized quantitative analyses. Our theoretical framework links static algebraic decision theory with observed variability in behavioral binary choice data. The paper is supplemented with a custom-designed public-domain statistical analysis package, the QTest software. We illustrate our approach with a quantitative analysis using published laboratory data, including tests of novel versions of "Random Cumulative Prospect Theory." A major asset of the approach is the potential to distinguish decision makers who have a fixed preference and commit errors in observed choices from decision makers who waver in their preferences.

QTest: Quantitative Testing of Theories of Binary Choice

PubMed Central

Regenwetter, Michel; Davis-Stober, Clintin P.; Lim, Shiau Hong; Guo, Ying; Popova, Anna; Zwilling, Chris; Cha, Yun-Shil; Messner, William

2014-01-01

The goal of this paper is to make modeling and quantitative testing accessible to behavioral decision researchers interested in substantive questions. We provide a novel, rigorous, yet very general, quantitative diagnostic framework for testing theories of binary choice. This permits the nontechnical scholar to proceed far beyond traditionally rather superficial methods of analysis, and it permits the quantitatively savvy scholar to triage theoretical proposals before investing effort into complex and specialized quantitative analyses. Our theoretical framework links static algebraic decision theory with observed variability in behavioral binary choice data. The paper is supplemented with a custom-designed public-domain statistical analysis package, the QTest software. We illustrate our approach with a quantitative analysis using published laboratory data, including tests of novel versions of “Random Cumulative Prospect Theory.” A major asset of the approach is the potential to distinguish decision makers who have a fixed preference and commit errors in observed choices from decision makers who waver in their preferences. PMID:24999495

New optomechanical approach to quantitative characterization of fatigue behavior of dynamically loaded structures

NASA Astrophysics Data System (ADS)

Furlong, Cosme; Pryputniewicz, Ryszard J.

1995-06-01

The basic relationships between stress and strain under cyclic conditions of loading are not at present well understood. It would seem that information of this type is vital for a fundamental approach to understand the fatigue behavior of dynamically loaded structures. In this paper, experimental and computational methods are utilized to study the fatigue behavior of a thin aluminum cantilever plate subjected to dynamic loading. The studies are performed by combining optomechanical and finite element methods. The cantilever plate is loaded periodically by excitation set at a fixed amplitude and at a specific resonance frequency of the plate. By continuously applying this type of loading and using holographic interferometry, the behavior of the plate during a specific period of time is investigated. Quantitative information is obtained from laser vibrometry data which are utilized by a finite element program to calculate strains and stresses assuming a homogeneous and isotropic material and constant strain elements. It is shown that the use of experimental and computational hybrid methodologies allows identification of different zones of the plate that are fatigue critical. This optomechanical approach proves to be a viable tool for understanding of fatigue behavior of mechanical components and for performing optimization of structures subjected to fatigue conditions.

Quantitative multiplex detection of pathogen biomarkers

DOEpatents

Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I.; Martinez, Jennifer; Grace, Wynne K.

2016-02-09

The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

Quantitative multiplex detection of pathogen biomarkers

DOEpatents

Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I; Martinez, Jennifer; Grace, Wynne K

2014-10-14

The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

Resolving the Quantitative-Qualitative Dilemma: A Critical Realist Approach

ERIC Educational Resources Information Center

Scott, David

2007-01-01

The philosophical issues underpinning the quantitative-qualitative divide in educational research are examined. Three types of argument which support a resolution are considered: pragmatism, false duality and warranty through triangulation. In addition a number of proposed strategies--alignment, sequencing, translation and triangulation--are…

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.

A rapid, automated approach to optimisation of multiple reaction monitoring conditions for quantitative bioanalytical mass spectrometry.

PubMed

Higton, D M

2001-01-01

An improvement to the procedure for the rapid optimisation of mass spectrometry (PROMS), for the development of multiple reaction methods (MRM) for quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS), is presented. PROMS is an automated protocol that uses flow-injection analysis (FIA) and AppleScripts to create methods and acquire the data for optimisation. The protocol determines the optimum orifice potential, the MRM conditions for each compound, and finally creates the MRM methods needed for sample analysis. The sensitivities of the MRM methods created by PROMS approach those created manually. MRM method development using PROMS currently takes less than three minutes per compound compared to at least fifteen minutes manually. To further enhance throughput, approaches to MRM optimisation using one injection per compound, two injections per pool of five compounds and one injection per pool of five compounds have been investigated. No significant difference in the optimised instrumental parameters for MRM methods were found between the original PROMS approach and these new methods, which are up to ten times faster. The time taken for an AppleScript to determine the optimum conditions and build the MRM methods is the same with all approaches. Copyright 2001 John Wiley & Sons, Ltd.

Approaches to quantitating the results of differentially dyed cottons

USDA-ARS?s Scientific Manuscript database

The differential dyeing (DD) method has served as a subjective method for visually determining immature cotton fibers. In an attempt to quantitate the results of the differential dyeing method, and thus offer an efficient means of elucidating cotton maturity without visual discretion, image analysi...

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.

An approach for quantitative image quality analysis for CT

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

Understanding Pre-Quantitative Risk in Projects

NASA Technical Reports Server (NTRS)

Cooper, Lynne P.

2011-01-01

Standard approaches to risk management in projects depend on the ability of teams to identify risks and quantify the probabilities and consequences of these risks (e.g., the 5 x 5 risk matrix). However, long before quantification does - or even can - occur, and long after, teams make decisions based on their pre-quantitative understanding of risk. These decisions can have long-lasting impacts on the project. While significant research has looked at the process of how to quantify risk, our understanding of how teams conceive of and manage pre-quantitative risk is lacking. This paper introduces the concept of pre-quantitative risk and discusses the implications of addressing pre-quantitative risk in projects.

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

Quantitative aspects of inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Bulska, Ewa; Wagner, Barbara

2016-10-01

Accurate determination of elements in various kinds of samples is essential for many areas, including environmental science, medicine, as well as industry. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful tool enabling multi-elemental analysis of numerous matrices with high sensitivity and good precision. Various calibration approaches can be used to perform accurate quantitative measurements by ICP-MS. They include the use of pure standards, matrix-matched standards, or relevant certified reference materials, assuring traceability of the reported results. This review critically evaluates the advantages and limitations of different calibration approaches, which are used in quantitative analyses by ICP-MS. Examples of such analyses are provided. This article is part of the themed issue 'Quantitative mass spectrometry'.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

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

Deciphering Phosphotyrosine-Dependent Signaling Networks in Cancer by SH2 Profiling

PubMed Central

Machida, Kazuya; Khenkhar, Malik

2012-01-01

It has been a decade since the introduction of SH2 profiling, a modular domain-based molecular diagnostics tool. This review covers the original concept of SH2 profiling, different analytical platforms, and their applications, from the detailed analysis of single proteins to broad screening in translational research. Illustrated by practical examples, we discuss the uniqueness and advantages of the approach as well as its limitations and challenges. We provide guidance for basic researchers and oncologists who may consider SH2 profiling in their respective cancer research, especially for those focusing on tyrosine phosphoproteomics. SH2 profiling can serve as an alternative phosphoproteomics tool to dissect aberrant tyrosine kinase pathways responsible for individual malignancies, with the goal of facilitating personalized diagnostics for the treatment of cancer. PMID:23226573

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

Quantitative aspects of inductively coupled plasma mass spectrometry

PubMed Central

Wagner, Barbara

2016-01-01

Accurate determination of elements in various kinds of samples is essential for many areas, including environmental science, medicine, as well as industry. Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful tool enabling multi-elemental analysis of numerous matrices with high sensitivity and good precision. Various calibration approaches can be used to perform accurate quantitative measurements by ICP-MS. They include the use of pure standards, matrix-matched standards, or relevant certified reference materials, assuring traceability of the reported results. This review critically evaluates the advantages and limitations of different calibration approaches, which are used in quantitative analyses by ICP-MS. Examples of such analyses are provided. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644971

Redox environment in stem and differentiated cells: A quantitative approach.

PubMed

Lyublinskaya, O G; Ivanova, Ju S; Pugovkina, N A; Kozhukharova, I V; Kovaleva, Z V; Shatrova, A N; Aksenov, N D; Zenin, V V; Kaulin, Yu A; Gamaley, I A; Nikolsky, N N

2017-08-01

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H 2 DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

Energy Education: The Quantitative Voice

NASA Astrophysics Data System (ADS)

Wolfson, Richard

2010-02-01

A serious study of energy use and its consequences has to be quantitative. It makes little sense to push your favorite renewable energy source if it can't provide enough energy to make a dent in humankind's prodigious energy consumption. Conversely, it makes no sense to dismiss alternatives---solar in particular---that supply Earth with energy at some 10,000 times our human energy consumption rate. But being quantitative---especially with nonscience students or the general public---is a delicate business. This talk draws on the speaker's experience presenting energy issues to diverse audiences through single lectures, entire courses, and a textbook. The emphasis is on developing a quick, ``back-of-the-envelope'' approach to quantitative understanding of energy issues. )

A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

PubMed

Kubota, Kazuyuki; Mizukoshi, Toshimi; Miyano, Hiroshi

2013-10-01

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 μM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

Statistical shape analysis using 3D Poisson equation--A quantitatively validated approach.

PubMed

Gao, Yi; Bouix, Sylvain

2016-05-01

Statistical shape analysis has been an important area of research with applications in biology, anatomy, neuroscience, agriculture, paleontology, etc. Unfortunately, the proposed methods are rarely quantitatively evaluated, and as shown in recent studies, when they are evaluated, significant discrepancies exist in their outputs. In this work, we concentrate on the problem of finding the consistent location of deformation between two population of shapes. We propose a new shape analysis algorithm along with a framework to perform a quantitative evaluation of its performance. Specifically, the algorithm constructs a Signed Poisson Map (SPoM) by solving two Poisson equations on the volumetric shapes of arbitrary topology, and statistical analysis is then carried out on the SPoMs. The method is quantitatively evaluated on synthetic shapes and applied on real shape data sets in brain structures. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel approach to mixing qualitative and quantitative methods in HIV and STI prevention research.

PubMed

Penman-Aguilar, Ana; Macaluso, Maurizio; Peacock, Nadine; Snead, M Christine; Posner, Samuel F

2014-04-01

Mixed-method designs are increasingly used in sexually transmitted infection (STI) and HIV prevention research. The authors designed a mixedmethod approach and applied it to estimate and evaluate a predictor of continued female condom use (6+ uses, among those who used it at least once) in a 6-month prospective cohort study. The analysis included 402 women who received an intervention promoting use of female and male condoms for STI prevention and completed monthly quantitative surveys; 33 also completed a semistructured qualitative interview. The authors identified a qualitative theme (couples' female condom enjoyment [CFCE]), applied discriminant analysis techniques to estimate CFCE for all participants, and added CFCE to a multivariable logistic regression model of continued female condom use. CFCE related to comfort, naturalness, pleasure, feeling protected, playfulness, ease of use, intimacy, and feeling in control of protection. CFCE was associated with continued female condom use (adjusted odds ratio: 2.8, 95% confidence interval: 1.4-5.6) and significantly improved model fit (p < .001). CFCE predicted continued female condom use. Mixed-method approaches for "scaling up" qualitative findings from small samples to larger numbers of participants can benefit HIV and STI prevention research.

A template-based approach to semi-quantitative SPECT myocardial perfusion imaging: Independent of normal databases.

PubMed

Hughes, Tyler; Shcherbinin, Sergey; Celler, Anna

2011-07-01

Normal patient databases (NPDs) are used to distinguish between normal and abnormal perfusion in SPECT myocardial perfusion imaging (MPI) and have gained wide acceptance in the clinical environment, yet there are limitations to this approach. This study introduces a template-based method for semi-quantitative MPI, which attempts to overcome some of the NPD limitations. Our approach involves the construction of a 3D digital healthy heart template from the delineation of the patient's left ventricle in the SPECT image. This patient-specific template of the heart, filled with uniform activity, is then analytically projected and reconstructed using the same algorithm as the original image. Subsequent to generating bulls-eye maps for the patient image (PB) and the template image (TB), a ratio (PB/TB) is calculated, which produces a reconstruction-artifact corrected image (CB). Finally, a threshold is used to define defects within CB enabling measurements of the perfusion defect extent (EXT). The SPECT-based template (Ts) measurements were compared to those of a CT-based "ideal" template (TI). Twenty digital phantoms were simulated: male and female, each with one healthy heart and nine hearts with various defects. Four physical phantom studies were performed modeling a healthy heart and three hearts with different defects. The phantom represented a thorax with spine, lung, and left ventricle inserts. Images were acquired on General Electric's (GE) Infinia Hawkeye SPECT/CT camera using standard clinical MPI protocol. Finally, our method was applied to 14 patient MPI rest/stress studies acquired on the GE Infinia Hawkeye SPECT/CT camera and compared to the results obtained from Cedars-Sinai's QPS software. In the simulation studies, the true EXT correlated well with the TI (slope= 1.08; offset = -0.40%; r = 0.99) and Ts (slope = 0.90; offset = 0.27%; r = 0.99) methods with no significant differences between them. Similarly, strong correlations were measured for EXT

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.

Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: applications and considerations.

PubMed

Kim, Jaai; Lim, Juntaek; Lee, Changsoo

2013-12-01

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. Copyright © 2013 Elsevier Inc. All rights reserved.

Critically appraising qualitative research: a guide for clinicians more familiar with quantitative techniques.

PubMed

Kisely, Stephen; Kendall, Elizabeth

2011-08-01

Papers using qualitative methods are increasingly common in psychiatric journals. This overview is an introduction to critically appraising a qualitative paper for clinicians who are more familiar with quantitative methods. Qualitative research uses data from interviews (semi-structured or unstructured), focus groups, observations or written materials. Data analysis is inductive, allowing meaning to emerge from the data, rather than the more deductive, hypothesis centred approach of quantitative research. This overview compares and contrasts quantitative and qualitative research methods. Quantitative concepts such as reliability, validity, statistical power, bias and generalisability have qualitative equivalents. These include triangulation, trustworthiness, saturation, reflexivity and applicability. Reflexivity also shares features of transference. Qualitative approaches include: ethnography, action-assessment, grounded theory, case studies and mixed methods. Qualitative research can complement quantitative approaches. An understanding of both is useful in critically appraising the psychiatric literature.

pH-regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments.

PubMed

Levi Mortera, Stefano; Dioni, Ilaria; Greco, Viviana; Neri, Cristina; Rovero, Paolo; Urbani, Andrea

2014-05-01

Among the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH₃ features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde could be employed as well without a substantial increase in terms of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless, only a single paper reports about a diethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation.

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations

PubMed Central

Hägerling, René; Drees, Dominik; Scherzinger, Aaron; Dierkes, Cathrin; Martin-Almedina, Silvia; Butz, Stefan; Gordon, Kristiana; Schäfers, Michael; Hinrichs, Klaus; Vestweber, Dietmar; Goerge, Tobias; Mansour, Sahar; Mortimer, Peter S.

2017-01-01

BACKGROUND. Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. METHODS. VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy–based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. RESULTS. Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). CONCLUSION. VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. FUNDING. Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003. PMID

VIPAR, a quantitative approach to 3D histopathology applied to lymphatic malformations.

PubMed

Hägerling, René; Drees, Dominik; Scherzinger, Aaron; Dierkes, Cathrin; Martin-Almedina, Silvia; Butz, Stefan; Gordon, Kristiana; Schäfers, Michael; Hinrichs, Klaus; Ostergaard, Pia; Vestweber, Dietmar; Goerge, Tobias; Mansour, Sahar; Jiang, Xiaoyi; Mortimer, Peter S; Kiefer, Friedemann

2017-08-17

Lack of investigatory and diagnostic tools has been a major contributing factor to the failure to mechanistically understand lymphedema and other lymphatic disorders in order to develop effective drug and surgical therapies. One difficulty has been understanding the true changes in lymph vessel pathology from standard 2D tissue sections. VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction), a light-sheet microscopy-based approach for the analysis of tissue biopsies, is based on digital reconstruction and visualization of microscopic image stacks. VIPAR allows semiautomated segmentation of the vasculature and subsequent nonbiased extraction of characteristic vessel shape and connectivity parameters. We applied VIPAR to analyze biopsies from healthy lymphedematous and lymphangiomatous skin. Digital 3D reconstruction provided a directly visually interpretable, comprehensive representation of the lymphatic and blood vessels in the analyzed tissue volumes. The most conspicuous features were disrupted lymphatic vessels in lymphedematous skin and a hyperplasia (4.36-fold lymphatic vessel volume increase) in the lymphangiomatous skin. Both abnormalities were detected by the connectivity analysis based on extracted vessel shape and structure data. The quantitative evaluation of extracted data revealed a significant reduction of lymphatic segment length (51.3% and 54.2%) and straightness (89.2% and 83.7%) for lymphedematous and lymphangiomatous skin, respectively. Blood vessel length was significantly increased in the lymphangiomatous sample (239.3%). VIPAR is a volume-based tissue reconstruction data extraction and analysis approach that successfully distinguished healthy from lymphedematous and lymphangiomatous skin. Its application is not limited to the vascular systems or skin. Max Planck Society, DFG (SFB 656), and Cells-in-Motion Cluster of Excellence EXC 1003.

Identification and uncertainty estimation of vertical reflectivity profiles using a Lagrangian approach to support quantitative precipitation measurements by weather radar

NASA Astrophysics Data System (ADS)

Hazenberg, P.; Torfs, P. J. J. F.; Leijnse, H.; Delrieu, G.; Uijlenhoet, R.

2013-09-01

This paper presents a novel approach to estimate the vertical profile of reflectivity (VPR) from volumetric weather radar data using both a traditional Eulerian as well as a newly proposed Lagrangian implementation. For this latter implementation, the recently developed Rotational Carpenter Square Cluster Algorithm (RoCaSCA) is used to delineate precipitation regions at different reflectivity levels. A piecewise linear VPR is estimated for either stratiform or neither stratiform/convective precipitation. As a second aspect of this paper, a novel approach is presented which is able to account for the impact of VPR uncertainty on the estimated radar rainfall variability. Results show that implementation of the VPR identification and correction procedure has a positive impact on quantitative precipitation estimates from radar. Unfortunately, visibility problems severely limit the impact of the Lagrangian implementation beyond distances of 100 km. However, by combining this procedure with the global Eulerian VPR estimation procedure for a given rainfall type (stratiform and neither stratiform/convective), the quality of the quantitative precipitation estimates increases up to a distance of 150 km. Analyses of the impact of VPR uncertainty shows that this aspect accounts for a large fraction of the differences between weather radar rainfall estimates and rain gauge measurements.

Linking Yeast Gcn5p Catalytic Function and Gene Regulation Using a Quantitative, Graded Dominant Mutant Approach

PubMed Central

Lanza, Amanda M.; Blazeck, John J.; Crook, Nathan C.; Alper, Hal S.

2012-01-01

Establishing causative links between protein functional domains and global gene regulation is critical for advancements in genetics, biotechnology, disease treatment, and systems biology. This task is challenging for multifunctional proteins when relying on traditional approaches such as gene deletions since they remove all domains simultaneously. Here, we describe a novel approach to extract quantitative, causative links by modulating the expression of a dominant mutant allele to create a function-specific competitive inhibition. Using the yeast histone acetyltransferase Gcn5p as a case study, we demonstrate the utility of this approach and (1) find evidence that Gcn5p is more involved in cell-wide gene repression, instead of the accepted gene activation associated with HATs, (2) identify previously unknown gene targets and interactions for Gcn5p-based acetylation, (3) quantify the strength of some Gcn5p-DNA associations, (4) demonstrate that this approach can be used to correctly identify canonical chromatin modifications, (5) establish the role of acetyltransferase activity on synthetic lethal interactions, and (6) identify new functional classes of genes regulated by Gcn5p acetyltransferase activity—all six of these major conclusions were unattainable by using standard gene knockout studies alone. We recommend that a graded dominant mutant approach be utilized in conjunction with a traditional knockout to study multifunctional proteins and generate higher-resolution data that more accurately probes protein domain function and influence. PMID:22558379

Confidence estimation for quantitative photoacoustic imaging

NASA Astrophysics Data System (ADS)

Gröhl, Janek; Kirchner, Thomas; Maier-Hein, Lena

2018-02-01

Quantification of photoacoustic (PA) images is one of the major challenges currently being addressed in PA research. Tissue properties can be quantified by correcting the recorded PA signal with an estimation of the corresponding fluence. Fluence estimation itself, however, is an ill-posed inverse problem which usually needs simplifying assumptions to be solved with state-of-the-art methods. These simplifications, as well as noise and artifacts in PA images reduce the accuracy of quantitative PA imaging (PAI). This reduction in accuracy is often localized to image regions where the assumptions do not hold true. This impedes the reconstruction of functional parameters when averaging over entire regions of interest (ROI). Averaging over a subset of voxels with a high accuracy would lead to an improved estimation of such parameters. To achieve this, we propose a novel approach to the local estimation of confidence in quantitative reconstructions of PA images. It makes use of conditional probability densities to estimate confidence intervals alongside the actual quantification. It encapsulates an estimation of the errors introduced by fluence estimation as well as signal noise. We validate the approach using Monte Carlo generated data in combination with a recently introduced machine learning-based approach to quantitative PAI. Our experiments show at least a two-fold improvement in quantification accuracy when evaluating on voxels with high confidence instead of thresholding signal intensity.

IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs)

EPA Science Inventory

This report summarizes the discussion, conclusions, and points of consensus of the IWGT Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (QWG) based on a meeting in Foz do Iguaçu, Brazil October 31–November 2, 2013. Topics addressed incl...

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

Virtualising the Quantitative Research Methods Course: An Island-Based Approach

ERIC Educational Resources Information Center

Baglin, James; Reece, John; Baker, Jenalle

2015-01-01

Many recent improvements in pedagogical practice have been enabled by the rapid development of innovative technologies, particularly for teaching quantitative research methods and statistics. This study describes the design, implementation, and evaluation of a series of specialised computer laboratory sessions. The sessions combined the use of an…

A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model

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

Caruso, Joseph A.; Stemmer, Paul M.; Dombkowski, Alan

2014-04-01

Network and protein–protein interaction analyses of proteins undergoing Hg{sup 2+}-induced phosphorylation and dephosphorylation in Hg{sup 2+}-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sitesmore » for phosphorylation following mercury exposure. In order to determine how Hg{sup 2+} exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg{sup 2+}, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg{sup 2+} closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg{sup 2+} led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure. - Highlights: • Inorganic mercury (Hg{sup 2+}) induces changes in the WEHI-231 B cell phosphoproteome. • The B cell receptor (BCR) signaling pathway was the pathway most affected by Hg{sup 2+}. • The Src family phosphoprotein kinase Lyn

A novel approach for quantitative evaluation of the physicochemical interactions between rough membrane surface and sludge foulants in a submerged membrane bioreactor.

PubMed

Lin, Hongjun; Zhang, Meijia; Mei, Rongwu; Chen, Jianrong; Hong, Huachang

2014-11-01

This study proposed a novel approach for quantitative evaluation of the physicochemical interactions between a particle and rough surface. The approach adopts the composite Simpson's rule to numerically calculate the double integrals in the surface element integration of these physicochemical interactions. The calculation could be achieved by a MATLAB program based on this approach. This approach was then applied to assess the physicochemical interactions between rough membrane surface and sludge foulants in a submerged membrane bioreactor (MBR). The results showed that, as compared with smooth membrane surface, rough membrane surface had a much lower strength of interactions with sludge foulants. Meanwhile, membrane surface morphology significantly affected the strength and properties of the interactions. This study showed that the newly developed approach was feasible, and could serve as a primary tool for investigating membrane fouling in MBRs. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

correlated the results of the relative quantification with amounts estimated from the calibration curves. This approach represents a way to validate and verify biomarker candidates discovered from large-scale global phospho-proteomics analysis. The application of these modified immuno-MRM assays in lung adenocarcinoma cells provides proof-of concept for the feasibility of clinical applications. These assays may be used in prospective clinical studies of EGFR TKI treatment of EGFR mutant lung cancer to correlate treatment response and other clinical endpoints. Copyright © 2018. Published by Elsevier B.V.

The Next Frontier: Quantitative Biochemistry in Living Cells.

PubMed

Honigmann, Alf; Nadler, André

2018-01-09

Researchers striving to convert biology into an exact science foremost rely on structural biology and biochemical reconstitution approaches to obtain quantitative data. However, cell biological research is moving at an ever-accelerating speed into areas where these approaches lose much of their edge. Intrinsically unstructured proteins and biochemical interaction networks composed of interchangeable, multivalent, and unspecific interactions pose unique challenges to quantitative biology, as do processes that occur in discrete cellular microenvironments. Here we argue that a conceptual change in our way of conducting biochemical experiments is required to take on these new challenges. We propose that reconstitution of cellular processes in vitro should be much more focused on mimicking the cellular environment in vivo, an approach that requires detailed knowledge of the material properties of cellular compartments, essentially requiring a material science of the cell. In a similar vein, we suggest that quantitative biochemical experiments in vitro should be accompanied by corresponding experiments in vivo, as many newly relevant cellular processes are highly context-dependent. In essence, this constitutes a call for chemical biologists to convert their discipline from a proof-of-principle science to an area that could rightfully be called quantitative biochemistry in living cells. In this essay, we discuss novel techniques and experimental strategies with regard to their potential to fulfill such ambitious aims.

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses.

PubMed

Kasprowicz, Richard; Rand, Emma; O'Toole, Peter J; Signoret, Nathalie

2018-05-22

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4 + T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4 + T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.

Quantitative analysis of the human AKR family members in cancer cell lines using the mTRAQ/MRM approach.

PubMed

Zhang, Shenyan; Wen, Bo; Zhou, Baojin; Yang, Lei; Cha, Chao; Xu, Shaoxing; Qiu, Xuemei; Wang, Quanhui; Sun, Haidan; Lou, Xiaomin; Zi, Jin; Zhang, Yong; Lin, Liang; Liu, Siqi

2013-05-03

Members of human aldo-keto reductase (AKR) superfamily have been reported to be involved in cancer progression, whereas the final conclusion is not generally accepted. Herein, we propose a quantitative method to measure human AKR proteins in cells using mTRAQ-based multiple reaction monitoring (MRM). AKR peptides with multiple transitions were carefully selected upon tryptic digestion of the recombinant AKR proteins, while AKR proteins were identified by SDS-PAGE fractionation coupled with LC-MS/MS. Utilizing mTRAQ triplex labeling to produce the derivative peptides, calibration curves were generated using the mixed lysate as background, and no significantly different quantification of AKRs was elicited from the two sets of calibration curves under the mixed and single lysate as background. We employed this approach to quantitatively determine the 6 AKR proteins, AKR1A1, AKR1B1, AKR1B10, AKR1C1/C2, AKR1C3, and AKR1C4, in 7 different cancer cell lines and for the first time to obtain the absolute quantities of all the AKR proteins in each cell. The cluster plot revealed that AKR1A and AKR1B were widely distributed in most cancer cells with relatively stable abundances, whereas AKR1Cs were unevenly detected among these cells with diverse dynamic abundances. The AKR quantitative distribution in different cancer cells, therefore, may assist further exploration toward how the AKR proteins are involved in tumorigenesis.

Quantitative Reasoning in Problem Solving

ERIC Educational Resources Information Center

Ramful, Ajay; Ho, Siew Yin

2015-01-01

In this article, Ajay Ramful and Siew Yin Ho explain the meaning of quantitative reasoning, describing how it is used in the to solve mathematical problems. They also describe a diagrammatic approach to represent relationships among quantities and provide examples of problems and their solutions.

A multiplexed quantitative proteomics approach for investigating protein expression in the developing central nervous system.

PubMed

Orme, Rowan P; Gates, Monte A; Fricker-Gates, Rosemary A

2010-08-15

Cell transplantation using stem cell-derived neurons is commonly viewed as a candidate therapy for neurodegenerative diseases. However, methods for differentiating stem cells into homogenous populations of neurons suitable for transplant remain elusive. This suggests that there are as yet unknown signalling factors working in vivo to specify neuronal cell fate during development. These factors could be manipulated to better differentiate stem cells into neural populations useful for therapeutic transplantation. Here a quantitative proteomics approach is described for investigating cell signalling in the developing central nervous system (CNS), using the embryonic ventral mesencephalon as a model. Briefly, total protein was extracted from embryonic ventral midbrain tissue before, during and after the birth of dopaminergic neurons, and digested using trypsin. Two-dimensional liquid chromatography, coupled with tandem mass spectrometry, was then used to identify proteins from the tryptic peptides. Isobaric tagging for relative and absolute quantification (iTRAQ) reagents were used to label the tryptic peptides and facilitate relative quantitative analysis. The success of the experiment was confirmed by the identification of proteins known to be expressed in the developing ventral midbrain, as well as by Western blotting, and immunolabelling of embryonic tissue sections. This method of protein discovery improves upon previous attempts to identify novel signalling factors through microarray analysis. Importantly, the methods described here could be applied to virtually any aspect of development. (c) 2010 Elsevier B.V. All rights reserved.

A quantitative microscopic approach to predict local recurrence based on in vivo intraoperative imaging of sarcoma tumor margins

PubMed Central

Mueller, Jenna L.; Fu, Henry L.; Mito, Jeffrey K.; Whitley, Melodi J.; Chitalia, Rhea; Erkanli, Alaattin; Dodd, Leslie; Cardona, Diana M.; Geradts, Joseph; Willett, Rebecca M.; Kirsch, David G.; Ramanujam, Nimmi

2015-01-01

The goal of resection of soft tissue sarcomas located in the extremity is to preserve limb function while completely excising the tumor with a margin of normal tissue. With surgery alone, one-third of patients with soft tissue sarcoma of the extremity will have local recurrence due to microscopic residual disease in the tumor bed. Currently, a limited number of intraoperative pathology-based techniques are used to assess margin status; however, few have been widely adopted due to sampling error and time constraints. To aid in intraoperative diagnosis, we developed a quantitative optical microscopy toolbox, which includes acriflavine staining, fluorescence microscopy, and analytic techniques called sparse component analysis and circle transform to yield quantitative diagnosis of tumor margins. A series of variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82% and 75%. The utility of this approach was tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78% and 82%. For comparison, if pathology was used to predict local recurrence in this data set, it would achieve a sensitivity of 29% and a specificity of 71%. These results indicate a robust approach for detecting microscopic residual disease, which is an effective predictor of local recurrence. PMID:25994353

Overview of Student Affairs Research Methods: Qualitative and Quantitative.

ERIC Educational Resources Information Center

Perl, Emily J.; Noldon, Denise F.

2000-01-01

Reviews the strengths and weaknesses of quantitative and qualitative research in student affairs research, noting that many student affairs professionals question the value of more traditional quantitative approaches to research, though they typically have very good people skills that they have applied to being good qualitative researchers.…

Exhaust-System Leak Test : Quantitative Procedure

DOT National Transportation Integrated Search

1974-01-01

A quantitative, periodic motor vehicle safety-inspection test for determining the leakage rate of engine exhaust from an automotive exhaust system was investigated. Two technical approaches were evaluated, and the better one was selected for developm...

Genomic Quantitative Genetics to Study Evolution in the Wild.

PubMed

Gienapp, Phillip; Fior, Simone; Guillaume, Frédéric; Lasky, Jesse R; Sork, Victoria L; Csilléry, Katalin

2017-12-01

Quantitative genetic theory provides a means of estimating the evolutionary potential of natural populations. However, this approach was previously only feasible in systems where the genetic relatedness between individuals could be inferred from pedigrees or experimental crosses. The genomic revolution opened up the possibility of obtaining the realized proportion of genome shared among individuals in natural populations of virtually any species, which could promise (more) accurate estimates of quantitative genetic parameters in virtually any species. Such a 'genomic' quantitative genetics approach relies on fewer assumptions, offers a greater methodological flexibility, and is thus expected to greatly enhance our understanding of evolution in natural populations, for example, in the context of adaptation to environmental change, eco-evolutionary dynamics, and biodiversity conservation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative biology: where modern biology meets physical sciences.

PubMed

Shekhar, Shashank; Zhu, Lian; Mazutis, Linas; Sgro, Allyson E; Fai, Thomas G; Podolski, Marija

2014-11-05

Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines. © 2014 Shekhar, Zhu, Mazutis, Sgro, Fai, and Podolski. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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.

Estimating background-subtracted fluorescence transients in calcium imaging experiments: a quantitative approach.

PubMed

Joucla, Sébastien; Franconville, Romain; Pippow, Andreas; Kloppenburg, Peter; Pouzat, Christophe

2013-08-01

Calcium imaging has become a routine technique in neuroscience for subcellular to network level investigations. The fast progresses in the development of new indicators and imaging techniques call for dedicated reliable analysis methods. In particular, efficient and quantitative background fluorescence subtraction routines would be beneficial to most of the calcium imaging research field. A background-subtracted fluorescence transients estimation method that does not require any independent background measurement is therefore developed. This method is based on a fluorescence model fitted to single-trial data using a classical nonlinear regression approach. The model includes an appropriate probabilistic description of the acquisition system's noise leading to accurate confidence intervals on all quantities of interest (background fluorescence, normalized background-subtracted fluorescence time course) when background fluorescence is homogeneous. An automatic procedure detecting background inhomogeneities inside the region of interest is also developed and is shown to be efficient on simulated data. The implementation and performances of the proposed method on experimental recordings from the mouse hypothalamus are presented in details. This method, which applies to both single-cell and bulk-stained tissues recordings, should help improving the statistical comparison of fluorescence calcium signals between experiments and studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantitative Research in Written Composition.

ERIC Educational Resources Information Center

Gebhard, Ann O.

Offered as an introductory guide to teachers interested in approaching written English as a "second dialect" that students must master, this review covers quantitative investigations of written language. The first section deals with developmental studies, describing how a variety of researchers have related written structure to writer maturity.…

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.

Quantitative analysis of glycoprotein glycans.

PubMed

Orlando, Ron

2013-01-01

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

Qualitative Research? Quantitative Research? What's the Problem? Resolving the Dilemma via a Postconstructivist Approach.

ERIC Educational Resources Information Center

Shank, Gary

It is argued that the debate between qualitative and quantitative research for educational researchers is actually an argument between constructivism and positivism. Positivism has been the basis for most quantitative research in education. Two different things are actually meant when constructivism is discussed (constructivism and…

Quantitative proteomics in cardiovascular research: global and targeted strategies

PubMed Central

Shen, Xiaomeng; Young, Rebeccah; Canty, John M.; Qu, Jun

2014-01-01

Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation. PMID:24920501

Quantitative architectural analysis: a new approach to cortical mapping.

PubMed

Schleicher, A; Palomero-Gallagher, N; Morosan, P; Eickhoff, S B; Kowalski, T; de Vos, K; Amunts, K; Zilles, K

2005-12-01

Recent progress in anatomical and functional MRI has revived the demand for a reliable, topographic map of the human cerebral cortex. Till date, interpretations of specific activations found in functional imaging studies and their topographical analysis in a spatial reference system are, often, still based on classical architectonic maps. The most commonly used reference atlas is that of Brodmann and his successors, despite its severe inherent drawbacks. One obvious weakness in traditional, architectural mapping is the subjective nature of localising borders between cortical areas, by means of a purely visual, microscopical examination of histological specimens. To overcome this limitation, more objective, quantitative mapping procedures have been established in the past years. The quantification of the neocortical, laminar pattern by defining intensity line profiles across the cortical layers, has a long tradition. During the last years, this method has been extended to enable a reliable, reproducible mapping of the cortex based on image analysis and multivariate statistics. Methodological approaches to such algorithm-based, cortical mapping were published for various architectural modalities. In our contribution, principles of algorithm-based mapping are described for cyto- and receptorarchitecture. In a cytoarchitectural parcellation of the human auditory cortex, using a sliding window procedure, the classical areal pattern of the human superior temporal gyrus was modified by a replacing of Brodmann's areas 41, 42, 22 and parts of area 21, with a novel, more detailed map. An extension and optimisation of the sliding window procedure to the specific requirements of receptorarchitectonic mapping, is also described using the macaque central sulcus and adjacent superior parietal lobule as a second, biologically independent example. Algorithm-based mapping procedures, however, are not limited to these two architectural modalities, but can be applied to all images in

Young People's Attitudes to Religious Diversity: Quantitative Approaches from Social Psychology and Empirical Theology

ERIC Educational Resources Information Center

Francis, Leslie J.; Croft, Jennifer S.; Pyke, Alice; Robbins, Mandy

2012-01-01

This essay discusses the design of the quantitative component of the "Young People's Attitudes to Religious Diversity" project, conceived by Professor Robert Jackson within the Warwick Religions and Education Research Unit, and presents some preliminary findings from the data. The quantitative component followed and built on the…

A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

PubMed

Tassy, Olivier; Daian, Fabrice; Hudson, Clare; Bertrand, Vincent; Lemaire, Patrick

2006-02-21

The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates. We generated a collection of 19 interactive ascidian embryos between the 2- and 44-cell stages. We characterized the evolution with time, and in different cell lineages, of the volume of cells and of eight mathematical descriptors of their geometry, and we measured the surface of contact between neighboring blastomeres. These analyses first revealed that early embryonic blastomeres adopt a surprising variety of shapes, which appeared to be under strict and dynamic developmental control. Second, we found novel asymmetric cell divisions in the posterior vegetal lineages, which gave birth to sister cells with different fates. Third, during neural induction, differences in the area of contact between individual competent animal cells and inducing vegetal blastomeres appeared important to select the induced cells. In addition to novel insight into both cell-autonomous and inductive processes controlling early ascidian development, we establish a generic conceptual framework for the quantitative analysis of embryo geometry that can be applied to other model organisms.

On Quantitative Comparative Research in Communication and Language Evolution

PubMed Central

Oller, D. Kimbrough; Griebel, Ulrike

2014-01-01

Quantitative comparison of human language and natural animal communication requires improved conceptualizations. We argue that an infrastructural approach to development and evolution incorporating an extended interpretation of the distinctions among illocution, perlocution, and meaning (Austin 1962; Oller and Griebel 2008) can help place the issues relevant to quantitative comparison in perspective. The approach can illuminate the controversy revolving around the notion of functional referentiality as applied to alarm calls, for example in the vervet monkey. We argue that referentiality offers a poor point of quantitative comparison across language and animal communication in the wild. Evidence shows that even newborn human cry could be deemed to show functional referentiality according to the criteria typically invoked by advocates of referentiality in animal communication. Exploring the essence of the idea of illocution, we illustrate an important realm of commonality among animal communication systems and human language, a commonality that opens the door to more productive, quantifiable comparisons. Finally, we delineate two examples of infrastructural communicative capabilities that should be particularly amenable to direct quantitative comparison across humans and our closest relatives. PMID:25285057

On Quantitative Comparative Research in Communication and Language Evolution.

PubMed

Oller, D Kimbrough; Griebel, Ulrike

2014-09-01

Quantitative comparison of human language and natural animal communication requires improved conceptualizations. We argue that an infrastructural approach to development and evolution incorporating an extended interpretation of the distinctions among illocution, perlocution, and meaning (Austin 1962; Oller and Griebel 2008) can help place the issues relevant to quantitative comparison in perspective. The approach can illuminate the controversy revolving around the notion of functional referentiality as applied to alarm calls, for example in the vervet monkey. We argue that referentiality offers a poor point of quantitative comparison across language and animal communication in the wild. Evidence shows that even newborn human cry could be deemed to show functional referentiality according to the criteria typically invoked by advocates of referentiality in animal communication. Exploring the essence of the idea of illocution, we illustrate an important realm of commonality among animal communication systems and human language, a commonality that opens the door to more productive, quantifiable comparisons. Finally, we delineate two examples of infrastructural communicative capabilities that should be particularly amenable to direct quantitative comparison across humans and our closest relatives.

Closing Down the Conversation: The End of the Quantitative-Qualitative Debate among Educational Inquirers.

ERIC Educational Resources Information Center

Smith, John K.; Heshusius, Lous

Educational researchers have claimed that the quantitative and qualitative approaches to educational inquiry are, indeed, compatible. However, it would be unfortunate to discontinue this debate. The quantitative-qualitative debate began with the interpretive approach to social inquiry. Dilthey argued that since cultural/moral sciences differ from…

A bottom-up approach in estimating the measurement uncertainty and other important considerations for quantitative analyses in drug testing for horses.

PubMed

Leung, Gary N W; Ho, Emmie N M; Kwok, W Him; Leung, David K K; Tang, Francis P W; Wan, Terence S M; Wong, April S Y; Wong, Colton H F; Wong, Jenny K Y; Yu, Nola H

2007-09-07

Quantitative determination, particularly for threshold substances in biological samples, is much more demanding than qualitative identification. A proper assessment of any quantitative determination is the measurement uncertainty (MU) associated with the determined value. The International Standard ISO/IEC 17025, "General requirements for the competence of testing and calibration laboratories", has more prescriptive requirements on the MU than its superseded document, ISO/IEC Guide 25. Under the 2005 or 1999 versions of the new standard, an estimation of the MU is mandatory for all quantitative determinations. To comply with the new requirement, a protocol was established in the authors' laboratory in 2001. The protocol has since evolved based on our practical experience, and a refined version was adopted in 2004. This paper describes our approach in establishing the MU, as well as some other important considerations, for the quantification of threshold substances in biological samples as applied in the area of doping control for horses. The testing of threshold substances can be viewed as a compliance test (or testing to a specified limit). As such, it should only be necessary to establish the MU at the threshold level. The steps in a "Bottom-Up" approach adopted by us are similar to those described in the EURACHEM/CITAC guide, "Quantifying Uncertainty in Analytical Measurement". They involve first specifying the measurand, including the relationship between the measurand and the input quantities upon which it depends. This is followed by identifying all applicable uncertainty contributions using a "cause and effect" diagram. The magnitude of each uncertainty component is then calculated and converted to a standard uncertainty. A recovery study is also conducted to determine if the method bias is significant and whether a recovery (or correction) factor needs to be applied. All standard uncertainties with values greater than 30% of the largest one are then used to

Quantiprot - a Python package for quantitative analysis of protein sequences.

PubMed

Konopka, Bogumił M; Marciniak, Marta; Dyrka, Witold

2017-07-17

The field of protein sequence analysis is dominated by tools rooted in substitution matrices and alignments. A complementary approach is provided by methods of quantitative characterization. A major advantage of the approach is that quantitative properties defines a multidimensional solution space, where sequences can be related to each other and differences can be meaningfully interpreted. Quantiprot is a software package in Python, which provides a simple and consistent interface to multiple methods for quantitative characterization of protein sequences. The package can be used to calculate dozens of characteristics directly from sequences or using physico-chemical properties of amino acids. Besides basic measures, Quantiprot performs quantitative analysis of recurrence and determinism in the sequence, calculates distribution of n-grams and computes the Zipf's law coefficient. We propose three main fields of application of the Quantiprot package. First, quantitative characteristics can be used in alignment-free similarity searches, and in clustering of large and/or divergent sequence sets. Second, a feature space defined by quantitative properties can be used in comparative studies of protein families and organisms. Third, the feature space can be used for evaluating generative models, where large number of sequences generated by the model can be compared to actually observed sequences.

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

Quantitative descriptions of generalized arousal, an elementary function of the vertebrate brain

PubMed Central

Quinkert, Amy Wells; Vimal, Vivek; Weil, Zachary M.; Reeke, George N.; Schiff, Nicholas D.; Banavar, Jayanth R.; Pfaff, Donald W.

2011-01-01

We review a concept of the most primitive, fundamental function of the vertebrate CNS, generalized arousal (GA). Three independent lines of evidence indicate the existence of GA: statistical, genetic, and mechanistic. Here we ask, is this concept amenable to quantitative analysis? Answering in the affirmative, four quantitative approaches have proven useful: (i) factor analysis, (ii) information theory, (iii) deterministic chaos, and (iv) application of a Gaussian equation. It strikes us that, to date, not just one but at least four different quantitative approaches seem necessary for describing different aspects of scientific work on GA. PMID:21555568

How to Measure the Intervention Process? An Assessment of Qualitative and Quantitative Approaches to Data Collection in the Process Evaluation of Organizational Interventions

PubMed Central

Abildgaard, Johan S.; Saksvik, Per Ø.; Nielsen, Karina

2016-01-01

Organizational interventions aiming at improving employee health and wellbeing have proven to be challenging to evaluate. To analyze intervention processes two methodological approaches have widely been used: quantitative (often questionnaire data), or qualitative (often interviews). Both methods are established tools, but their distinct epistemological properties enable them to illuminate different aspects of organizational interventions. In this paper, we use the quantitative and qualitative process data from an organizational intervention conducted in a national postal service, where the Intervention Process Measure questionnaire (N = 285) as well as an extensive interview study (N = 50) were used. We analyze what type of knowledge about intervention processes these two methodologies provide and discuss strengths and weaknesses as well as potentials for mixed methods evaluation methodologies. PMID:27713707

How to Measure the Intervention Process? An Assessment of Qualitative and Quantitative Approaches to Data Collection in the Process Evaluation of Organizational Interventions.

PubMed

Abildgaard, Johan S; Saksvik, Per Ø; Nielsen, Karina

2016-01-01

Organizational interventions aiming at improving employee health and wellbeing have proven to be challenging to evaluate. To analyze intervention processes two methodological approaches have widely been used: quantitative (often questionnaire data), or qualitative (often interviews). Both methods are established tools, but their distinct epistemological properties enable them to illuminate different aspects of organizational interventions. In this paper, we use the quantitative and qualitative process data from an organizational intervention conducted in a national postal service, where the Intervention Process Measure questionnaire ( N = 285) as well as an extensive interview study ( N = 50) were used. We analyze what type of knowledge about intervention processes these two methodologies provide and discuss strengths and weaknesses as well as potentials for mixed methods evaluation methodologies.

Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

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

Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha

2007-10-12

To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation sitemore » and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.« less

Quantitation of Met tyrosine phosphorylation using MRM-MS.

PubMed

Meng, Zhaojing; Srivastava, Apurva K; Zhou, Ming; Veenstra, Timothy

2013-01-01

Phosphorylation has long been accepted as a key cellular regulator of cell signaling pathways. The recent development of multiple-reaction monitoring mass spectrometry (MRM-MS) provides a useful tool for measuring the absolute quantity of phosphorylation occupancy at pivotal sites within signaling proteins, even when the phosphorylation sites are in close proximity. Here, we described a targeted quantitation approach to measure the absolute phosphorylation occupancy at Y1234 and Y1235 of Met. The approach is utilized to obtain absolute occupancy of the two phosphorylation sites in the full-length recombinant Met. It is further applied to quantitate the phosphorylation state of these two sites in SNU-5 cells treated with a Met inhibitor.

A New Approach for Quantitative Evaluation of Ultrasonic Wave Attenuation in Composites

NASA Astrophysics Data System (ADS)

Ni, Qing-Qing; Li, Ran; Xia, Hong

2017-02-01

When ultrasonic waves propagate in composite materials, the propagation behaviors result from the combination effects of various factors, such as material anisotropy and viscoelastic property, internal microstructure and defects, incident wave characteristics and interface condition between composite components. It is essential to make it clear how these factors affect the ultrasonic wave propagation and attenuation characteristics, and how they mutually interact on each other. In the present paper, based on a newly developed time-domain finite element analysis code, PZflex, a unique approach for clarifying the detailed influence mechanism of aforementioned factors is proposed, in which each attenuation component can be extracted from the overall attenuation and analyzed respectively. By taking into consideration the interrelation between each individual attenuation component, the variation behaviors of each component and internal dynamic stress distribution against material anisotropy and matrix viscosity are separately and quantitatively evaluated. From the detailed analysis results of each attenuation component, the energy dissipation at interface is a major component in ultrasonic wave attenuation characteristics, which can provide a maximum contribution rate of 68.2 % to the overall attenuation, and each attenuation component is closely related to the material anisotropy and viscoelasticity. The results clarify the correlation between ultrasonic wave propagation characteristics and material viscoelastic properties, which will be useful in the further development of ultrasonic technology in defect detection.

Quantitative characterisation of sedimentary grains

NASA Astrophysics Data System (ADS)

Tunwal, Mohit; Mulchrone, Kieran F.; Meere, Patrick A.

2016-04-01

Analysis of sedimentary texture helps in determining the formation, transportation and deposition processes of sedimentary rocks. Grain size analysis is traditionally quantitative, whereas grain shape analysis is largely qualitative. A semi-automated approach to quantitatively analyse shape and size of sand sized sedimentary grains is presented. Grain boundaries are manually traced from thin section microphotographs in the case of lithified samples and are automatically identified in the case of loose sediments. Shape and size paramters can then be estimated using a software package written on the Mathematica platform. While automated methodology already exists for loose sediment analysis, the available techniques for the case of lithified samples are limited to cases of high definition thin section microphotographs showing clear contrast between framework grains and matrix. Along with the size of grain, shape parameters such as roundness, angularity, circularity, irregularity and fractal dimension are measured. A new grain shape parameter developed using Fourier descriptors has also been developed. To test this new approach theoretical examples were analysed and produce high quality results supporting the accuracy of the algorithm. Furthermore sandstone samples from known aeolian and fluvial environments from the Dingle Basin, County Kerry, Ireland were collected and analysed. Modern loose sediments from glacial till from County Cork, Ireland and aeolian sediments from Rajasthan, India have also been collected and analysed. A graphical summary of the data is presented and allows for quantitative distinction between samples extracted from different sedimentary environments.

Using Live-Crown Ratio to Control Wood Quality: An Example of Quantitative Silviculture

Treesearch

Thomas J. Dean

1999-01-01

Quantitative silviculture is the application of biological relationships in meeting specific, quantitative management objectives. It is a two-sided approach requiring the identification and application of biological relationships. An example of quantitative silviculture is presented that uses a relationship between average-live crown ratio and relative stand density...

Mixing qualitative and quantitative research in developmental science: uses and methodological choices.

PubMed

Yoshikawa, Hirokazu; Weisner, Thomas S; Kalil, Ariel; Way, Niobe

2008-03-01

Multiple methods are vital to understanding development as a dynamic, transactional process. This article focuses on the ways in which quantitative and qualitative methodologies can be combined to enrich developmental science and the study of human development, focusing on the practical questions of "when" and "how." Research situations that may be especially suited to mixing qualitative and quantitative approaches are described. The authors also discuss potential choices for using mixed quantitative- qualitative approaches in study design, sampling, construction of measures or interview protocols, collaborations, and data analysis relevant to developmental science. Finally, they discuss some common pitfalls that occur in mixing these methods and include suggestions for surmounting them.

The selected reaction monitoring/multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases.

PubMed

Gianazza, Erica; Tremoli, Elena; Banfi, Cristina

2014-12-01

Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.

Allelic-based gene-gene interaction associated with quantitative traits.

PubMed

Jung, Jeesun; Sun, Bin; Kwon, Deukwoo; Koller, Daniel L; Foroud, Tatiana M

2009-05-01

Recent studies have shown that quantitative phenotypes may be influenced not only by multiple single nucleotide polymorphisms (SNPs) within a gene but also by the interaction between SNPs at unlinked genes. We propose a new statistical approach that can detect gene-gene interactions at the allelic level which contribute to the phenotypic variation in a quantitative trait. By testing for the association of allelic combinations at multiple unlinked loci with a quantitative trait, we can detect the SNP allelic interaction whether or not it can be detected as a main effect. Our proposed method assigns a score to unrelated subjects according to their allelic combination inferred from observed genotypes at two or more unlinked SNPs, and then tests for the association of the allelic score with a quantitative trait. To investigate the statistical properties of the proposed method, we performed a simulation study to estimate type I error rates and power and demonstrated that this allelic approach achieves greater power than the more commonly used genotypic approach to test for gene-gene interaction. As an example, the proposed method was applied to data obtained as part of a candidate gene study of sodium retention by the kidney. We found that this method detects an interaction between the calcium-sensing receptor gene (CaSR), the chloride channel gene (CLCNKB) and the Na, K, 2Cl cotransporter gene (CLC12A1) that contributes to variation in diastolic blood pressure.

Integrated Environmental Modeling: Quantitative Microbial Risk Assessment

EPA Science Inventory

The presentation discusses the need for microbial assessments and presents a road map associated with quantitative microbial risk assessments, through an integrated environmental modeling approach. A brief introduction and the strengths of the current knowledge are illustrated. W...

Quantitative approaches to information recovery from black holes

NASA Astrophysics Data System (ADS)

Balasubramanian, Vijay; Czech, Bartłomiej

2011-08-01

The evaporation of black holes into apparently thermal radiation poses a serious conundrum for theoretical physics: at face value, it appears that in the presence of a black hole, quantum evolution is non-unitary and destroys information. This information loss paradox has its seed in the presence of a horizon causally separating the interior and asymptotic regions in a black hole spacetime. A quantitative resolution of the paradox could take several forms: (a) a precise argument that the underlying quantum theory is unitary, and that information loss must be an artifact of approximations in the derivation of black hole evaporation, (b) an explicit construction showing how information can be recovered by the asymptotic observer, (c) a demonstration that the causal disconnection of the black hole interior from infinity is an artifact of the semiclassical approximation. This review summarizes progress on all these fronts.

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk

EPA Science Inventory

This is the second of two reports from the International Workshops on Genotoxicity Testing (IWGT) Working Group on Quantitative Approaches to Genetic Toxicology Risk Assessment (the QWG). The first report summarized the discussions and recommendations of the QWG related to the ne...

Optimization of Robust HPLC Method for Quantitation of Ambroxol Hydrochloride and Roxithromycin Using a DoE Approach.

PubMed

Patel, Rashmin B; Patel, Nilay M; Patel, Mrunali R; Solanki, Ajay B

2017-03-01

The aim of this work was to develop and optimize a robust HPLC method for the separation and quantitation of ambroxol hydrochloride and roxithromycin utilizing Design of Experiment (DoE) approach. The Plackett-Burman design was used to assess the impact of independent variables (concentration of organic phase, mobile phase pH, flow rate and column temperature) on peak resolution, USP tailing and number of plates. A central composite design was utilized to evaluate the main, interaction, and quadratic effects of independent variables on the selected dependent variables. The optimized HPLC method was validated based on ICH Q2R1 guideline and was used to separate and quantify ambroxol hydrochloride and roxithromycin in tablet formulations. The findings showed that DoE approach could be effectively applied to optimize a robust HPLC method for quantification of ambroxol hydrochloride and roxithromycin in tablet formulations. Statistical comparison between results of proposed and reported HPLC method revealed no significant difference; indicating the ability of proposed HPLC method for analysis of ambroxol hydrochloride and roxithromycin in pharmaceutical formulations. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Proteomic profiling in MPTP monkey model for early Parkinson disease biomarker discovery

PubMed Central

Lin, Xiangmin; Shi, Min; Gunasingh Masilamoni, Jeyaraj; Dator, Romel; Movius, James; Aro, Patrick; Smith, Yoland; Zhang, Jing

2015-01-01

Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology. PMID:25617661

Absolute activity quantitation from projections using an analytical approach: comparison with iterative methods in Tc-99m and I-123 brain SPECT

NASA Astrophysics Data System (ADS)

Fakhri, G. El; Kijewski, M. F.; Moore, S. C.

2001-06-01

Estimates of SPECT activity within certain deep brain structures could be useful for clinical tasks such as early prediction of Alzheimer's disease with Tc-99m or Parkinson's disease with I-123; however, such estimates are biased by poor spatial resolution and inaccurate scatter and attenuation corrections. We compared an analytical approach (AA) of more accurate quantitation to a slower iterative approach (IA). Monte Carlo simulated projections of 12 normal and 12 pathologic Tc-99m perfusion studies, as well as 12, normal and 12 pathologic I-123 neurotransmission studies, were generated using a digital brain phantom and corrected for scatter by a multispectral fitting procedure. The AA included attenuation correction by a modified Metz-Fan algorithm and activity estimation by a technique that incorporated Metz filtering to compensate for variable collimator response (VCR), IA-modeled attenuation, and VCR in the projector/backprojector of an ordered subsets-expectation maximization (OSEM) algorithm. Bias and standard deviation over the 12 normal and 12 pathologic patients were calculated with respect to the reference values in the corpus callosum, caudate nucleus, and putamen. The IA and AA yielded similar quantitation results in both Tc-99m and I-123 studies in all brain structures considered in both normal and pathologic patients. The bias with respect to the reference activity distributions was less than 7% for Tc-99m studies, but greater than 30% for I-123 studies, due to partial volume effect in the striata. Our results were validated using I-123 physical acquisitions of an anthropomorphic brain phantom. The IA yielded quantitation accuracy comparable to that obtained with IA, while requiring much less processing time. However, in most conditions, IA yielded lower noise for the same bias than did AA.

Quantitative Predictive Models for Systemic Toxicity (SOT)

EPA Science Inventory

Models to identify systemic and specific target organ toxicity were developed to help transition the field of toxicology towards computational models. By leveraging multiple data sources to incorporate read-across and machine learning approaches, a quantitative model of systemic ...

Quantitative Determination of Aluminum in Deodorant Brands: A Guided Inquiry Learning Experience in Quantitative Analysis Laboratory

ERIC Educational Resources Information Center

Sedwick, Victoria; Leal, Anne; Turner, Dea; Kanu, A. Bakarr

2018-01-01

The monitoring of metals in commercial products is essential for protecting public health against the hazards of metal toxicity. This article presents a guided inquiry (GI) experimental lab approach in a quantitative analysis lab class that enabled students' to determine the levels of aluminum in deodorant brands. The utility of a GI experimental…

Analyzing the impacts of global trade and investment on non-communicable diseases and risk factors: a critical review of methodological approaches used in quantitative analyses.

PubMed

Cowling, Krycia; Thow, Anne Marie; Pollack Porter, Keshia

2018-05-24

A key mechanism through which globalization has impacted health is the liberalization of trade and investment, yet relatively few studies to date have used quantitative methods to investigate the impacts of global trade and investment policies on non-communicable diseases and risk factors. Recent reviews of this literature have found heterogeneity in results and a range of quality across studies, which may be in part attributable to a lack of conceptual clarity and methodological inconsistencies. This study is a critical review of methodological approaches used in the quantitative literature on global trade and investment and diet, tobacco, alcohol, and related health outcomes, with the objective of developing recommendations and providing resources to guide future robust, policy relevant research. A review of reviews, expert review, and reference tracing were employed to identify relevant studies, which were evaluated using a novel quality assessment tool designed for this research. Eight review articles and 34 quantitative studies were identified for inclusion. Important ways to improve this literature were identified and discussed: clearly defining exposures of interest and not conflating trade and investment; exploring mechanisms of broader relationships; increasing the use of individual-level data; ensuring consensus and consistency in key confounding variables; utilizing more sector-specific versus economy-wide trade and investment indicators; testing and adequately adjusting for autocorrelation and endogeneity when using longitudinal data; and presenting results from alternative statistical models and sensitivity analyses. To guide the development of future analyses, recommendations for international data sources for selected trade and investment indicators, as well as key gaps in the literature, are presented. More methodologically rigorous and consistent approaches in future quantitative studies on the impacts of global trade and investment policies on non

Applying quantitative benefit-risk analysis to aid regulatory decision making in diagnostic imaging: methods, challenges, and opportunities.

PubMed

Agapova, Maria; Devine, Emily Beth; Bresnahan, Brian W; Higashi, Mitchell K; Garrison, Louis P

2014-09-01

Health agencies making regulatory marketing-authorization decisions use qualitative and quantitative approaches to assess expected benefits and expected risks associated with medical interventions. There is, however, no universal standard approach that regulatory agencies consistently use to conduct benefit-risk assessment (BRA) for pharmaceuticals or medical devices, including for imaging technologies. Economics, health services research, and health outcomes research use quantitative approaches to elicit preferences of stakeholders, identify priorities, and model health conditions and health intervention effects. Challenges to BRA in medical devices are outlined, highlighting additional barriers in radiology. Three quantitative methods--multi-criteria decision analysis, health outcomes modeling and stated-choice survey--are assessed using criteria that are important in balancing benefits and risks of medical devices and imaging technologies. To be useful in regulatory BRA, quantitative methods need to: aggregate multiple benefits and risks, incorporate qualitative considerations, account for uncertainty, and make clear whose preferences/priorities are being used. Each quantitative method performs differently across these criteria and little is known about how BRA estimates and conclusions vary by approach. While no specific quantitative method is likely to be the strongest in all of the important areas, quantitative methods may have a place in BRA of medical devices and radiology. Quantitative BRA approaches have been more widely applied in medicines, with fewer BRAs in devices. Despite substantial differences in characteristics of pharmaceuticals and devices, BRA methods may be as applicable to medical devices and imaging technologies as they are to pharmaceuticals. Further research to guide the development and selection of quantitative BRA methods for medical devices and imaging technologies is needed. Copyright © 2014 AUR. Published by Elsevier Inc. All rights

A resilience-oriented approach for quantitatively assessing recurrent spatial-temporal congestion on urban roads.

PubMed

Tang, Junqing; Heinimann, Hans Rudolf

2018-01-01

Traffic congestion brings not only delay and inconvenience, but other associated national concerns, such as greenhouse gases, air pollutants, road safety issues and risks. Identification, measurement, tracking, and control of urban recurrent congestion are vital for building a livable and smart community. A considerable amount of works has made contributions to tackle the problem. Several methods, such as time-based approaches and level of service, can be effective for characterizing congestion on urban streets. However, studies with systemic perspectives have been minor in congestion quantification. Resilience, on the other hand, is an emerging concept that focuses on comprehensive systemic performance and characterizes the ability of a system to cope with disturbance and to recover its functionality. In this paper, we symbolized recurrent congestion as internal disturbance and proposed a modified metric inspired by the well-applied "R4" resilience-triangle framework. We constructed the metric with generic dimensions from both resilience engineering and transport science to quantify recurrent congestion based on spatial-temporal traffic patterns and made the comparison with other two approaches in freeway and signal-controlled arterial cases. Results showed that the metric can effectively capture congestion patterns in the study area and provides a quantitative benchmark for comparison. Also, it suggested not only a good comparative performance in measuring strength of proposed metric, but also its capability of considering the discharging process in congestion. The sensitivity tests showed that proposed metric possesses robustness against parameter perturbation in Robustness Range (RR), but the number of identified congestion patterns can be influenced by the existence of ϵ. In addition, the Elasticity Threshold (ET) and the spatial dimension of cell-based platform differ the congestion results significantly on both the detected number and intensity. By tackling

Targeted Quantitation of Proteins by Mass Spectrometry

PubMed Central

2013-01-01

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332

Targeted quantitation of proteins by mass spectrometry.

PubMed

Liebler, Daniel C; Zimmerman, Lisa J

2013-06-04

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement.

The Functional Resonance Analysis Method for a systemic risk based environmental auditing in a sinter plant: A semi-quantitative approach

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

Patriarca, Riccardo, E-mail: riccardo.patriarca@uniroma1.it; Di Gravio, Giulio; Costantino, Francesco

Environmental auditing is a main issue for any production plant and assessing environmental performance is crucial to identify risks factors. The complexity of current plants arises from interactions among technological, human and organizational system components, which are often transient and not easily detectable. The auditing thus requires a systemic perspective, rather than focusing on individual behaviors, as emerged in recent research in the safety domain for socio-technical systems. We explore the significance of modeling the interactions of system components in everyday work, by the application of a recent systemic method, i.e. the Functional Resonance Analysis Method (FRAM), in order tomore » define dynamically the system structure. We present also an innovative evolution of traditional FRAM following a semi-quantitative approach based on Monte Carlo simulation. This paper represents the first contribution related to the application of FRAM in the environmental context, moreover considering a consistent evolution based on Monte Carlo simulation. The case study of an environmental risk auditing in a sinter plant validates the research, showing the benefits in terms of identifying potential critical activities, related mitigating actions and comprehensive environmental monitoring indicators. - Highlights: • We discuss the relevance of a systemic risk based environmental audit. • We present FRAM to represent functional interactions of the system. • We develop a semi-quantitative FRAM framework to assess environmental risks. • We apply the semi-quantitative FRAM framework to build a model for a sinter plant.« less

Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).

PubMed

Xiao, Kunhong; Sun, Jinpeng; Kim, Jihee; Rajagopal, Sudarshan; Zhai, Bo; Villén, Judit; Haas, Wilhelm; Kovacs, Jeffrey J; Shukla, Arun K; Hara, Makoto R; Hernandez, Marylens; Lachmann, Alexander; Zhao, Shan; Lin, Yuan; Cheng, Yishan; Mizuno, Kensaku; Ma'ayan, Avi; Gygi, Steven P; Lefkowitz, Robert J

2010-08-24

beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area

Toward best practices in data processing and analysis for intact biotherapeutics by MS in quantitative bioanalysis.

PubMed

Kellie, John F; Kehler, Jonathan R; Karlinsey, Molly Z; Summerfield, Scott G

2017-12-01

Typically, quantitation of biotherapeutics from biological matrices by LC-MS is based on a surrogate peptide approach to determine molecule concentration. Recent efforts have focused on quantitation of the intact protein molecules or larger mass subunits of monoclonal antibodies. To date, there has been limited guidance for large or intact protein mass quantitation for quantitative bioanalysis. Intact- and subunit-level analyses of biotherapeutics from biological matrices are performed at 12-25 kDa mass range with quantitation data presented. Linearity, bias and other metrics are presented along with recommendations made on the viability of existing quantitation approaches. This communication is intended to start a discussion around intact protein data analysis and processing, recognizing that other published contributions will be required.

Family-centred approaches to healthcare interventions in chronic diseases in adults: a quantitative systematic review.

PubMed

Deek, Hiba; Hamilton, Sandra; Brown, Nicola; Inglis, Sally C; Digiacomo, Michelle; Newton, Phillip J; Noureddine, Samar; MacDonald, Peter S; Davidson, Patricia M

2016-05-01

Increasingly there is a focus on self-care strategies for both malignant and non-malignant conditions. Models of self-care interventions have focussed on the individual and less on the broader context of family and society. In many societies, decision-making and health seeking behaviours, involve family members. To identify elements of effective family-centred self-care interventions that are likely to improve outcomes of adults living with chronic conditions. Review paper. MEDLINE (Ovid), CINAHL, Academic Search Complete, PsychInfo and Scopus between 2000-2014. Quantitative studies targeting patient outcomes through family-centred interventions in adults were retrieved using systematic methods in January, 2015. Search terms used were: 'family', 'spouse', 'carer', 'caregiver', 'chronic', 'chronic disease', 'self-care', 'self-management' and 'self-efficacy'. Reference lists were reviewed. Risk of bias assessment was performed using the Cochrane Collaboration's tool. Data were reported using a narrative summary approach. Ten studies were identified. Improvements were noted in readmission rates, emergency department presentations, and anxiety levels using family-centred interventions compared with controls. Elements of effective interventions used were a family-centred approach, active learning strategy and transitional care with appropriate follow-up. Involving the family in self-care has shown some positive results for patients with chronic conditions. The benefits of family-centred care may be more likely in specific socio-cultural contexts. The review has year limits and further research needs to identify support for both the patients and family caregivers. © 2016 John Wiley & Sons Ltd.

Novel quantitative calibration approach for multi-configuration electromagnetic induction (EMI) systems using data acquired at multiple elevations

NASA Astrophysics Data System (ADS)

Tan, Xihe; Mester, Achim; von Hebel, Christian; van der Kruk, Jan; Zimmermann, Egon; Vereecken, Harry; van Waasen, Stefan

2017-04-01

Electromagnetic induction (EMI) systems offer a great potential to obtain highly resolved layered electrical conductivity models of the shallow subsurface. State-of-the-art inversion procedures require quantitative calibration of EMI data, especially for short-offset EMI systems where significant data shifts are often observed. These shifts are caused by external influences such as the presence of the operator, zero-leveling procedures, the field setup used to move the EMI system and/or cables close by. Calibrations can be performed by using collocated electrical resistivity measurements or taking soil samples, however, these two methods take a lot of time in the field. To improve the calibration in a fast and concise way, we introduce a novel on-site calibration method using a series of apparent electrical conductivity (ECa) values acquired at multiple elevations for a multi-configuration EMI system. No additional instrument or pre-knowledge of the subsurface is needed to acquire quantitative ECa data. By using this calibration method, we correct each coil configuration, i.e., transmitter and receiver coil separation and the horizontal or vertical coplanar (HCP or VCP) coil orientation with a unique set of calibration parameters. A multi-layer soil structure at the corresponding measurement location is inverted together with the calibration parameters using full-solution Maxwell equations for the forward modelling within the shuffled complex evolution (SCE) algorithm to find the optimum solution under a user-defined parameter space. Synthetic data verified the feasibility for calibrating HCP and VCP measurements of a custom made six-coil EMI system with coil offsets between 0.35 m and 1.8 m for quantitative data inversions. As a next step, we applied the calibration approach on acquired experimental data from a bare soil test field (Selhausen, Germany) for the considered EMI system. The obtained calibration parameters were applied to measurements over a 30 m

Subjective Quantitative Studies of Human Agency

ERIC Educational Resources Information Center

Alkire, Sabina

2005-01-01

Amartya Sen's writings have articulated the importance of human agency, and identified the need for information on agency freedom to inform our evaluation of social arrangements. Many approaches to poverty reduction stress the need for empowerment. This paper reviews "subjective quantitative measures of human agency at the individual level." It…

Differences between genders in colorectal morphology on CT colonography using a quantitative approach: a pilot study.

PubMed

Weber, Charles N; Poff, Jason A; Lev-Toaff, Anna S; Levine, Marc S; Zafar, Hanna M

To explore quantitative differences between genders in morphologic colonic metrics and determine metric reproducibility. Quantitative colonic metrics from 20 male and 20 female CTC datasets were evaluated twice by two readers; all exams were performed after incomplete optical colonoscopy. Intra-/inter-reader reliability was measured with intraclass correlation coefficient (ICC) and concordance correlation coefficient (CCC). Women had overall decreased colonic volume, increased tortuosity and compactness and lower sigmoid apex height on CTC compared to men (p<0.0001,all). Quantitative measurements in colonic metrics were highly reproducible (ICC=0.9989 and 0.9970; CCC=0.9945). Quantitative morphologic differences between genders can be reproducibility measured. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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.

Approaches to acceptable risk

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

Whipple, C

Several alternative approaches to address the question {open_quotes}How safe is safe enough?{close_quotes} are reviewed and an attempt is made to apply the reasoning behind these approaches to the issue of acceptability of radiation exposures received in space. The approaches to the issue of the acceptability of technological risk described here are primarily analytical, and are drawn from examples in the management of environmental health risks. These include risk-based approaches, in which specific quantitative risk targets determine the acceptability of an activity, and cost-benefit and decision analysis, which generally focus on the estimation and evaluation of risks, benefits and costs, inmore » a framework that balances these factors against each other. These analytical methods tend by their quantitative nature to emphasize the magnitude of risks, costs and alternatives, and to downplay other factors, especially those that are not easily expressed in quantitative terms, that affect acceptance or rejection of risk. Such other factors include the issues of risk perceptions and how and by whom risk decisions are made.« less

Self-organisation in Cellular Automata with Coalescent Particles: Qualitative and Quantitative Approaches

NASA Astrophysics Data System (ADS)

Hellouin de Menibus, Benjamin; Sablik, Mathieu

2017-06-01

This article introduces new tools to study self-organisation in a family of simple cellular automata which contain some particle-like objects with good collision properties (coalescence) in their time evolution. We draw an initial configuration at random according to some initial shift-ergodic measure, and use the limit measure to describe the asymptotic behaviour of the automata. We first take a qualitative approach, i.e. we obtain information on the limit measure(s). We prove that only particles moving in one particular direction can persist asymptotically. This provides some previously unknown information on the limit measures of various deterministic and probabilistic cellular automata: 3 and 4-cyclic cellular automata [introduced by Fisch (J Theor Probab 3(2):311-338, 1990; Phys D 45(1-3):19-25, 1990)], one-sided captive cellular automata [introduced by Theyssier (Captive Cellular Automata, 2004)], the majority-traffic cellular automaton, a self stabilisation process towards a discrete line [introduced by Regnault and Rémila (in: Mathematical Foundations of Computer Science 2015—40th International Symposium, MFCS 2015, Milan, Italy, Proceedings, Part I, 2015)]. In a second time we restrict our study to a subclass, the gliders cellular automata. For this class we show quantitative results, consisting in the asymptotic law of some parameters: the entry times [generalising K ůrka et al. (in: Proceedings of AUTOMATA, 2011)], the density of particles and the rate of convergence to the limit measure.

Post-event reviews: Using a quantitative approach for analysing incident response to demonstrate the value of business continuity programmes and increase planning efficiency.

PubMed

Vaidyanathan, Karthik

2017-01-01

Business continuity management is often thought of as a proactive planning process for minimising impact from large-scale incidents and disasters. While this is true, and it is critical to plan for the worst, consistently validating plan effectiveness against smaller disruptions can enable an organisation to gain key insights about its business continuity readiness, drive programme improvements, reduce costs and provide an opportunity to quantitatively demonstrate the value of the programme to management. This paper describes a post mortem framework which is used as a continuous improvement mechanism for tracking, reviewing and learning from real-world events at Microsoft Customer Service & Support. This approach was developed and adopted because conducting regular business continuity exercises proved difficult and expensive in a complex and distributed operations environment with high availability requirements. Using a quantitative approach to measure response to incidents, and categorising outcomes based on such responses, enables business continuity teams to provide data-driven insights to leadership, change perceptions of incident root cause, and instil a higher level of confidence towards disaster response readiness and incident management. The scope of the framework discussed here is specific to reviewing and driving improvements from operational incidents. However, the concept can be extended to learning and evolving readiness plans for other types of incidents.

A transformative model for undergraduate quantitative biology education.

PubMed

Usher, David C; Driscoll, Tobin A; Dhurjati, Prasad; Pelesko, John A; Rossi, Louis F; Schleiniger, Gilberto; Pusecker, Kathleen; White, Harold B

2010-01-01

The BIO2010 report recommended that students in the life sciences receive a more rigorous education in mathematics and physical sciences. The University of Delaware approached this problem by (1) developing a bio-calculus section of a standard calculus course, (2) embedding quantitative activities into existing biology courses, and (3) creating a new interdisciplinary major, quantitative biology, designed for students interested in solving complex biological problems using advanced mathematical approaches. To develop the bio-calculus sections, the Department of Mathematical Sciences revised its three-semester calculus sequence to include differential equations in the first semester and, rather than using examples traditionally drawn from application domains that are most relevant to engineers, drew models and examples heavily from the life sciences. The curriculum of the B.S. degree in Quantitative Biology was designed to provide students with a solid foundation in biology, chemistry, and mathematics, with an emphasis on preparation for research careers in life sciences. Students in the program take core courses from biology, chemistry, and physics, though mathematics, as the cornerstone of all quantitative sciences, is given particular prominence. Seminars and a capstone course stress how the interplay of mathematics and biology can be used to explain complex biological systems. To initiate these academic changes required the identification of barriers and the implementation of solutions.

A Transformative Model for Undergraduate Quantitative Biology Education

PubMed Central

Driscoll, Tobin A.; Dhurjati, Prasad; Pelesko, John A.; Rossi, Louis F.; Schleiniger, Gilberto; Pusecker, Kathleen; White, Harold B.

2010-01-01

The BIO2010 report recommended that students in the life sciences receive a more rigorous education in mathematics and physical sciences. The University of Delaware approached this problem by (1) developing a bio-calculus section of a standard calculus course, (2) embedding quantitative activities into existing biology courses, and (3) creating a new interdisciplinary major, quantitative biology, designed for students interested in solving complex biological problems using advanced mathematical approaches. To develop the bio-calculus sections, the Department of Mathematical Sciences revised its three-semester calculus sequence to include differential equations in the first semester and, rather than using examples traditionally drawn from application domains that are most relevant to engineers, drew models and examples heavily from the life sciences. The curriculum of the B.S. degree in Quantitative Biology was designed to provide students with a solid foundation in biology, chemistry, and mathematics, with an emphasis on preparation for research careers in life sciences. Students in the program take core courses from biology, chemistry, and physics, though mathematics, as the cornerstone of all quantitative sciences, is given particular prominence. Seminars and a capstone course stress how the interplay of mathematics and biology can be used to explain complex biological systems. To initiate these academic changes required the identification of barriers and the implementation of solutions. PMID:20810949

A Quantitative and Combinatorial Approach to Non-Linear Meanings of Multiplication

ERIC Educational Resources Information Center

Tillema, Erik; Gatza, Andrew

2016-01-01

We provide a conceptual analysis of how combinatorics problems have the potential to support students to establish non-linear meanings of multiplication (NLMM). The problems we analyze we have used in a series of studies with 6th, 8th, and 10th grade students. We situate the analysis in prior work on students' quantitative and multiplicative…

A network-based approach for semi-quantitative knowledge mining and its application to yield variability

NASA Astrophysics Data System (ADS)

Schauberger, Bernhard; Rolinski, Susanne; Müller, Christoph

2016-12-01

Variability of crop yields is detrimental for food security. Under climate change its amplitude is likely to increase, thus it is essential to understand the underlying causes and mechanisms. Crop models are the primary tool to project future changes in crop yields under climate change. A systematic overview of drivers and mechanisms of crop yield variability (YV) can thus inform crop model development and facilitate improved understanding of climate change impacts on crop yields. Yet there is a vast body of literature on crop physiology and YV, which makes a prioritization of mechanisms for implementation in models challenging. Therefore this paper takes on a novel approach to systematically mine and organize existing knowledge from the literature. The aim is to identify important mechanisms lacking in models, which can help to set priorities in model improvement. We structure knowledge from the literature in a semi-quantitative network. This network consists of complex interactions between growing conditions, plant physiology and crop yield. We utilize the resulting network structure to assign relative importance to causes of YV and related plant physiological processes. As expected, our findings confirm existing knowledge, in particular on the dominant role of temperature and precipitation, but also highlight other important drivers of YV. More importantly, our method allows for identifying the relevant physiological processes that transmit variability in growing conditions to variability in yield. We can identify explicit targets for the improvement of crop models. The network can additionally guide model development by outlining complex interactions between processes and by easily retrieving quantitative information for each of the 350 interactions. We show the validity of our network method as a structured, consistent and scalable dictionary of literature. The method can easily be applied to many other research fields.

A new systematic and quantitative approach to characterization of surface nanostructures using fuzzy logic

NASA Astrophysics Data System (ADS)

Al-Mousa, Amjed A.

Thin films are essential constituents of modern electronic devices and have a multitude of applications in such devices. The impact of the surface morphology of thin films on the device characteristics where these films are used has generated substantial attention to advanced film characterization techniques. In this work, we present a new approach to characterize surface nanostructures of thin films by focusing on isolating nanostructures and extracting quantitative information, such as the shape and size of the structures. This methodology is applicable to any Scanning Probe Microscopy (SPM) data, such as Atomic Force Microscopy (AFM) data which we are presenting here. The methodology starts by compensating the AFM data for some specific classes of measurement artifacts. After that, the methodology employs two distinct techniques. The first, which we call the overlay technique, proceeds by systematically processing the raster data that constitute the scanning probe image in both vertical and horizontal directions. It then proceeds by classifying points in each direction separately. Finally, the results from both the horizontal and the vertical subsets are overlaid, where a final decision on each surface point is made. The second technique, based on fuzzy logic, relies on a Fuzzy Inference Engine (FIE) to classify the surface points. Once classified, these points are clustered into surface structures. The latter technique also includes a mechanism which can consistently distinguish crowded surfaces from those with sparsely distributed structures and then tune the fuzzy technique system uniquely for that surface. Both techniques have been applied to characterize organic semiconductor thin films of pentacene on different substrates. Also, we present a case study to demonstrate the effectiveness of our methodology to identify quantitatively particle sizes of two specimens of gold nanoparticles of different nominal dimensions dispersed on a mica surface. A comparison

Past, Present, and Future of Critical Quantitative Research in Higher Education

ERIC Educational Resources Information Center

Wells, Ryan S.; Stage, Frances K.

2014-01-01

This chapter discusses the evolution of the critical quantitative paradigm with an emphasis on extending this approach to new populations and new methods. Along with this extension of critical quantitative work, however, come continued challenges and tensions for researchers. This chapter recaps and responds to each chapter in the volume, and…

Quantitative analysis of arm movement smoothness

NASA Astrophysics Data System (ADS)

Szczesna, Agnieszka; Błaszczyszyn, Monika

2017-07-01

The paper deals with the problem of motion data quantitative smoothness analysis. We investigated values of movement unit, fluidity and jerk for healthy and paralyzed arm of patients with hemiparesis after stroke. Patients were performing drinking task. To validate the approach, movement of 24 patients were captured using optical motion capture system.

Quantitative maps of genetic interactions in yeast - comparative evaluation and integrative analysis.

PubMed

Lindén, Rolf O; Eronen, Ville-Pekka; Aittokallio, Tero

2011-03-24

High-throughput genetic screening approaches have enabled systematic means to study how interactions among gene mutations contribute to quantitative fitness phenotypes, with the aim of providing insights into the functional wiring diagrams of genetic interaction networks on a global scale. However, it is poorly known how well these quantitative interaction measurements agree across the screening approaches, which hinders their integrated use toward improving the coverage and quality of the genetic interaction maps in yeast and other organisms. Using large-scale data matrices from epistatic miniarray profiling (E-MAP), genetic interaction mapping (GIM), and synthetic genetic array (SGA) approaches, we carried out here a systematic comparative evaluation among these quantitative maps of genetic interactions in yeast. The relatively low association between the original interaction measurements or their customized scores could be improved using a matrix-based modelling framework, which enables the use of single- and double-mutant fitness estimates and measurements, respectively, when scoring genetic interactions. Toward an integrative analysis, we show how the detections from the different screening approaches can be combined to suggest novel positive and negative interactions which are complementary to those obtained using any single screening approach alone. The matrix approximation procedure has been made available to support the design and analysis of the future screening studies. We have shown here that even if the correlation between the currently available quantitative genetic interaction maps in yeast is relatively low, their comparability can be improved by means of our computational matrix approximation procedure, which will enable integrative analysis and detection of a wider spectrum of genetic interactions using data from the complementary screening approaches.

Effectiveness of a systematic approach to promote intersectoral collaboration in comprehensive school health promotion-a multiple-case study using quantitative and qualitative data.

PubMed

Pucher, Katharina K; Candel, Math J J M; Krumeich, Anja; Boot, Nicole M W M; De Vries, Nanne K

2015-07-05

We report on the longitudinal quantitative and qualitative data resulting from a two-year trajectory (2008-2011) based on the DIagnosis of Sustainable Collaboration (DISC) model. This trajectory aimed to support regional coordinators of comprehensive school health promotion (CSHP) in systematically developing change management and project management to establish intersectoral collaboration. Multilevel analyses of quantitative data on the determinants of collaborations according to the DISC model were done, with 90 respondents (response 57 %) at pretest and 69 respondents (52 %) at posttest. Nvivo analyses of the qualitative data collected during the trajectory included minutes of monthly/bimonthly personal/telephone interviews (N = 65) with regional coordinators, and documents they produced about their activities. Quantitative data showed major improvements in change management and project management. There were also improvements in consensus development, commitment formation, formalization of the CSHP, and alignment of policies, although organizational problems within the collaboration increased. Content analyses of qualitative data identified five main management styles, including (1) facilitating active involvement of relevant parties; (2) informing collaborating parties; (3) controlling and (4) supporting their task accomplishment; and (5) coordinating the collaborative processes. We have contributed to the fundamental understanding of the development of intersectoral collaboration by combining qualitative and quantitative data. Our results support a systematic approach to intersectoral collaboration using the DISC model. They also suggest five main management styles to improve intersectoral collaboration in the initial stage. The outcomes are useful for health professionals involved in similar ventures.

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

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.

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.

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.

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

Multiple-Strain Approach and Probabilistic Modeling of Consumer Habits in Quantitative Microbial Risk Assessment: A Quantitative Assessment of Exposure to Staphylococcal Enterotoxin A in Raw Milk.

PubMed

Crotta, Matteo; Rizzi, Rita; Varisco, Giorgio; Daminelli, Paolo; Cunico, Elena Cosciani; Luini, Mario; Graber, Hans Ulrich; Paterlini, Franco; Guitian, Javier

2016-03-01

Quantitative microbial risk assessment (QMRA) models are extensively applied to inform management of a broad range of food safety risks. Inevitably, QMRA modeling involves an element of simplification of the biological process of interest. Two features that are frequently simplified or disregarded are the pathogenicity of multiple strains of a single pathogen and consumer behavior at the household level. In this study, we developed a QMRA model with a multiple-strain approach and a consumer phase module (CPM) based on uncertainty distributions fitted from field data. We modeled exposure to staphylococcal enterotoxin A in raw milk in Lombardy; a specific enterotoxin production module was thus included. The model is adaptable and could be used to assess the risk related to other pathogens in raw milk as well as other staphylococcal enterotoxins. The multiplestrain approach, implemented as a multinomial process, allowed the inclusion of variability and uncertainty with regard to pathogenicity at the bacterial level. Data from 301 questionnaires submitted to raw milk consumers were used to obtain uncertainty distributions for the CPM. The distributions were modeled to be easily updatable with further data or evidence. The sources of uncertainty due to the multiple-strain approach and the CPM were identified, and their impact on the output was assessed by comparing specific scenarios to the baseline. When the distributions reflecting the uncertainty in consumer behavior were fixed to the 95th percentile, the risk of exposure increased up to 160 times. This reflects the importance of taking into consideration the diversity of consumers' habits at the household level and the impact that the lack of knowledge about variables in the CPM can have on the final QMRA estimates. The multiple-strain approach lends itself to use in other food matrices besides raw milk and allows the model to better capture the complexity of the real world and to be capable of geographical

RECENT ADVANCES IN QUANTITATIVE NEUROPROTEOMICS

PubMed Central

Craft, George E; Chen, Anshu; Nairn, Angus C

2014-01-01

The field of proteomics is undergoing rapid development in a number of different areas including improvements in mass spectrometric platforms, peptide identification algorithms and bioinformatics. In particular, new and/or improved approaches have established robust methods that not only allow for in-depth and accurate peptide and protein identification and modification, but also allow for sensitive measurement of relative or absolute quantitation. These methods are beginning to be applied to the area of neuroproteomics, but the central nervous system poses many specific challenges in terms of quantitative proteomics, given the large number of different neuronal cell types that are intermixed and that exhibit distinct patterns of gene and protein expression. This review highlights the recent advances that have been made in quantitative neuroproteomics, with a focus on work published over the last five years that applies emerging methods to normal brain function as well as to various neuropsychiatric disorders including schizophrenia and drug addiction as well as of neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease. While older methods such as two-dimensional polyacrylamide electrophoresis continued to be used, a variety of more in-depth MS-based approaches including both label (ICAT, iTRAQ, TMT, SILAC, SILAM), label-free (label-free, MRM, SWATH) and absolute quantification methods, are rapidly being applied to neurobiological investigations of normal and diseased brain tissue as well as of cerebrospinal fluid (CSF). While the biological implications of many of these studies remain to be clearly established, that there is a clear need for standardization of experimental design and data analysis, and that the analysis of protein changes in specific neuronal cell types in the central nervous system remains a serious challenge, it appears that the quality and depth of the more recent quantitative proteomics studies is beginning to

Recent advances in quantitative neuroproteomics.

PubMed

Craft, George E; Chen, Anshu; Nairn, Angus C

2013-06-15

The field of proteomics is undergoing rapid development in a number of different areas including improvements in mass spectrometric platforms, peptide identification algorithms and bioinformatics. In particular, new and/or improved approaches have established robust methods that not only allow for in-depth and accurate peptide and protein identification and modification, but also allow for sensitive measurement of relative or absolute quantitation. These methods are beginning to be applied to the area of neuroproteomics, but the central nervous system poses many specific challenges in terms of quantitative proteomics, given the large number of different neuronal cell types that are intermixed and that exhibit distinct patterns of gene and protein expression. This review highlights the recent advances that have been made in quantitative neuroproteomics, with a focus on work published over the last five years that applies emerging methods to normal brain function as well as to various neuropsychiatric disorders including schizophrenia and drug addiction as well as of neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. While older methods such as two-dimensional polyacrylamide electrophoresis continued to be used, a variety of more in-depth MS-based approaches including both label (ICAT, iTRAQ, TMT, SILAC, SILAM), label-free (label-free, MRM, SWATH) and absolute quantification methods, are rapidly being applied to neurobiological investigations of normal and diseased brain tissue as well as of cerebrospinal fluid (CSF). While the biological implications of many of these studies remain to be clearly established, that there is a clear need for standardization of experimental design and data analysis, and that the analysis of protein changes in specific neuronal cell types in the central nervous system remains a serious challenge, it appears that the quality and depth of the more recent quantitative proteomics studies is beginning to shed

Quantitative, spectrally-resolved intraoperative fluorescence imaging

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Jacobs, Valerie L.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2012-01-01

Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

Quantitative Modeling of Earth Surface Processes

NASA Astrophysics Data System (ADS)

Pelletier, Jon D.

This textbook describes some of the most effective and straightforward quantitative techniques for modeling Earth surface processes. By emphasizing a core set of equations and solution techniques, the book presents state-of-the-art models currently employed in Earth surface process research, as well as a set of simple but practical research tools. Detailed case studies demonstrate application of the methods to a wide variety of processes including hillslope, fluvial, aeolian, glacial, tectonic, and climatic systems. Exercises at the end of each chapter begin with simple calculations and then progress to more sophisticated problems that require computer programming. All the necessary computer codes are available online at www.cambridge.org/9780521855976. Assuming some knowledge of calculus and basic programming experience, this quantitative textbook is designed for advanced geomorphology courses and as a reference book for professional researchers in Earth and planetary science looking for a quantitative approach to Earth surface processes.

Design and analysis issues in quantitative proteomics studies.

PubMed

Karp, Natasha A; Lilley, Kathryn S

2007-09-01

Quantitative proteomics is the comparison of distinct proteomes which enables the identification of protein species which exhibit changes in expression or post-translational state in response to a given stimulus. Many different quantitative techniques are being utilized and generate large datasets. Independent of the technique used, these large datasets need robust data analysis to ensure valid conclusions are drawn from such studies. Approaches to address the problems that arise with large datasets are discussed to give insight into the types of statistical analyses of data appropriate for the various experimental strategies that can be employed by quantitative proteomic studies. This review also highlights the importance of employing a robust experimental design and highlights various issues surrounding the design of experiments. The concepts and examples discussed within will show how robust design and analysis will lead to confident results that will ensure quantitative proteomics delivers.

[Access to health care for an induced abortion: qualitative and quantitative approaches].

PubMed

Bajos, N; Moreau, C; Ferrand, M; Bouyer, J

2003-12-01

Despite recent studies showing evidence that the organisation of the French health care system raises some problems concerning access to abortion, far little is known on the reality of access conditions and the views of women on the difficulties they experience when they attend an abortion clinic. In this article, we discuss the complementarity of materials from two surveys one qualitative, the other quantitative in the study of patterns of care for an abortion. The qualitative survey included 51 women who reported a history of induced abortion, selected from a qualitative study on unintended pregnancy in France. The quantitative survey included 480 women, who had an abortion in the past 10 years. These women were selected from a representative sample of 2863 women aged 18 to 44, who participated in a study on contraception and abortion. The variety of patterns of care for an abortion, the rareness of dysfunctions in the health care system and the importance of the first professional women contacted, demonstrated in the qualitative survey, were confirmed in the quantitative survey. The quantitative survey enabled quantifying the distribution of the different patterns of care. It also permitted to identify factors associated with the choice of first professional contacted and with the type of subsequent patterns of care. The qualitative survey permitted to explore these patterns of care and to highlight the interaction between the women's request and the representation of the legitimacy of their request. Difficulties of access seemed to be linked to the lack of support women experienced in the process of finding an abortion clinic. Results suggest that general practitioners are less well informed of the procedures required for an abortion than other professionals. However, the qualitative survey also shows that problems of access cannot be reduced to the lack of information of professionals, as their practice was also linked to their own representation of abortion

Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach

PubMed Central

Bennett, Bryson D; Yuan, Jie; Kimball, Elizabeth H; Rabinowitz, Joshua D

2009-01-01

This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with 13C-labeled carbon sources, with detection of 13C-assimilation by liquid chromatography–tandem MS. It enables absolute quantitation of several dozen metabolites over ~1 week of work. PMID:18714298

Towards quantitative assessment of calciphylaxis

NASA Astrophysics Data System (ADS)

Deserno, Thomas M.; Sárándi, István.; Jose, Abin; Haak, Daniel; Jonas, Stephan; Specht, Paula; Brandenburg, Vincent

2014-03-01

Calciphylaxis is a rare disease that has devastating conditions associated with high morbidity and mortality. Calciphylaxis is characterized by systemic medial calcification of the arteries yielding necrotic skin ulcerations. In this paper, we aim at supporting the installation of multi-center registries for calciphylaxis, which includes a photographic documentation of skin necrosis. However, photographs acquired in different centers under different conditions using different equipment and photographers cannot be compared quantitatively. For normalization, we use a simple color pad that is placed into the field of view, segmented from the image, and its color fields are analyzed. In total, 24 colors are printed on that scale. A least-squares approach is used to determine the affine color transform. Furthermore, the card allows scale normalization. We provide a case study for qualitative assessment. In addition, the method is evaluated quantitatively using 10 images of two sets of different captures of the same necrosis. The variability of quantitative measurements based on free hand photography is assessed regarding geometric and color distortions before and after our simple calibration procedure. Using automated image processing, the standard deviation of measurements is significantly reduced. The coefficients of variations yield 5-20% and 2-10% for geometry and color, respectively. Hence, quantitative assessment of calciphylaxis becomes practicable and will impact a better understanding of this rare but fatal disease.

Integrating Quantitative and Ethnographic Methods to Describe the Classroom. Report No. 5083.

ERIC Educational Resources Information Center

Malitz, David; And Others

The debate between proponents of ethnographic and quantitative methodology in classroom observation is reviewed, and the respective strengths and weaknesses of the two approaches are discussed. These methodologies are directly compared in a study that conducted simultaneous ethnographic and quantitative observations on nine classrooms. It is…

Behavioral Assembly Required: Particularly for Quantitative Courses

ERIC Educational Resources Information Center

Mazen, Abdelmagid

2008-01-01

This article integrates behavioral approaches into the teaching and learning of quantitative subjects with application to statistics. Focusing on the emotional component of learning, the article presents a system dynamic model that provides descriptive and prescriptive accounts of learners' anxiety. Metaphors and the metaphorizing process are…

Quantitative Approach to Failure Mode and Effect Analysis for Linear Accelerator Quality Assurance

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

O'Daniel, Jennifer C., E-mail: jennifer.odaniel@duke.edu; Yin, Fang-Fang

Purpose: To determine clinic-specific linear accelerator quality assurance (QA) TG-142 test frequencies, to maximize physicist time efficiency and patient treatment quality. Methods and Materials: A novel quantitative approach to failure mode and effect analysis is proposed. Nine linear accelerator-years of QA records provided data on failure occurrence rates. The severity of test failure was modeled by introducing corresponding errors into head and neck intensity modulated radiation therapy treatment plans. The relative risk of daily linear accelerator QA was calculated as a function of frequency of test performance. Results: Although the failure severity was greatest for daily imaging QA (imaging vsmore » treatment isocenter and imaging positioning/repositioning), the failure occurrence rate was greatest for output and laser testing. The composite ranking results suggest that performing output and lasers tests daily, imaging versus treatment isocenter and imaging positioning/repositioning tests weekly, and optical distance indicator and jaws versus light field tests biweekly would be acceptable for non-stereotactic radiosurgery/stereotactic body radiation therapy linear accelerators. Conclusions: Failure mode and effect analysis is a useful tool to determine the relative importance of QA tests from TG-142. Because there are practical time limitations on how many QA tests can be performed, this analysis highlights which tests are the most important and suggests the frequency of testing based on each test's risk priority number.« less

Using Active Learning to Teach Concepts and Methods in Quantitative Biology.

PubMed

Waldrop, Lindsay D; Adolph, Stephen C; Diniz Behn, Cecilia G; Braley, Emily; Drew, Joshua A; Full, Robert J; Gross, Louis J; Jungck, John A; Kohler, Brynja; Prairie, Jennifer C; Shtylla, Blerta; Miller, Laura A

2015-11-01

This article provides a summary of the ideas discussed at the 2015 Annual Meeting of the Society for Integrative and Comparative Biology society-wide symposium on Leading Students and Faculty to Quantitative Biology through Active Learning. It also includes a brief review of the recent advancements in incorporating active learning approaches into quantitative biology classrooms. We begin with an overview of recent literature that shows that active learning can improve students' outcomes in Science, Technology, Engineering and Math Education disciplines. We then discuss how this approach can be particularly useful when teaching topics in quantitative biology. Next, we describe some of the recent initiatives to develop hands-on activities in quantitative biology at both the graduate and the undergraduate levels. Throughout the article we provide resources for educators who wish to integrate active learning and technology into their classrooms. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Comparison of Maximum Likelihood Estimation Approach and Regression Approach in Detecting Quantitative Trait Lco Using RAPD Markers

Treesearch

Changren Weng; Thomas L. Kubisiak; C. Dana Nelson; James P. Geaghan; Michael Stine

1999-01-01

Single marker regression and single marker maximum likelihood estimation were tied to detect quantitative trait loci (QTLs) controlling the early height growth of longleaf pine and slash pine using a ((longleaf pine x slash pine) x slash pine) BC, population consisting of 83 progeny. Maximum likelihood estimation was found to be more power than regression and could...

Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

PubMed

Gizak, Agnieszka; Rakus, Dariusz

2016-01-11

Molecular and cellular biology methodology is traditionally based on the reasoning called "the mechanistic explanation". In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems' complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites), and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

What Are We Doing When We Translate from Quantitative Models?

PubMed Central

Critchfield, Thomas S; Reed, Derek D

2009-01-01

Although quantitative analysis (in which behavior principles are defined in terms of equations) has become common in basic behavior analysis, translational efforts often examine everyday events through the lens of narrative versions of laboratory-derived principles. This approach to translation, although useful, is incomplete because equations may convey concepts that are difficult to capture in words. To support this point, we provide a nontechnical introduction to selected aspects of quantitative analysis; consider some issues that translational investigators (and, potentially, practitioners) confront when attempting to translate from quantitative models; and discuss examples of relevant translational studies. We conclude that, where behavior-science translation is concerned, the quantitative features of quantitative models cannot be ignored without sacrificing conceptual precision, scientific and practical insights, and the capacity of the basic and applied wings of behavior analysis to communicate effectively. PMID:22478533

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

PubMed

Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir

2018-05-15

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several

The Quantitative Preparation of Future Geoscience Graduate Students

NASA Astrophysics Data System (ADS)

Manduca, C. A.; Hancock, G. S.

2006-12-01

Modern geoscience is a highly quantitative science. In February, a small group of faculty and graduate students from across the country met to discuss the quantitative preparation of geoscience majors for graduate school. The group included ten faculty supervising graduate students in quantitative areas spanning the earth, atmosphere, and ocean sciences; five current graduate students in these areas; and five faculty teaching undergraduate students in the spectrum of institutions preparing students for graduate work. Discussion focused in four key ares: Are incoming graduate students adequately prepared for the quantitative aspects of graduate geoscience programs? What are the essential quantitative skills are that are required for success in graduate school? What are perceived as the important courses to prepare students for the quantitative aspects of graduate school? What programs/resources would be valuable in helping faculty/departments improve the quantitative preparation of students? The participants concluded that strengthening the quantitative preparation of undergraduate geoscience majors would increase their opportunities in graduate school. While specifics differed amongst disciplines, a special importance was placed on developing the ability to use quantitative skills to solve geoscience problems. This requires the ability to pose problems so they can be addressed quantitatively, understand the relationship between quantitative concepts and physical representations, visualize mathematics, test the reasonableness of quantitative results, creatively move forward from existing models/techniques/approaches, and move between quantitative and verbal descriptions. A list of important quantitative competencies desirable in incoming graduate students includes mechanical skills in basic mathematics, functions, multi-variate analysis, statistics and calculus, as well as skills in logical analysis and the ability to learn independently in quantitative ways

Chemoenzymatic method for glycomics: isolation, identification, and quantitation

PubMed Central

Yang, Shuang; Rubin, Abigail; Eshghi, Shadi Toghi; Zhang, Hui

2015-01-01

Over the past decade, considerable progress has been made with respect to the analytical methods for analysis of glycans from biological sources. Regardless of the specific methods that are used, glycan analysis includes isolation, identification, and quantitation. Derivatization is indispensable to increase their identification. Derivatization of glycans can be performed by permethylation or carbodiimide coupling / esterification. By introducing a fluorophore or chromophore at their reducing end, glycans can be separated by electrophoresis or chromatography. The fluorogenically labeled glycans can be quantitated using fluorescent detection. The recently developed approaches using solid-phase such as glycoprotein immobilization for glycan extraction and on-tissue glycan mass spectrometry imaging demonstrate advantages over methods performed in solution. Derivatization of sialic acids is favorably implemented on the solid support using carbodiimide coupling, and the released glycans can be further modified at the reducing end or permethylated for quantitative analysis. In this review, methods for glycan isolation, identification, and quantitation are discussed. PMID:26390280

Measuring Filament Orientation: A New Quantitative, Local Approach

NASA Astrophysics Data System (ADS)

Green, C.-E.; Dawson, J. R.; Cunningham, M. R.; Jones, P. A.; Novak, G.; Fissel, L. M.

2017-09-01

The relative orientation between filamentary structures in molecular clouds and the ambient magnetic field provides insight into filament formation and stability. To calculate the relative orientation, a measurement of filament orientation is first required. We propose a new method to calculate the orientation of the one-pixel-wide filament skeleton that is output by filament identification algorithms such as filfinder. We derive the local filament orientation from the direction of the intensity gradient in the skeleton image using the Sobel filter and a few simple post-processing steps. We call this the “Sobel-gradient method.” The resulting filament orientation map can be compared quantitatively on a local scale with the magnetic field orientation map to then find the relative orientation of the filament with respect to the magnetic field at each point along the filament. It can also be used for constructing radial profiles for filament width fitting. The proposed method facilitates automation in analyses of filament skeletons, which is imperative in this era of “big data.”

Targeted methods for quantitative analysis of protein glycosylation

PubMed Central

Goldman, Radoslav; Sanda, Miloslav

2018-01-01

Quantification of proteins by LC-MS/MS-MRM has become a standard method with broad projected clinical applicability. MRM quantification of protein modifications is, however, far less utilized, especially in the case of glycoproteins. This review summarizes current methods for quantitative analysis of protein glycosylation with a focus on MRM methods. We describe advantages of this quantitative approach, analytical parameters that need to be optimized to achieve reliable measurements, and point out the limitations. Differences between major classes of N- and O-glycopeptides are described and class-specific glycopeptide assays are demonstrated. PMID:25522218

A qualitative and quantitative assessment for a bone marrow harvest simulator.

PubMed

Machado, Liliane S; Moraes, Ronei M

2009-01-01

Several approaches to perform assessment in training simulators based on virtual reality have been proposed. There are two kinds of assessment methods: offline and online. The main requirements related to online training assessment methodologies applied to virtual reality systems are the low computational complexity and the high accuracy. In the literature it can be found several approaches for general cases which can satisfy such requirements. An inconvenient about those approaches is related to an unsatisfactory solution for specific cases, as in some medical procedures, where there are quantitative and qualitative information available to perform the assessment. In this paper, we present an approach to online training assessment based on a Modified Naive Bayes which can manipulate qualitative and quantitative variables simultaneously. A special medical case was simulated in a bone marrow harvest simulator. The results obtained were satisfactory and evidenced the applicability of the method.

Liquid crystal-based biosensor with backscattering interferometry: A quantitative approach.

PubMed

Khan, Mashooq; Park, Soo-Young

2017-01-15

We developed a new technology that uses backscattering interferometry (BSI) to quantitatively measure nematic liquid crystal (NLC)-based biosensors, those usually relied on texture reading for on/off signals. The LC-based BSI comprised an octadecyltrichlorosilane (OTS)-coated square capillary filled with 4-cyano-4'-pentylbiphenyl (5CB, a nematic LC at room temperature). The LC/water interface in the capillary was functionalized by a coating of poly(acrylicacid-b-4-cyanobiphenyl-4'-oxyundecylacrylate) (PAA-b-LCP) and immobilized with the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) through covalent linkage to the PAA chains (5CB PAA-GOx:HRP ) for glucose detection. Laser irradiation of the LC near the LC/water interface resulted in backscattered fringes with high contrast. The change in the spatial position of the fringes (because of the change in the orientation of the LC caused by the GOx:HRP enzymatic reaction of glucose) altered the output voltage of the photodetector when its active area was aligned with the edge of one of the fringes. The change in the intensity at the photodetector allowed the detection limit of the instrument to be as low as 0.008mM with a linear range of 0.02-9mM in a short response time (~60s). This LC-based BSI technique allows for quantitative, sensitive, selective, reproducible, easily obtainable, and interference-free detection in a large linear dynamic range and for practical applications with human serum. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative Decision Support Requires Quantitative User Guidance

NASA Astrophysics Data System (ADS)

Smith, L. A.

2009-12-01

Is it conceivable that models run on 2007 computer hardware could provide robust and credible probabilistic information for decision support and user guidance at the ZIP code level for sub-daily meteorological events in 2060? In 2090? Retrospectively, how informative would output from today’s models have proven in 2003? or the 1930’s? Consultancies in the United Kingdom, including the Met Office, are offering services to “future-proof” their customers from climate change. How is a US or European based user or policy maker to determine the extent to which exciting new Bayesian methods are relevant here? or when a commercial supplier is vastly overselling the insights of today’s climate science? How are policy makers and academic economists to make the closely related decisions facing them? How can we communicate deep uncertainty in the future at small length-scales without undermining the firm foundation established by climate science regarding global trends? Three distinct aspects of the communication of the uses of climate model output targeting users and policy makers, as well as other specialist adaptation scientists, are discussed. First, a brief scientific evaluation of the length and time scales at which climate model output is likely to become uninformative is provided, including a note on the applicability the latest Bayesian methodology to current state-of-the-art general circulation models output. Second, a critical evaluation of the language often employed in communication of climate model output, a language which accurately states that models are “better”, have “improved” and now “include” and “simulate” relevant meteorological processed, without clearly identifying where the current information is thought to be uninformative and misleads, both for the current climate and as a function of the state of the (each) climate simulation. And thirdly, a general approach for evaluating the relevance of quantitative climate model output

Quantitative diagnosis of breast tumors by morphometric classification of microenvironmental myoepithelial cells using a machine learning approach

PubMed Central

Yamamoto, Yoichiro; Saito, Akira; Tateishi, Ayako; Shimojo, Hisashi; Kanno, Hiroyuki; Tsuchiya, Shinichi; Ito, Ken-ichi; Cosatto, Eric; Graf, Hans Peter; Moraleda, Rodrigo R.; Eils, Roland; Grabe, Niels

2017-01-01

Machine learning systems have recently received increased attention for their broad applications in several fields. In this study, we show for the first time that histological types of breast tumors can be classified using subtle morphological differences of microenvironmental myoepithelial cell nuclei without any direct information about neoplastic tumor cells. We quantitatively measured 11661 nuclei on the four histological types: normal cases, usual ductal hyperplasia and low/high grade ductal carcinoma in situ (DCIS). Using a machine learning system, we succeeded in classifying the four histological types with 90.9% accuracy. Electron microscopy observations suggested that the activity of typical myoepithelial cells in DCIS was lowered. Through these observations as well as meta-analytic database analyses, we developed a paracrine cross-talk-based biological mechanism of DCIS progressing to invasive cancer. Our observations support novel approaches in clinical computational diagnostics as well as in therapy development against progression. PMID:28440283

Infrared thermography quantitative image processing

NASA Astrophysics Data System (ADS)

Skouroliakou, A.; Kalatzis, I.; Kalyvas, N.; Grivas, TB

2017-11-01

Infrared thermography is an imaging technique that has the ability to provide a map of temperature distribution of an object’s surface. It is considered for a wide range of applications in medicine as well as in non-destructive testing procedures. One of its promising medical applications is in orthopaedics and diseases of the musculoskeletal system where temperature distribution of the body’s surface can contribute to the diagnosis and follow up of certain disorders. Although the thermographic image can give a fairly good visual estimation of distribution homogeneity and temperature pattern differences between two symmetric body parts, it is important to extract a quantitative measurement characterising temperature. Certain approaches use temperature of enantiomorphic anatomical points, or parameters extracted from a Region of Interest (ROI). A number of indices have been developed by researchers to that end. In this study a quantitative approach in thermographic image processing is attempted based on extracting different indices for symmetric ROIs on thermograms of the lower back area of scoliotic patients. The indices are based on first order statistical parameters describing temperature distribution. Analysis and comparison of these indices result in evaluating the temperature distribution pattern of the back trunk expected in healthy, regarding spinal problems, subjects.

Advancing the study of violence against women using mixed methods: integrating qualitative methods into a quantitative research program.

PubMed

Testa, Maria; Livingston, Jennifer A; VanZile-Tamsen, Carol

2011-02-01

A mixed methods approach, combining quantitative with qualitative data methods and analysis, offers a promising means of advancing the study of violence. Integrating semi-structured interviews and qualitative analysis into a quantitative program of research on women's sexual victimization has resulted in valuable scientific insight and generation of novel hypotheses for testing. This mixed methods approach is described and recommendations for integrating qualitative data into quantitative research are provided.

Public and patient involvement in quantitative health research: A statistical perspective.

PubMed

Hannigan, Ailish

2018-06-19

The majority of studies included in recent reviews of impact for public and patient involvement (PPI) in health research had a qualitative design. PPI in solely quantitative designs is underexplored, particularly its impact on statistical analysis. Statisticians in practice have a long history of working in both consultative (indirect) and collaborative (direct) roles in health research, yet their perspective on PPI in quantitative health research has never been explicitly examined. To explore the potential and challenges of PPI from a statistical perspective at distinct stages of quantitative research, that is sampling, measurement and statistical analysis, distinguishing between indirect and direct PPI. Statistical analysis is underpinned by having a representative sample, and a collaborative or direct approach to PPI may help achieve that by supporting access to and increasing participation of under-represented groups in the population. Acknowledging and valuing the role of lay knowledge of the context in statistical analysis and in deciding what variables to measure may support collective learning and advance scientific understanding, as evidenced by the use of participatory modelling in other disciplines. A recurring issue for quantitative researchers, which reflects quantitative sampling methods, is the selection and required number of PPI contributors, and this requires further methodological development. Direct approaches to PPI in quantitative health research may potentially increase its impact, but the facilitation and partnership skills required may require further training for all stakeholders, including statisticians. © 2018 The Authors Health Expectations published by John Wiley & Sons Ltd.

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

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.

Research essentials. How to critique quantitative research.

PubMed

Clarke, Sharon; Collier, Sue

2015-11-01

QUANTITATIVE RESEARCH is a systematic approach to investigating numerical data and involves measuring or counting attributes, that is quantities. Through a process of transforming information that is collected or observed, the researcher can often describes a situation or event, answering the 'what' and 'how many' questions about a situation ( Parahoo 2014 ).

Quantitative fluorescence microscopy and image deconvolution.

PubMed

Swedlow, Jason R

2013-01-01

Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used

Quantitative Approach to Collaborative Learning: Performance Prediction, Individual Assessment, and Group Composition

ERIC Educational Resources Information Center

Cen, Ling; Ruta, Dymitr; Powell, Leigh; Hirsch, Benjamin; Ng, Jason

2016-01-01

The benefits of collaborative learning, although widely reported, lack the quantitative rigor and detailed insight into the dynamics of interactions within the group, while individual contributions and their impacts on group members and their collaborative work remain hidden behind joint group assessment. To bridge this gap we intend to address…

Mixed methods in gerontological research: Do the qualitative and quantitative data “touch”?

PubMed Central

Happ, Mary Beth

2010-01-01

This paper distinguishes between parallel and integrated mixed methods research approaches. Barriers to integrated mixed methods approaches in gerontological research are discussed and critiqued. The author presents examples of mixed methods gerontological research to illustrate approaches to data integration at the levels of data analysis, interpretation, and research reporting. As a summary of the methodological literature, four basic levels of mixed methods data combination are proposed. Opportunities for mixing qualitative and quantitative data are explored using contemporary examples from published studies. Data transformation and visual display, judiciously applied, are proposed as pathways to fuller mixed methods data integration and analysis. Finally, practical strategies for mixing qualitative and quantitative data types are explicated as gerontological research moves beyond parallel mixed methods approaches to achieve data integration. PMID:20077973

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

Quantitative optical scanning tests of complex microcircuits

NASA Technical Reports Server (NTRS)

Erickson, J. J.

1980-01-01

An approach for the development of the optical scanner as a screening inspection instrument for microcircuits involves comparing the quantitative differences in photoresponse images and then correlating them with electrical parameter differences in test devices. The existing optical scanner was modified so that the photoresponse data could be recorded and subsequently digitized. A method was devised for applying digital image processing techniques to the digitized photoresponse data in order to quantitatively compare the data. Electrical tests were performed and photoresponse images were recorded before and following life test intervals on two groups of test devices. Correlations were made between differences or changes in the electrical parameters of the test devices.

2D Hydrodynamic Based Logic Modeling Tool for River Restoration Decision Analysis: A Quantitative Approach to Project Prioritization

NASA Astrophysics Data System (ADS)

Bandrowski, D.; Lai, Y.; Bradley, N.; Gaeuman, D. A.; Murauskas, J.; Som, N. A.; Martin, A.; Goodman, D.; Alvarez, J.

2014-12-01

In the field of river restoration sciences there is a growing need for analytical modeling tools and quantitative processes to help identify and prioritize project sites. 2D hydraulic models have become more common in recent years and with the availability of robust data sets and computing technology, it is now possible to evaluate large river systems at the reach scale. The Trinity River Restoration Program is now analyzing a 40 mile segment of the Trinity River to determine priority and implementation sequencing for its Phase II rehabilitation projects. A comprehensive approach and quantitative tool has recently been developed to analyze this complex river system referred to as: 2D-Hydrodynamic Based Logic Modeling (2D-HBLM). This tool utilizes various hydraulic output parameters combined with biological, ecological, and physical metrics at user-defined spatial scales. These metrics and their associated algorithms are the underpinnings of the 2D-HBLM habitat module used to evaluate geomorphic characteristics, riverine processes, and habitat complexity. The habitat metrics are further integrated into a comprehensive Logic Model framework to perform statistical analyses to assess project prioritization. The Logic Model will analyze various potential project sites by evaluating connectivity using principal component methods. The 2D-HBLM tool will help inform management and decision makers by using a quantitative process to optimize desired response variables with balancing important limiting factors in determining the highest priority locations within the river corridor to implement restoration projects. Effective river restoration prioritization starts with well-crafted goals that identify the biological objectives, address underlying causes of habitat change, and recognizes that social, economic, and land use limiting factors may constrain restoration options (Bechie et. al. 2008). Applying natural resources management actions, like restoration prioritization, is

Natural enamel caries in polarized light microscopy: differences in histopathological features derived from a qualitative versus a quantitative approach to interpret enamel birefringence.

PubMed

De Medeiros, R C G; Soares, J D; De Sousa, F B

2012-05-01

Lesion area measurement of enamel caries using polarized light microscopy (PLM) is currently performed in a large number of studies, but measurements are based mainly on a mislead qualitative interpretation of enamel birefringence in a single immersion medium. Here, five natural enamel caries lesions are analysed by microradiography and in PLM, and the differences in their histopathological features derived from a qualitative versus a quantitative interpretation of enamel birefringence are described. Enamel birefringence in different immersion media (air, water and quinoline) is interpreted by both qualitative and quantitative approaches, the former leading to an underestimation of the depth of enamel caries mainly when the criterion of validating sound enamel as a negatively birefringent area in immersion in water is used (a current common practice in dental research). Procedures to avoid the shortcomings of a qualitative interpretation of enamel birefringence are presented and discussed. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

DRIFTSEL: an R package for detecting signals of natural selection in quantitative traits.

PubMed

Karhunen, M; Merilä, J; Leinonen, T; Cano, J M; Ovaskainen, O

2013-07-01

Approaches and tools to differentiate between natural selection and genetic drift as causes of population differentiation are of frequent demand in evolutionary biology. Based on the approach of Ovaskainen et al. (2011), we have developed an R package (DRIFTSEL) that can be used to differentiate between stabilizing selection, diversifying selection and random genetic drift as causes of population differentiation in quantitative traits when neutral marker and quantitative genetic data are available. Apart from illustrating the use of this method and the interpretation of results using simulated data, we apply the package on data from three-spined sticklebacks (Gasterosteus aculeatus) to highlight its virtues. DRIFTSEL can also be used to perform usual quantitative genetic analyses in common-garden study designs. © 2013 John Wiley & Sons Ltd.

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

Bayesian parameter estimation in spectral quantitative photoacoustic tomography

NASA Astrophysics Data System (ADS)

Pulkkinen, Aki; Cox, Ben T.; Arridge, Simon R.; Kaipio, Jari P.; Tarvainen, Tanja

2016-03-01

Photoacoustic tomography (PAT) is an imaging technique combining strong contrast of optical imaging to high spatial resolution of ultrasound imaging. These strengths are achieved via photoacoustic effect, where a spatial absorption of light pulse is converted into a measurable propagating ultrasound wave. The method is seen as a potential tool for small animal imaging, pre-clinical investigations, study of blood vessels and vasculature, as well as for cancer imaging. The goal in PAT is to form an image of the absorbed optical energy density field via acoustic inverse problem approaches from the measured ultrasound data. Quantitative PAT (QPAT) proceeds from these images and forms quantitative estimates of the optical properties of the target. This optical inverse problem of QPAT is illposed. To alleviate the issue, spectral QPAT (SQPAT) utilizes PAT data formed at multiple optical wavelengths simultaneously with optical parameter models of tissue to form quantitative estimates of the parameters of interest. In this work, the inverse problem of SQPAT is investigated. Light propagation is modelled using the diffusion equation. Optical absorption is described with chromophore concentration weighted sum of known chromophore absorption spectra. Scattering is described by Mie scattering theory with an exponential power law. In the inverse problem, the spatially varying unknown parameters of interest are the chromophore concentrations, the Mie scattering parameters (power law factor and the exponent), and Gruneisen parameter. The inverse problem is approached with a Bayesian method. It is numerically demonstrated, that estimation of all parameters of interest is possible with the approach.

ADVANCING THE STUDY OF VIOLENCE AGAINST WOMEN USING MIXED METHODS: INTEGRATING QUALITATIVE METHODS INTO A QUANTITATIVE RESEARCH PROGRAM

PubMed Central

Testa, Maria; Livingston, Jennifer A.; VanZile-Tamsen, Carol

2011-01-01

A mixed methods approach, combining quantitative with qualitative data methods and analysis, offers a promising means of advancing the study of violence. Integrating semi-structured interviews and qualitative analysis into a quantitative program of research on women’s sexual victimization has resulted in valuable scientific insight and generation of novel hypotheses for testing. This mixed methods approach is described and recommendations for integrating qualitative data into quantitative research are provided. PMID:21307032

A quantitative benefit-risk assessment approach to improve decision making in drug development: Application of a multicriteria decision analysis model in the development of combination therapy for overactive bladder.

PubMed

de Greef-van der Sandt, I; Newgreen, D; Schaddelee, M; Dorrepaal, C; Martina, R; Ridder, A; van Maanen, R

2016-04-01

A multicriteria decision analysis (MCDA) approach was developed and used to estimate the benefit-risk of solifenacin and mirabegron and their combination in the treatment of overactive bladder (OAB). The objectives were 1) to develop an MCDA tool to compare drug effects in OAB quantitatively, 2) to establish transparency in the evaluation of the benefit-risk profile of various dose combinations, and 3) to quantify the added value of combination use compared to monotherapies. The MCDA model was developed using efficacy, safety, and tolerability attributes and the results of a phase II factorial design combination study were evaluated. Combinations of solifenacin 5 mg and mirabegron 25 mg and mirabegron 50 (5+25 and 5+50) scored the highest clinical utility and supported combination therapy development of solifenacin and mirabegron for phase III clinical development at these dose regimens. This case study underlines the benefit of using a quantitative approach in clinical drug development programs. © 2015 The American Society for Clinical Pharmacology and Therapeutics.

QUALITATIVE AND QUANTITATIVE METHODS OF SUICIDE RESEARCH IN OLD AGE.

PubMed

Ojagbemi, A

2017-06-01

This paper examines the merits of the qualitative and quantitative methods of suicide research in the elderly using two studies identified through a free search of the Pubmed database for articles that might have direct bearing on suicidality in the elderly. The studies have been purposively selected for critical appraisal because they meaningfully reflect the quantitative and qualitative divide as well as the social, economic, and cultural boundaries between the elderly living in sub-Saharan Africa and Europe. The paper concludes that an integration of both the qualitative and quantitative research approaches may provide a better platform for unraveling the complex phenomenon of suicide in the elderly.

Mapping Quantitative Traits in Unselected Families: Algorithms and Examples

PubMed Central

Dupuis, Josée; Shi, Jianxin; Manning, Alisa K.; Benjamin, Emelia J.; Meigs, James B.; Cupples, L. Adrienne; Siegmund, David

2009-01-01

Linkage analysis has been widely used to identify from family data genetic variants influencing quantitative traits. Common approaches have both strengths and limitations. Likelihood ratio tests typically computed in variance component analysis can accommodate large families but are highly sensitive to departure from normality assumptions. Regression-based approaches are more robust but their use has primarily been restricted to nuclear families. In this paper, we develop methods for mapping quantitative traits in moderately large pedigrees. Our methods are based on the score statistic which in contrast to the likelihood ratio statistic, can use nonparametric estimators of variability to achieve robustness of the false positive rate against departures from the hypothesized phenotypic model. Because the score statistic is easier to calculate than the likelihood ratio statistic, our basic mapping methods utilize relatively simple computer code that performs statistical analysis on output from any program that computes estimates of identity-by-descent. This simplicity also permits development and evaluation of methods to deal with multivariate and ordinal phenotypes, and with gene-gene and gene-environment interaction. We demonstrate our methods on simulated data and on fasting insulin, a quantitative trait measured in the Framingham Heart Study. PMID:19278016

An Efficient Audio Coding Scheme for Quantitative and Qualitative Large Scale Acoustic Monitoring Using the Sensor Grid Approach

PubMed Central

Gontier, Félix; Lagrange, Mathieu; Can, Arnaud; Lavandier, Catherine

2017-01-01

The spreading of urban areas and the growth of human population worldwide raise societal and environmental concerns. To better address these concerns, the monitoring of the acoustic environment in urban as well as rural or wilderness areas is an important matter. Building on the recent development of low cost hardware acoustic sensors, we propose in this paper to consider a sensor grid approach to tackle this issue. In this kind of approach, the crucial question is the nature of the data that are transmitted from the sensors to the processing and archival servers. To this end, we propose an efficient audio coding scheme based on third octave band spectral representation that allows: (1) the estimation of standard acoustic indicators; and (2) the recognition of acoustic events at state-of-the-art performance rate. The former is useful to provide quantitative information about the acoustic environment, while the latter is useful to gather qualitative information and build perceptually motivated indicators using for example the emergence of a given sound source. The coding scheme is also demonstrated to transmit spectrally encoded data that, reverted to the time domain using state-of-the-art techniques, are not intelligible, thus protecting the privacy of citizens. PMID:29186021

Quantitative image processing in fluid mechanics

NASA Technical Reports Server (NTRS)

Hesselink, Lambertus; Helman, James; Ning, Paul

1992-01-01

The current status of digital image processing in fluid flow research is reviewed. In particular, attention is given to a comprehensive approach to the extraction of quantitative data from multivariate databases and examples of recent developments. The discussion covers numerical simulations and experiments, data processing, generation and dissemination of knowledge, traditional image processing, hybrid processing, fluid flow vector field topology, and isosurface analysis using Marching Cubes.

Challenge in Enhancing the Teaching and Learning of Variable Measurements in Quantitative Research

ERIC Educational Resources Information Center

Kee, Chang Peng; Osman, Kamisah; Ahmad, Fauziah

2013-01-01

Statistical analysis is one component that cannot be avoided in a quantitative research. Initial observations noted that students in higher education institution faced difficulty analysing quantitative data which were attributed to the confusions of various variable measurements. This paper aims to compare the outcomes of two approaches applied in…

Industrial ecology: Quantitative methods for exploring a lower carbon future

NASA Astrophysics Data System (ADS)

Thomas, Valerie M.

2015-03-01

Quantitative methods for environmental and cost analyses of energy, industrial, and infrastructure systems are briefly introduced and surveyed, with the aim of encouraging broader utilization and development of quantitative methods in sustainable energy research. Material and energy flow analyses can provide an overall system overview. The methods of engineering economics and cost benefit analysis, such as net present values, are the most straightforward approach for evaluating investment options, with the levelized cost of energy being a widely used metric in electricity analyses. Environmental lifecycle assessment has been extensively developed, with both detailed process-based and comprehensive input-output approaches available. Optimization methods provide an opportunity to go beyond engineering economics to develop detailed least-cost or least-impact combinations of many different choices.

Does Quantitative Research in Child Maltreatment Tell the Whole Story? The Need for Mixed-Methods Approaches to Explore the Effects of Maltreatment in Infancy.

PubMed

Glass, Samuel; Gajwani, Ruchika; Turner-Halliday, Fiona

Background and Aims. Research on child maltreatment has largely overlooked the under-five age group and focuses primarily on quantitative measurement. This mixed-methods study of maltreated children (N = 92) entering care (age 6-60 months) combines a quantitative focus on the associations between care journey characteristics and mental health outcomes with a qualitative exploration of maltreatment in four different families. Methods. Care journey data was obtained from social care records; mental health and attachment assessments were carried out following entry to care; qualitative data comprised semistructured interviews with professionals, foster carers, and parents. Results. Significant associations were found between suspected sexual abuse and increased DAI inhibited attachment symptoms (p = 0.001) and between reported domestic violence and decreased DAI inhibited (p = 0.016) and disinhibited (p = 0.004) attachment symptoms. Qualitative results: two themes demonstrate the complexity of assessing maltreatment: (1) overlapping maltreatment factors occur in most cases and (2) maltreatment effects may be particularly challenging to isolate. Conclusions. Qualitative exploration has underscored the complexity of assessing maltreatment, indicating why expected associations were not found in this study and posing questions for the quantitative measurement of maltreatment in general. We therefore suggest a new categorisation of maltreatment and call for the complimentary research lenses of further mixed-methods approaches.

Studying learning in the healthcare setting: the potential of quantitative diary methods.

PubMed

Ciere, Yvette; Jaarsma, Debbie; Visser, Annemieke; Sanderman, Robbert; Snippe, Evelien; Fleer, Joke

2015-08-01

Quantitative diary methods are longitudinal approaches that involve the repeated measurement of aspects of peoples' experience of daily life. In this article, we outline the main characteristics and applications of quantitative diary methods and discuss how their use may further research in the field of medical education. Quantitative diary methods offer several methodological advantages, such as measuring aspects of learning with great detail, accuracy and authenticity. Moreover, they enable researchers to study how and under which conditions learning in the health care setting occurs and in which way learning can be promoted. Hence, quantitative diary methods may contribute to theory development and the optimization of teaching methods in medical education.

On the Distinction Between Quantitative and Qualitative Research.

ERIC Educational Resources Information Center

Smith, P. L.

Quantitative and qualitative research are differing modes of measurement, one using numbers and the other not. The assignment of numerals to represent properties enables a researcher to distinguish minutely between different properties. The major issue dividing these approaches to empirical research represents a philosophical dispute which has…

An introduction to quantitative remote sensing. [data processing

NASA Technical Reports Server (NTRS)

Lindenlaub, J. C.; Russell, J.

1974-01-01

The quantitative approach to remote sensing is discussed along with the analysis of remote sensing data. Emphasis is placed on the application of pattern recognition in numerically oriented remote sensing systems. A common background and orientation for users of the LARS computer software system is provided.

Genetic toxicology at the crossroads-from qualitative hazard evaluation to quantitative risk assessment.

PubMed

White, Paul A; Johnson, George E

2016-05-01

Applied genetic toxicology is undergoing a transition from qualitative hazard identification to quantitative dose-response analysis and risk assessment. To facilitate this change, the Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC) sponsored a workshop held in Lancaster, UK on July 10-11, 2014. The event included invited speakers from several institutions and the contents was divided into three themes-1: Point-of-departure Metrics for Quantitative Dose-Response Analysis in Genetic Toxicology; 2: Measurement and Estimation of Exposures for Better Extrapolation to Humans and 3: The Use of Quantitative Approaches in Genetic Toxicology for human health risk assessment (HHRA). A host of pertinent issues were discussed relating to the use of in vitro and in vivo dose-response data, the development of methods for in vitro to in vivo extrapolation and approaches to use in vivo dose-response data to determine human exposure limits for regulatory evaluations and decision-making. This Special Issue, which was inspired by the workshop, contains a series of papers that collectively address topics related to the aforementioned themes. The Issue includes contributions that collectively evaluate, describe and discuss in silico, in vitro, in vivo and statistical approaches that are facilitating the shift from qualitative hazard evaluation to quantitative risk assessment. The use and application of the benchmark dose approach was a central theme in many of the workshop presentations and discussions, and the Special Issue includes several contributions that outline novel applications for the analysis and interpretation of genetic toxicity data. Although the contents of the Special Issue constitutes an important step towards the adoption of quantitative methods for regulatory assessment of genetic toxicity, formal acceptance of quantitative methods for HHRA and regulatory decision-making will require consensus regarding the

A quantitative approach to evaluating caring in nursing simulation.

PubMed

Eggenberger, Terry L; Keller, Kathryn B; Chase, Susan K; Payne, Linda

2012-01-01

This study was designed to test a quantitative method of measuring caring in the simulated environment. Since competency in caring is central to nursing practice, ways of including caring concepts in designing scenarios and in evaluation of performance need to be developed. Coates' Caring Efficacy scales were adapted for simulation and named the Caring Efficacy Scale-Simulation Student Version (CES-SSV) and Caring Efficacy Scale-Simulation Faculty Version (CES-SFV). A correlational study was designed to compare student self-ratings with faculty ratings on caring efficacy during an adult acute simulation experience with traditional and accelerated baccalaureate students in a nursing program grounded in caring theory. Student self-ratings were significantly correlated with objective ratings (r = 0.345, 0.356). Both the CES-SSV and the CES-SFV were found to have excellent internal consistency and significantly correlated interrater reliability. They were useful in measuring caring in the simulated learning environment.

A Qualitative-Quantitative H-NMR Experiment for the Instrumental Analysis Laboratory.

ERIC Educational Resources Information Center

Phillips, John S.; Leary, James J.

1986-01-01

Describes an experiment combining qualitative and quantitative information from hydrogen nuclear magnetic resonance spectra. Reviews theory, discusses the experimental approach, and provides sample results. (JM)

A QUANTITATIVE APPROACH FOR ESTIMATING EXPOSURE TO PESTICIDES IN THE AGRICULTURAL HEALTH STUDY

EPA Science Inventory

We developed a quantitative method to estimate chemical-specific pesticide exposures in a large prospective cohort study of over 58,000 pesticide applicators in North Carolina and Iowa. An enrollment questionnaire was administered to applicators to collect basic time- and inten...

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

PubMed

Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

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, ...

A quantitative approach to aquifer vulnerability mapping

NASA Astrophysics Data System (ADS)

Connell, L. D.; Daele, Gerd van den

2003-05-01

This paper presents a procedure for calculating the transport to groundwater of surface-released contaminants. The approach is derived from a series of analytical and semi-analytical solutions to the advection-dispersion equation that include root zone and unsaturated water movement effects on the transport process. The steady-state form of these equations provides an efficient means of calculating the maximum concentration at the watertable and therefore has potential for use in vulnerability mapping. A two-layer approach is used in the solutions to represent the unsaturated profile, with the root zone corresponding to the upper layer where evapotranspiration can occur and transport properties can be in contrast to the rest of the profile. A novel transformation is applied to the advection-dispersion equation that considerably simplifies the way in which water movement is represented. To provide a combined flow and transport model an approximate procedure for water movement, using averages of the infiltration and transpiration rates with a novel, simple, quasi-steady state solution, is presented that can be used in conjunction with the solutions to the advection-dispersion equation. This quasi-steady state approximation for water movement allows for layering in the soil profile and root water uptake. Results from the combined quasi-steady state water movement and semi-analytical solute transport procedure compare well with numerical solutions to the coupled unsaturated flow and solute transport equations in a series of hypothetical simulations.

Quantitative PCR for genetic markers of human fecal pollution

EPA Science Inventory

Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for enumeration of two recently described hum...

Quantitative criticism of literary relationships

PubMed Central

Dexter, Joseph P.; Katz, Theodore; Tripuraneni, Nilesh; Dasgupta, Tathagata; Kannan, Ajay; Brofos, James A.; Bonilla Lopez, Jorge A.; Schroeder, Lea A.; Casarez, Adriana; Rabinovich, Maxim; Haimson Lushkov, Ayelet; Chaudhuri, Pramit

2017-01-01

Authors often convey meaning by referring to or imitating prior works of literature, a process that creates complex networks of literary relationships (“intertextuality”) and contributes to cultural evolution. In this paper, we use techniques from stylometry and machine learning to address subjective literary critical questions about Latin literature, a corpus marked by an extraordinary concentration of intertextuality. Our work, which we term “quantitative criticism,” focuses on case studies involving two influential Roman authors, the playwright Seneca and the historian Livy. We find that four plays related to but distinct from Seneca’s main writings are differentiated from the rest of the corpus by subtle but important stylistic features. We offer literary interpretations of the significance of these anomalies, providing quantitative data in support of hypotheses about the use of unusual formal features and the interplay between sound and meaning. The second part of the paper describes a machine-learning approach to the identification and analysis of citational material that Livy loosely appropriated from earlier sources. We extend our approach to map the stylistic topography of Latin prose, identifying the writings of Caesar and his near-contemporary Livy as an inflection point in the development of Latin prose style. In total, our results reflect the integration of computational and humanistic methods to investigate a diverse range of literary questions. PMID:28373557

Quantitative criticism of literary relationships.

PubMed

Dexter, Joseph P; Katz, Theodore; Tripuraneni, Nilesh; Dasgupta, Tathagata; Kannan, Ajay; Brofos, James A; Bonilla Lopez, Jorge A; Schroeder, Lea A; Casarez, Adriana; Rabinovich, Maxim; Haimson Lushkov, Ayelet; Chaudhuri, Pramit

2017-04-18

Authors often convey meaning by referring to or imitating prior works of literature, a process that creates complex networks of literary relationships ("intertextuality") and contributes to cultural evolution. In this paper, we use techniques from stylometry and machine learning to address subjective literary critical questions about Latin literature, a corpus marked by an extraordinary concentration of intertextuality. Our work, which we term "quantitative criticism," focuses on case studies involving two influential Roman authors, the playwright Seneca and the historian Livy. We find that four plays related to but distinct from Seneca's main writings are differentiated from the rest of the corpus by subtle but important stylistic features. We offer literary interpretations of the significance of these anomalies, providing quantitative data in support of hypotheses about the use of unusual formal features and the interplay between sound and meaning. The second part of the paper describes a machine-learning approach to the identification and analysis of citational material that Livy loosely appropriated from earlier sources. We extend our approach to map the stylistic topography of Latin prose, identifying the writings of Caesar and his near-contemporary Livy as an inflection point in the development of Latin prose style. In total, our results reflect the integration of computational and humanistic methods to investigate a diverse range of literary questions.

Quantitative methods to direct exploration based on hydrogeologic information

USGS Publications Warehouse

Graettinger, A.J.; Lee, J.; Reeves, H.W.; Dethan, D.

2006-01-01

Quantitatively Directed Exploration (QDE) approaches based on information such as model sensitivity, input data covariance and model output covariance are presented. Seven approaches for directing exploration are developed, applied, and evaluated on a synthetic hydrogeologic site. The QDE approaches evaluate input information uncertainty, subsurface model sensitivity and, most importantly, output covariance to identify the next location to sample. Spatial input parameter values and covariances are calculated with the multivariate conditional probability calculation from a limited number of samples. A variogram structure is used during data extrapolation to describe the spatial continuity, or correlation, of subsurface information. Model sensitivity can be determined by perturbing input data and evaluating output response or, as in this work, sensitivities can be programmed directly into an analysis model. Output covariance is calculated by the First-Order Second Moment (FOSM) method, which combines the covariance of input information with model sensitivity. A groundwater flow example, modeled in MODFLOW-2000, is chosen to demonstrate the seven QDE approaches. MODFLOW-2000 is used to obtain the piezometric head and the model sensitivity simultaneously. The seven QDE approaches are evaluated based on the accuracy of the modeled piezometric head after information from a QDE sample is added. For the synthetic site used in this study, the QDE approach that identifies the location of hydraulic conductivity that contributes the most to the overall piezometric head variance proved to be the best method to quantitatively direct exploration. ?? IWA Publishing 2006.

Quantitative Ultrasound for Nondestructive Characterization of Engineered Tissues and Biomaterials

PubMed Central

Dalecki, Diane; Mercado, Karla P.; Hocking, Denise C.

2015-01-01

Non-invasive, non-destructive technologies for imaging and quantitatively monitoring the development of artificial tissues are critical for the advancement of tissue engineering. Current standard techniques for evaluating engineered tissues, including histology, biochemical assays and mechanical testing, are destructive approaches. Ultrasound is emerging as a valuable tool for imaging and quantitatively monitoring the properties of engineered tissues and biomaterials longitudinally during fabrication and post-implantation. Ultrasound techniques are rapid, non-invasive, non-destructive and can be easily integrated into sterile environments necessary for tissue engineering. Furthermore, high-frequency quantitative ultrasound techniques can enable volumetric characterization of the structural, biological, and mechanical properties of engineered tissues during fabrication and post-implantation. This review provides an overview of ultrasound imaging, quantitative ultrasound techniques, and elastography, with representative examples of applications of these ultrasound-based techniques to the field of tissue engineering. PMID:26581347

Beyond Math Skills: Measuring Quantitative Reasoning in Context

ERIC Educational Resources Information Center

Grawe, Nathan D.

2011-01-01

It might be argued that quantitative and qualitative analyses are merely two alternative reflections of an overarching critical thinking. For instance, just as instructors of numeracy warn their charges to consider the construction of variables, teachers of qualitative approaches caution students to define terms. Similarly, an advocate of…

Integrated Analysis and Tools for Land Subsidence Surveying and Monitoring: a Semi-Quantitative Approach

NASA Astrophysics Data System (ADS)

Mosconi, A.; Pozzoli, A.; Meroni, A.; Gagliano, S.

2015-10-01

This paper presents an integrated approach for land subsidence monitoring using measures coming from different sensors. Eni S.p.A., the main Italian oil and gas company, constantly surveys the land with all the state of the art and innovative techniques, and a method able to integrate the results is an important and actual topic. Nowadays the world is a multi-sensor platform, and measure integration is strictly necessary. Combining the different data sources should be done in a clever way, taking advantages from the best performances of each technique. An integrated analysis allows the interpretation of simultaneous temporal series of data, coming from different sources, and try to separate subsidence contributions. With this purpose Exelis VIS in collaboration with Eni S.p.A. customize PISAV (Permanent Interferometric Scatterometer Analysis and Visualization), an ENVI extension able to capitalize on and combine all the different data collected in the surveys. In this article are presented some significant examples to show the potential of this tool in oil and gas activity: a hydrocarbon storage field where the comparison between SAR and production volumes emphasise a correlation between the two measures in few steps; and a hydrocarbon production field with the Satellite Survey Unit (S.S.U.), where SAR, CGPS, piezometers and assestimeters measure in the same area at the same time, giving the opportunity to analyse data contextually. In the integrated analysis performed with PISAV not always a mathematical rigorous study is possible, and a semi-quantitative approach is the only method for results interpretation. As a result, in the first test case strong correlation between injected hydrocarbon volume and vertical displacement were highlighted; in the second one the integrated analysis has different advantages in monitoring the land subsidence: permits a first qualitative "differentiation" of the natural and anthropic component of subsidence, and also gives more

Modeling the Effect of Polychromatic Light in Quantitative Absorbance Spectroscopy

ERIC Educational Resources Information Center

Smith, Rachel; Cantrell, Kevin

2007-01-01

Laboratory experiment is conducted to give the students practical experience with the principles of electronic absorbance spectroscopy. This straightforward approach creates a powerful tool for exploring many of the aspects of quantitative absorbance spectroscopy.

A robust quantitative near infrared modeling approach for blend monitoring.

PubMed

Mohan, Shikhar; Momose, Wataru; Katz, Jeffrey M; Hossain, Md Nayeem; Velez, Natasha; Drennen, James K; Anderson, Carl A

2018-01-30

This study demonstrates a material sparing Near-Infrared modeling approach for powder blend monitoring. In this new approach, gram scale powder mixtures are subjected to compression loads to simulate the effect of scale using an Instron universal testing system. Models prepared by the new method development approach (small-scale method) and by a traditional method development (blender-scale method) were compared by simultaneously monitoring a 1kg batch size blend run. Both models demonstrated similar model performance. The small-scale method strategy significantly reduces the total resources expended to develop Near-Infrared calibration models for on-line blend monitoring. Further, this development approach does not require the actual equipment (i.e., blender) to which the method will be applied, only a similar optical interface. Thus, a robust on-line blend monitoring method can be fully developed before any large-scale blending experiment is viable, allowing the blend method to be used during scale-up and blend development trials. Copyright © 2017. Published by Elsevier B.V.

Quantitative evaluation of muscle synergy models: a single-trial task decoding approach

PubMed Central

Delis, Ioannis; Berret, Bastien; Pozzo, Thierry; Panzeri, Stefano

2013-01-01

Muscle synergies, i.e., invariant coordinated activations of groups of muscles, have been proposed as building blocks that the central nervous system (CNS) uses to construct the patterns of muscle activity utilized for executing movements. Several efficient dimensionality reduction algorithms that extract putative synergies from electromyographic (EMG) signals have been developed. Typically, the quality of synergy decompositions is assessed by computing the Variance Accounted For (VAF). Yet, little is known about the extent to which the combination of those synergies encodes task-discriminating variations of muscle activity in individual trials. To address this question, here we conceive and develop a novel computational framework to evaluate muscle synergy decompositions in task space. Unlike previous methods considering the total variance of muscle patterns (VAF based metrics), our approach focuses on variance discriminating execution of different tasks. The procedure is based on single-trial task decoding from muscle synergy activation features. The task decoding based metric evaluates quantitatively the mapping between synergy recruitment and task identification and automatically determines the minimal number of synergies that captures all the task-discriminating variability in the synergy activations. In this paper, we first validate the method on plausibly simulated EMG datasets. We then show that it can be applied to different types of muscle synergy decomposition and illustrate its applicability to real data by using it for the analysis of EMG recordings during an arm pointing task. We find that time-varying and synchronous synergies with similar number of parameters are equally efficient in task decoding, suggesting that in this experimental paradigm they are equally valid representations of muscle synergies. Overall, these findings stress the effectiveness of the decoding metric in systematically assessing muscle synergy decompositions in task space. PMID

A Quantitative Study of Empathy in Pakistani Medical Students: A Multicentered Approach.

PubMed

Tariq, Nabia; Rasheed, Tariq; Tavakol, Mohsen

2017-10-01

To examine the empathy level of undergraduate medical students in Pakistan. Three hypotheses are developed based on the literature review. (1) Female medical students have a higher level of empathy than do male students. (2) Empathy scores vary during the medical school years in Pakistani students. (3) Medical students interested in people-oriented specialties would score higher than the students interested in technology-oriented specialties. This is a quantitative inquiry approach using a cross-sectional design of 1453 students from 8 Pakistani medical schools, both private and state. The sample consists of 41.1% (n = 597) male students and 58.9% (n = 856) female students. Empirical data are collected using the Jefferson Scale of Physician Empathy (JSPE), a well-validated self-administered questionnaire. The mean empathy score among students is 4.77 with a standard deviation of 0.72. The results show that there is no statistically significant association between the empathy scores and gender, t(1342.36) = -0.053, P = .95). There is a statistically significant difference between the empathy scores and the years of medical school, F(14, 1448) = 4.95, P = .01. Concerning the specialty interests, there is no statistically significant difference between the empathy score and specialty interests. The findings of this study showed that in Western countries, medical students performed better than Pakistani medical students on the empathy scale. This finding has important implications for Pakistani medical educators to improve the interpersonal skills of medical students in the context of patient care. Inconsistent with our expectations and experiences, our findings do not support that female medical students scored better than their male counterparts on the empathy scale. Because of the nature of a cross-sectional study, it is impossible to argue the decline of empathy during medical school training.

1, 2, 3, 4: infusing quantitative literacy into introductory biology.

PubMed

Speth, Elena Bray; Momsen, Jennifer L; Moyerbrailean, Gregory A; Ebert-May, Diane; Long, Tammy M; Wyse, Sara; Linton, Debra

2010-01-01

Biology of the twenty-first century is an increasingly quantitative science. Undergraduate biology education therefore needs to provide opportunities for students to develop fluency in the tools and language of quantitative disciplines. Quantitative literacy (QL) is important for future scientists as well as for citizens, who need to interpret numeric information and data-based claims regarding nearly every aspect of daily life. To address the need for QL in biology education, we incorporated quantitative concepts throughout a semester-long introductory biology course at a large research university. Early in the course, we assessed the quantitative skills that students bring to the introductory biology classroom and found that students had difficulties in performing simple calculations, representing data graphically, and articulating data-driven arguments. In response to students' learning needs, we infused the course with quantitative concepts aligned with the existing course content and learning objectives. The effectiveness of this approach is demonstrated by significant improvement in the quality of students' graphical representations of biological data. Infusing QL in introductory biology presents challenges. Our study, however, supports the conclusion that it is feasible in the context of an existing course, consistent with the goals of college biology education, and promotes students' development of important quantitative skills.

1, 2, 3, 4: Infusing Quantitative Literacy into Introductory Biology

PubMed Central

Momsen, Jennifer L.; Moyerbrailean, Gregory A.; Ebert-May, Diane; Long, Tammy M.; Wyse, Sara; Linton, Debra

2010-01-01

Biology of the twenty-first century is an increasingly quantitative science. Undergraduate biology education therefore needs to provide opportunities for students to develop fluency in the tools and language of quantitative disciplines. Quantitative literacy (QL) is important for future scientists as well as for citizens, who need to interpret numeric information and data-based claims regarding nearly every aspect of daily life. To address the need for QL in biology education, we incorporated quantitative concepts throughout a semester-long introductory biology course at a large research university. Early in the course, we assessed the quantitative skills that students bring to the introductory biology classroom and found that students had difficulties in performing simple calculations, representing data graphically, and articulating data-driven arguments. In response to students' learning needs, we infused the course with quantitative concepts aligned with the existing course content and learning objectives. The effectiveness of this approach is demonstrated by significant improvement in the quality of students' graphical representations of biological data. Infusing QL in introductory biology presents challenges. Our study, however, supports the conclusion that it is feasible in the context of an existing course, consistent with the goals of college biology education, and promotes students' development of important quantitative skills. PMID:20810965

A Transformative Model for Undergraduate Quantitative Biology Education

ERIC Educational Resources Information Center

Usher, David C.; Driscoll, Tobin A.; Dhurjati, Prasad; Pelesko, John A.; Rossi, Louis F.; Schleiniger, Gilberto; Pusecker, Kathleen; White, Harold B.

2010-01-01

The "BIO2010" report recommended that students in the life sciences receive a more rigorous education in mathematics and physical sciences. The University of Delaware approached this problem by (1) developing a bio-calculus section of a standard calculus course, (2) embedding quantitative activities into existing biology courses, and (3)…

Para-Quantitative Methodology: Reclaiming Experimentalism in Educational Research

ERIC Educational Resources Information Center

Shabani Varaki, Bakhtiar; Floden, Robert E.; Javidi Kalatehjafarabadi, Tahereh

2015-01-01

This article focuses on the criticisms of current approaches in educational research methodology. It summarizes rationales for mixed methods and argues that the mixing quantitative paradigm and qualitative paradigm is problematic due to practical and philosophical arguments. It is also indicated that the current rise of mixed methods work has…

Quantitative multimodality imaging in cancer research and therapy.

PubMed

Yankeelov, Thomas E; Abramson, Richard G; Quarles, C Chad

2014-11-01

Advances in hardware and software have enabled the realization of clinically feasible, quantitative multimodality imaging of tissue pathophysiology. Earlier efforts relating to multimodality imaging of cancer have focused on the integration of anatomical and functional characteristics, such as PET-CT and single-photon emission CT (SPECT-CT), whereas more-recent advances and applications have involved the integration of multiple quantitative, functional measurements (for example, multiple PET tracers, varied MRI contrast mechanisms, and PET-MRI), thereby providing a more-comprehensive characterization of the tumour phenotype. The enormous amount of complementary quantitative data generated by such studies is beginning to offer unique insights into opportunities to optimize care for individual patients. Although important technical optimization and improved biological interpretation of multimodality imaging findings are needed, this approach can already be applied informatively in clinical trials of cancer therapeutics using existing tools. These concepts are discussed herein.

The linearized multistage model and the future of quantitative risk assessment.

PubMed

Crump, K S

1996-10-01

The linearized multistage (LMS) model has for over 15 years been the default dose-response model used by the U.S. Environmental Protection Agency (USEPA) and other federal and state regulatory agencies in the United States for calculating quantitative estimates of low-dose carcinogenic risks from animal data. The LMS model is in essence a flexible statistical model that can describe both linear and non-linear dose-response patterns, and that produces an upper confidence bound on the linear low-dose slope of the dose-response curve. Unlike its namesake, the Armitage-Doll multistage model, the parameters of the LMS do not correspond to actual physiological phenomena. Thus the LMS is 'biological' only to the extent that the true biological dose response is linear at low dose and that low-dose slope is reflected in the experimental data. If the true dose response is non-linear the LMS upper bound may overestimate the true risk by many orders of magnitude. However, competing low-dose extrapolation models, including those derived from 'biologically-based models' that are capable of incorporating additional biological information, have not shown evidence to date of being able to produce quantitative estimates of low-dose risks that are any more accurate than those obtained from the LMS model. Further, even if these attempts were successful, the extent to which more accurate estimates of low-dose risks in a test animal species would translate into improved estimates of human risk is questionable. Thus, it does not appear possible at present to develop a quantitative approach that would be generally applicable and that would offer significant improvements upon the crude bounding estimates of the type provided by the LMS model. Draft USEPA guidelines for cancer risk assessment incorporate an approach similar to the LMS for carcinogens having a linear mode of action. However, under these guidelines quantitative estimates of low-dose risks would not be developed for

Advancing tuberculosis drug regimen development through innovative quantitative translational pharmacology methods and approaches.

PubMed

Hanna, Debra; Romero, Klaus; Schito, Marco

2017-03-01

The development of novel tuberculosis (TB) multi-drug regimens that are more efficacious and of shorter duration requires a robust drug development pipeline. Advances in quantitative modeling and simulation can be used to maximize the utility of patient-level data from prior and contemporary clinical trials, thus optimizing study design for anti-TB regimens. This perspective article highlights the work of seven project teams developing first-in-class translational and quantitative methodologies that aim to inform drug development decision-making, dose selection, trial design, and safety assessments, in order to achieve shorter and safer therapies for patients in need. These tools offer the opportunity to evaluate multiple hypotheses and provide a means to identify, quantify, and understand relevant sources of variability, to optimize translation and clinical trial design. When incorporated into the broader regulatory sciences framework, these efforts have the potential to transform the development paradigm for TB combination development, as well as other areas of global health. Copyright © 2016. Published by Elsevier Ltd.

Quantitative Diagnosis of Continuous-Valued, Stead-State Systems

NASA Technical Reports Server (NTRS)

Rouquette, N.

1995-01-01

Quantitative diagnosis involves numerically estimating the values of unobservable parameters that best explain the observed parameter values. We consider quantitative diagnosis for continuous, lumped- parameter, steady-state physical systems because such models are easy to construct and the diagnosis problem is considerably simpler than that for corresponding dynamic models. To further tackle the difficulties of numerically inverting a simulation model to compute a diagnosis, we propose to decompose a physical system model in terms of feedback loops. This decomposition reduces the dimension of the problem and consequently decreases the diagnosis search space. We illustrate this approach on a model of thermal control system studied in earlier research.

DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

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

SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

2004-03-24

Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNAmore » populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.« less

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.