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.

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

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

PubMed

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

2015-10-13

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2014-02-01

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

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

PubMed

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

2017-10-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed Central

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

2016-01-01

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

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 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.

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

PubMed

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

2015-01-01

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

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

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.

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.

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

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

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

Informatics methods to enable sharing of quantitative imaging research data.

PubMed

Levy, Mia A; Freymann, John B; Kirby, Justin S; Fedorov, Andriy; Fennessy, Fiona M; Eschrich, Steven A; Berglund, Anders E; Fenstermacher, David A; Tan, Yongqiang; Guo, Xiaotao; Casavant, Thomas L; Brown, Bartley J; Braun, Terry A; Dekker, Andre; Roelofs, Erik; Mountz, James M; Boada, Fernando; Laymon, Charles; Oborski, Matt; Rubin, Daniel L

2012-11-01

The National Cancer Institute Quantitative Research Network (QIN) is a collaborative research network whose goal is to share data, algorithms and research tools to accelerate quantitative imaging research. A challenge is the variability in tools and analysis platforms used in quantitative imaging. Our goal was to understand the extent of this variation and to develop an approach to enable sharing data and to promote reuse of quantitative imaging data in the community. We performed a survey of the current tools in use by the QIN member sites for representation and storage of their QIN research data including images, image meta-data and clinical data. We identified existing systems and standards for data sharing and their gaps for the QIN use case. We then proposed a system architecture to enable data sharing and collaborative experimentation within the QIN. There are a variety of tools currently used by each QIN institution. We developed a general information system architecture to support the QIN goals. We also describe the remaining architecture gaps we are developing to enable members to share research images and image meta-data across the network. As a research network, the QIN will stimulate quantitative imaging research by pooling data, algorithms and research tools. However, there are gaps in current functional requirements that will need to be met by future informatics development. Special attention must be given to the technical requirements needed to translate these methods into the clinical research workflow to enable validation and qualification of these novel imaging biomarkers. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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.

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

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

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.

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

PubMed

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

2012-04-01

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

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

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.

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

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.

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.

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.

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.

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

SimPhospho: a software tool enabling confident phosphosite assignment.

PubMed

Suni, Veronika; Suomi, Tomi; Tsubosaka, Tomoya; Imanishi, Susumu Y; Elo, Laura L; Corthals, Garry L

2018-03-27

Mass spectrometry combined with enrichment strategies for phosphorylated peptides has been successfully employed for two decades to identify sites of phosphorylation. However, unambiguous phosphosite assignment is considered challenging. Given that site-specific phosphorylation events function as different molecular switches, validation of phosphorylation sites is of utmost importance. In our earlier study we developed a method based on simulated phosphopeptide spectral libraries, which enables highly sensitive and accurate phosphosite assignments. To promote more widespread use of this method, we here introduce a software implementation with improved usability and performance. We present SimPhospho, a fast and user-friendly tool for accurate simulation of phosphopeptide tandem mass spectra. Simulated phosphopeptide spectral libraries are used to validate and supplement database search results, with a goal to improve reliable phosphoproteome identification and reporting. The presented program can be easily used together with the Trans-Proteomic Pipeline and integrated in a phosphoproteomics data analysis workflow. SimPhospho is available for Windows, Linux and Mac operating systems at https://sourceforge.net/projects/simphospho/. It is open source and implemented in C ++. A user's manual with detailed description of data analysis using SimPhospho as well as test data can be found as supplementary material of this article. Supplementary data are available at https://www.btk.fi/research/ computational-biomedicine/software/.

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.

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.

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

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

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

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

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

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

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

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

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

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

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 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

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

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.

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

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

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.

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

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.

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

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.

Quantitative nephelometry

MedlinePlus

... this page: //medlineplus.gov/ency/article/003545.htm Quantitative nephelometry test To use the sharing features on this page, please enable JavaScript. Quantitative nephelometry is a lab test to quickly and ...

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.

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

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

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

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

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

Preclinical evaluation of spatial frequency domain-enabled wide-field quantitative imaging for enhanced glioma resection

NASA Astrophysics Data System (ADS)

Sibai, Mira; Fisher, Carl; Veilleux, Israel; Elliott, Jonathan T.; Leblond, Frederic; Roberts, David W.; Wilson, Brian C.

2017-07-01

5-Aminolevelunic acid-induced protoporphyrin IX (PpIX) fluorescence-guided resection (FGR) enables maximum safe resection of glioma by providing real-time tumor contrast. However, the subjective visual assessment and the variable intrinsic optical attenuation of tissue limit this technique to reliably delineating only high-grade tumors that display strong fluorescence. We have previously shown, using a fiber-optic probe, that quantitative assessment using noninvasive point spectroscopic measurements of the absolute PpIX concentration in tissue further improves the accuracy of FGR, extending it to surgically curable low-grade glioma. More recently, we have shown that implementing spatial frequency domain imaging with a fluorescent-light transport model enables recovery of two-dimensional images of [PpIX], alleviating the need for time-consuming point sampling of the brain surface. We present first results of this technique modified for in vivo imaging on an RG2 rat brain tumor model. Despite the moderate errors in retrieving the absorption and reduced scattering coefficients in the subdiffusive regime of 14% and 19%, respectively, the recovered [PpIX] maps agree within 10% of the point [PpIX] values measured by the fiber-optic probe, validating its potential as an extension or an alternative to point sampling during glioma resection.

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 Glycomics Strategies*

PubMed Central

Mechref, Yehia; Hu, Yunli; Desantos-Garcia, Janie L.; Hussein, Ahmed; Tang, Haixu

2013-01-01

The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. PMID:23325767

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.

Image velocimetry and spectral analysis enable quantitative characterization of larval zebrafish gut motility.

PubMed

Ganz, J; Baker, R P; Hamilton, M K; Melancon, E; Diba, P; Eisen, J S; Parthasarathy, R

2018-05-02

Normal gut function requires rhythmic and coordinated movements that are affected by developmental processes, physical and chemical stimuli, and many debilitating diseases. The imaging and characterization of gut motility, especially regarding periodic, propagative contractions driving material transport, are therefore critical goals. Previous image analysis approaches have successfully extracted properties related to the temporal frequency of motility modes, but robust measures of contraction magnitude, especially from in vivo image data, remain challenging to obtain. We developed a new image analysis method based on image velocimetry and spectral analysis that reveals temporal characteristics such as frequency and wave propagation speed, while also providing quantitative measures of the amplitude of gut motion. We validate this approach using several challenges to larval zebrafish, imaged with differential interference contrast microscopy. Both acetylcholine exposure and feeding increase frequency and amplitude of motility. Larvae lacking enteric nervous system gut innervation show the same average motility frequency, but reduced and less variable amplitude compared to wild types. Our image analysis approach enables insights into gut dynamics in a wide variety of developmental and physiological contexts and can also be extended to analyze other types of cell movements. © 2018 John Wiley & Sons Ltd.

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

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.

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.

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

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.

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

High-Resolution Enabled 12-Plex DiLeu Isobaric Tags for Quantitative Proteomics

PubMed Central

2015-01-01

Multiplex isobaric tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantification (iTRAQ)) are a valuable tool for high-throughput mass spectrometry based quantitative proteomics. We have developed our own multiplex isobaric tags, DiLeu, that feature quantitative performance on par with commercial offerings but can be readily synthesized in-house as a cost-effective alternative. In this work, we achieve a 3-fold increase in the multiplexing capacity of the DiLeu reagent without increasing structural complexity by exploiting mass defects that arise from selective incorporation of 13C, 15N, and 2H stable isotopes in the reporter group. The inclusion of eight new reporter isotopologues that differ in mass from the existing four reporters by intervals of 6 mDa yields a 12-plex isobaric set that preserves the synthetic simplicity and quantitative performance of the original implementation. We show that the new reporter variants can be baseline-resolved in high-resolution higher-energy C-trap dissociation (HCD) spectra, and we demonstrate accurate 12-plex quantitation of a DiLeu-labeled Saccharomyces cerevisiae lysate digest via high-resolution nano liquid chromatography–tandem mass spectrometry (nanoLC–MS2) analysis on an Orbitrap Elite mass spectrometer. PMID:25405479

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.

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

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

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.

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.

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.

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

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.

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.

A peptide-retrieval strategy enables significant improvement of quantitative performance without compromising confidence of identification.

PubMed

Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

2017-01-30

Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches. We hypothesize that more quantifiable spectra and peptides in a protein, even including less confident peptides, could help reduce variations and improve protein quantification. Hence the peptide retrieval strategy was developed and evaluated in two spike-in sample sets with different LC-MS/MS variations using both MS1- and MS2-based quantitative approach. The list of confidently identified proteins using the standard target-decoy search strategy was fixed and more spectra/peptides with less

Quantitative secondary electron detection

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

Agrawal, Jyoti; Joy, David C.; Nayak, Subuhadarshi

Quantitative Secondary Electron Detection (QSED) using the array of solid state devices (SSD) based electron-counters enable critical dimension metrology measurements in materials such as semiconductors, nanomaterials, and biological samples (FIG. 3). Methods and devices effect a quantitative detection of secondary electrons with the array of solid state detectors comprising a number of solid state detectors. An array senses the number of secondary electrons with a plurality of solid state detectors, counting the number of secondary electrons with a time to digital converter circuit in counter mode.

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.

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

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.

Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities

PubMed Central

Bokulich, Nicholas A.

2013-01-01

Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities. PMID:23377949

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

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

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

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.

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

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

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

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

Systematic inference of functional phosphorylation events in yeast metabolism.

PubMed

Chen, Yu; Wang, Yonghong; Nielsen, Jens

2017-07-01

Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Quantitative proteomics in biological research.

PubMed

Wilm, Matthias

2009-10-01

Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article reviews the different methods used to extract quantitative information from mass spectra. It follows the technical developments aimed toward global proteomics, the attempt to characterize every expressed protein in a cell by at least one peptide. When applications of the technology are discussed, the focus is placed on yeast biology. In particular, differential quantitative proteomics, the comparison between an experiment and its control, is very discriminating for proteins involved in the process being studied. When trying to understand biological processes on a molecular level, differential quantitative proteomics tends to give a clearer picture than global transcription analyses. As a result, MS has become an even more indispensable tool for biochemically motivated biological research.

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…

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.

Quantitative mass spectrometry: an overview

NASA Astrophysics Data System (ADS)

Urban, Pawel L.

2016-10-01

Mass spectrometry (MS) is a mainstream chemical analysis technique in the twenty-first century. It has contributed to numerous discoveries in chemistry, physics and biochemistry. Hundreds of research laboratories scattered all over the world use MS every day to investigate fundamental phenomena on the molecular level. MS is also widely used by industry-especially in drug discovery, quality control and food safety protocols. In some cases, mass spectrometers are indispensable and irreplaceable by any other metrological tools. The uniqueness of MS is due to the fact that it enables direct identification of molecules based on the mass-to-charge ratios as well as fragmentation patterns. Thus, for several decades now, MS has been used in qualitative chemical analysis. To address the pressing need for quantitative molecular measurements, a number of laboratories focused on technological and methodological improvements that could render MS a fully quantitative metrological platform. In this theme issue, the experts working for some of those laboratories share their knowledge and enthusiasm about quantitative MS. I hope this theme issue will benefit readers, and foster fundamental and applied research based on quantitative MS measurements. This article is part of the themed issue 'Quantitative mass spectrometry'.

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

Impact of voice- and knowledge-enabled clinical reporting--US example.

PubMed

Bushko, Renata G; Havlicek, Penny L; Deppert, Edward; Epner, Stephen

2002-01-01

This study shows qualitative and quantitative estimates of the national and the clinic level impact of utilizing voice and knowledge enabled clinical reporting systems. Using common sense estimation methodology, we show that the delivery of health care can experience a dramatic improvement in four areas as a result of the broad use of voice and knowledge enabled clinical reporting: (1) Process Quality as measured by cost savings, (2) Organizational Quality as measured by compliance, (3) Clinical Quality as measured by clinical outcomes and (4) Service Quality as measured by patient satisfaction. If only 15 percent of US physicians replaced transcription with modem clinical reporting voice-based methodology, about one half billion dollars could be saved. $6.7 Billion could be saved annually if all medical reporting currently transcribed was handled with voice-and knowledge-enabled dictation and reporting systems.

PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

PubMed

Chaudhuri, Rima; Sadrieh, Arash; Hoffman, Nolan J; Parker, Benjamin L; Humphrey, Sean J; Stöckli, Jacqueline; Hill, Adam P; James, David E; Yang, Jean Yee Hwa

2015-08-19

Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community. Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

PubMed

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

2018-01-08

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

Optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Wu, Yi; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Lee, Sangwook; Isozaki, Akihiro; Li, Ming; Jiang, Yiyue; Yasumoto, Atsushi; Di Carlo, Dino; Tanaka, Yo; Yatomi, Yutaka; Ozeki, Yasuyuki; Goda, Keisuke

2018-03-01

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

A quantitative reconstruction software suite for SPECT imaging

NASA Astrophysics Data System (ADS)

Namías, Mauro; Jeraj, Robert

2017-11-01

Quantitative Single Photon Emission Tomography (SPECT) imaging allows for measurement of activity concentrations of a given radiotracer in vivo. Although SPECT has usually been perceived as non-quantitative by the medical community, the introduction of accurate CT based attenuation correction and scatter correction from hybrid SPECT/CT scanners has enabled SPECT systems to be as quantitative as Positron Emission Tomography (PET) systems. We implemented a software suite to reconstruct quantitative SPECT images from hybrid or dedicated SPECT systems with a separate CT scanner. Attenuation, scatter and collimator response corrections were included in an Ordered Subset Expectation Maximization (OSEM) algorithm. A novel scatter fraction estimation technique was introduced. The SPECT/CT system was calibrated with a cylindrical phantom and quantitative accuracy was assessed with an anthropomorphic phantom and a NEMA/IEC image quality phantom. Accurate activity measurements were achieved at an organ level. This software suite helps increasing quantitative accuracy of SPECT scanners.

Paper Capillary Enables Effective Sampling for Microfluidic Paper Analytical Devices.

PubMed

Shangguan, Jin-Wen; Liu, Yu; Wang, Sha; Hou, Yun-Xuan; Xu, Bi-Yi; Xu, Jing-Juan; Chen, Hong-Yuan

2018-06-06

Paper capillary is introduced to enable effective sampling on microfluidic paper analytical devices. By coupling mac-roscale capillary force of paper capillary and microscale capillary forces of native paper, fluid transport can be flexibly tailored with proper design. Subsequently, a hybrid-fluid-mode paper capillary device was proposed, which enables fast and reliable sampling in an arrayed form, with less surface adsorption and bias for different components. The resulting device thus well supports high throughput, quantitative, and repeatable assays all by hands operation. With all these merits, multiplex analysis of ions, proteins, and microbe have all been realized on this platform, which has paved the way to level-up analysis on μPADs.

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.

A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan

PubMed Central

Yoshida, Yoko; Miyata, Toshiyuki; Matsumoto, Masanori; Shirotani-Ikejima, Hiroko; Uchida, Yumiko; Ohyama, Yoshifumi; Kokubo, Tetsuro; Fujimura, Yoshihiro

2015-01-01

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients. PMID:25951460

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 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 prediction of phase transformations in silicon during nanoindentation

NASA Astrophysics Data System (ADS)

Zhang, Liangchi; Basak, Animesh

2013-08-01

This paper establishes the first quantitative relationship between the phases transformed in silicon and the shape characteristics of nanoindentation curves. Based on an integrated analysis using TEM and unit cell properties of phases, the volumes of the phases emerged in a nanoindentation are formulated as a function of pop-out size and depth of nanoindentation impression. This simple formula enables a fast, accurate and quantitative prediction of the phases in a nanoindentation cycle, which has been impossible before.

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.

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…

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

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

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.

Nanoplasmon-enabled macroscopic thermal management

PubMed Central

Jonsson, Gustav Edman; Miljkovic, Vladimir; Dmitriev, Alexandre

2014-01-01

In numerous applications of energy harvesting via transformation of light into heat the focus recently shifted towards highly absorptive nanoplasmonic materials. It is currently established that noble metals-based absorptive plasmonic platforms deliver significant light-capturing capability and can be viewed as super-absorbers of optical radiation. Naturally, approaches to the direct experimental probing of macroscopic temperature increase resulting from these absorbers are welcomed. Here we derive a general quantitative method of characterizing heat-generating properties of optically absorptive layers via macroscopic thermal imaging. We further monitor macroscopic areas that are homogeneously heated by several degrees with nanostructures that occupy a mere 8% of the surface, leaving it essentially transparent and evidencing significant heat generation capability of nanoplasmon-enabled light capture. This has a direct bearing to a large number of applications where thermal management is crucial. PMID:24870613

The Development of Mathematical Knowledge for Teaching for Quantitative Reasoning Using Video-Based Instruction

ERIC Educational Resources Information Center

Walters, Charles David

2017-01-01

Quantitative reasoning (P. W. Thompson, 1990, 1994) is a powerful mathematical tool that enables students to engage in rich problem solving across the curriculum. One way to support students' quantitative reasoning is to develop prospective secondary teachers' (PSTs) mathematical knowledge for teaching (MKT; Ball, Thames, & Phelps, 2008)…

Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

2010-05-01

Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

Quantitative force measurements using frequency modulation atomic force microscopy—theoretical foundations

NASA Astrophysics Data System (ADS)

Sader, John E.; Uchihashi, Takayuki; Higgins, Michael J.; Farrell, Alan; Nakayama, Yoshikazu; Jarvis, Suzanne P.

2005-03-01

Use of the atomic force microscope (AFM) in quantitative force measurements inherently requires a theoretical framework enabling conversion of the observed deflection properties of the cantilever to an interaction force. In this paper, the theoretical foundations of using frequency modulation atomic force microscopy (FM-AFM) in quantitative force measurements are examined and rigorously elucidated, with consideration being given to both 'conservative' and 'dissipative' interactions. This includes a detailed discussion of the underlying assumptions involved in such quantitative force measurements, the presentation of globally valid explicit formulae for evaluation of so-called 'conservative' and 'dissipative' forces, discussion of the origin of these forces, and analysis of the applicability of FM-AFM to quantitative force measurements in liquid.

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.

Microfluidic device enabled quantitative time-lapse microscopic-photography for phenotyping vegetative and reproductive phases in Fusarium virguliforme, which is pathogenic to soybean.

PubMed

Marshall, Jill; Qiao, Xuan; Baumbach, Jordan; Xie, Jingyu; Dong, Liang; Bhattacharyya, Madan K

2017-03-15

Time-lapse microscopic-photography allows in-depth phenotyping of microorganisms. Here we report development of such a system using a microfluidic device, generated from polydimethylsiloxane and glass slide, placed on a motorized stage of a microscope for conducting time-lapse microphotography of multiple observations in 20 channels simultaneously. We have demonstrated the utility of the device in studying growth, germination and sporulation in Fusarium virguliforme that causes sudden death syndrome in soybean. To measure the growth differences, we developed a polyamine oxidase fvpo1 mutant in this fungus that fails to grow in minimal medium containing polyamines as the sole nitrogen source. Using this system, we demonstrated that the conidiospores of the pathogen take an average of five hours to germinate. During sporulation, it takes an average of 10.5 h for a conidiospore to mature and get detached from its conidiophore for the first time. Conidiospores are developed in a single conidiophore one after another. The microfluidic device enabled quantitative time-lapse microphotography reported here should be suitable for screening compounds, peptides, micro-organisms to identify fungitoxic or antimicrobial agents for controlling serious plant pathogens. The device could also be applied in identifying suitable target genes for host-induced gene silencing in pathogens for generating novel disease resistance in crop plants.

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

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

Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com; Cromwell, Evan F., E-mail: evan.cromwell@moldev.com; Crittenden, Carole

2013-12-15

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca{sup 2+} flux readouts synchronous with beating, and cell viability. Amore » number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive “cardiosafety” ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development. - Highlights: • Induced pluripotent stem cell-derived cardiomyocytes are promising in vitro models. • We tested if

Enabling Quantitative Optical Imaging for In-die-capable Critical Dimension Targets

PubMed Central

Barnes, B.M.; Henn, M.-A.; Sohn, M. Y.; Zhou, H.; Silver, R. M.

2017-01-01

Dimensional scaling trends will eventually bring semiconductor critical dimensions (CDs) down to only a few atoms in width. New optical techniques are required to address the measurement and variability for these CDs using sufficiently small in-die metrology targets. Recently, Qin et al. [Light Sci Appl, 5, e16038 (2016)] demonstrated quantitative model-based measurements of finite sets of lines with features as small as 16 nm using 450 nm wavelength light. This paper uses simulation studies, augmented with experiments at 193 nm wavelength, to adapt and optimize the finite sets of features that work as in-die-capable metrology targets with minimal increases in parametric uncertainty. A finite element based solver for time-harmonic Maxwell's equations yields two- and three-dimensional simulations of the electromagnetic scattering for optimizing the design of such targets as functions of reduced line lengths, fewer number of lines, fewer focal positions, smaller critical dimensions, and shorter illumination wavelength. Metrology targets that exceeded performance requirements are as short as 3 μm for 193 nm light, feature as few as eight lines, and are extensible to sub-10 nm CDs. Target areas measured at 193 nm can be fifteen times smaller in area than current state-of-the-art scatterometry targets described in the literature. This new methodology is demonstrated to be a promising alternative for optical model-based in-die CD metrology. PMID:28757674

Quantitative self-assembly prediction yields targeted nanomedicines

NASA Astrophysics Data System (ADS)

Shamay, Yosi; Shah, Janki; Işık, Mehtap; Mizrachi, Aviram; Leibold, Josef; Tschaharganeh, Darjus F.; Roxbury, Daniel; Budhathoki-Uprety, Januka; Nawaly, Karla; Sugarman, James L.; Baut, Emily; Neiman, Michelle R.; Dacek, Megan; Ganesh, Kripa S.; Johnson, Darren C.; Sridharan, Ramya; Chu, Karen L.; Rajasekhar, Vinagolu K.; Lowe, Scott W.; Chodera, John D.; Heller, Daniel A.

2018-02-01

Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.

Current trends in quantitative proteomics - an update.

PubMed

Li, H; Han, J; Pan, J; Liu, T; Parker, C E; Borchers, C H

2017-05-01

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Advances in Surface Plasmon Resonance Imaging enable quantitative measurement of laterally heterogeneous coatings of nanoscale thickness

NASA Astrophysics Data System (ADS)

Raegen, Adam; Reiter, Kyle; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2013-03-01

The Surface Plasmon Resonance (SPR) phenomenon is routinely exploited to qualitatively probe changes to the optical properties of nanoscale coatings on thin metallic surfaces, for use in probes and sensors. Unfortunately, extracting truly quantitative information is usually limited to a select few cases - uniform absorption/desorption of small biomolecules and films, in which a continuous ``slab'' model is a good approximation. We present advancements in the SPR technique that expand the number of cases for which the technique can provide meaningful results. Use of a custom, angle-scanning SPR imaging system, together with a refined data analysis method, allow for quantitative kinetic measurements of laterally heterogeneous systems. We first demonstrate the directionally heterogeneous nature of the SPR phenomenon using a directionally ordered sample, then show how this allows for the calculation of the average coverage of a heterogeneous sample. Finally, the degradation of cellulose microfibrils and bundles of microfibrils due to the action of cellulolytic enzymes will be presented as an excellent example of the capabilities of the SPR imaging 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.

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

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

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…

Kinetic Modeling of Accelerated Stability Testing Enabled by Second Harmonic Generation Microscopy.

PubMed

Song, Zhengtian; Sarkar, Sreya; Vogt, Andrew D; Danzer, Gerald D; Smith, Casey J; Gualtieri, Ellen J; Simpson, Garth J

2018-04-03

The low limits of detection afforded by second harmonic generation (SHG) microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs). ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. Extensive stability testing is typically performed for ASD characterization, the time frame for which is often dictated by the earliest detectable onset of crystal formation. Here a study of accelerated stability testing on ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, has been conducted. Under the condition for accelerated stability testing at 50 °C/75%RH and 40 °C/75%RH, ritonavir crystallization kinetics from amorphous solid dispersions were monitored by SHG microscopy. SHG microscopy coupled by image analysis yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about 2 orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The four decade dynamic range of SHG microscopy enabled quantitative modeling with an established (JMAK) kinetic model. From the SHG images, nucleation and crystal growth rates were independently determined.

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.

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

MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

PubMed

Caetano, Fabiana A; Dirk, Brennan S; Tam, Joshua H K; Cavanagh, P Craig; Goiko, Maria; Ferguson, Stephen S G; Pasternak, Stephen H; Dikeakos, Jimmy D; de Bruyn, John R; Heit, Bryan

2015-12-01

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

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

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

NanoDrop Microvolume Quantitation of Nucleic Acids

PubMed Central

Desjardins, Philippe; Conklin, Deborah

2010-01-01

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation. The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results. The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer. Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of

Quantitative analysis of culture using millions of digitized books

PubMed Central

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

2011-01-01

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

Quantitative analysis of culture using millions of digitized books.

PubMed

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

2011-01-14

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

Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

PubMed Central

Yeh, Erh-Chia; Fu, Chi-Cheng; Hu, Lucy; Thakur, Rohan; Feng, Jeffrey; Lee, Luke P.

2017-01-01

Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays. PMID:28345028

Uncertainty of quantitative microbiological methods of pharmaceutical analysis.

PubMed

Gunar, O V; Sakhno, N G

2015-12-30

The total uncertainty of quantitative microbiological methods, used in pharmaceutical analysis, consists of several components. The analysis of the most important sources of the quantitative microbiological methods variability demonstrated no effect of culture media and plate-count techniques in the estimation of microbial count while the highly significant effect of other factors (type of microorganism, pharmaceutical product and individual reading and interpreting errors) was established. The most appropriate method of statistical analysis of such data was ANOVA which enabled not only the effect of individual factors to be estimated but also their interactions. Considering all the elements of uncertainty and combining them mathematically the combined relative uncertainty of the test results was estimated both for method of quantitative examination of non-sterile pharmaceuticals and microbial count technique without any product. These data did not exceed 35%, appropriated for a traditional plate count methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

PubMed

Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

2016-05-20

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

Towards quantitative mass spectrometry-based metabolomics in microbial and mammalian systems.

PubMed

Kapoore, Rahul Vijay; Vaidyanathan, Seetharaman

2016-10-28

Metabolome analyses are a suite of analytical approaches that enable us to capture changes in the metabolome (small molecular weight components, typically less than 1500 Da) in biological systems. Mass spectrometry (MS) has been widely used for this purpose. The key challenge here is to be able to capture changes in a reproducible and reliant manner that is representative of the events that take place in vivo Typically, the analysis is carried out in vitro, by isolating the system and extracting the metabolome. MS-based approaches enable us to capture metabolomic changes with high sensitivity and resolution. When developing the technique for different biological systems, there are similarities in challenges and differences that are specific to the system under investigation. Here, we review some of the challenges in capturing quantitative changes in the metabolome with MS based approaches, primarily in microbial and mammalian systems.This article is part of the themed issue 'Quantitative mass spectrometry'. © 2016 The Author(s).

An evolving Mars telecommunications network to enable exploration and increase science data return

NASA Technical Reports Server (NTRS)

Edwards, Chad; Komarek, Tomas A.; Noreen, Gary K.; Wilson, Gregory R.

2003-01-01

The coming decade of Mars exploration involves a variety of unique telecommunications challenges. Increasing spatial and spectral resolution of in situ science instruments drive the need for increased bandwidth. At the same time, many innovative and low-cost in situ mission concepts are enabled by energy-efficient relay communications. In response to these needs, the Mars Exploration Program has established a plan for an evolving orbital infrastructure that can provide enhancing and enabling telecommunications services to future Mars missions. We will present the evolving capabilities of this network over the coming decade in terms of specific quantitative metrics such as data volume per sol and required lander energy per Gb of returned data for representative classes of Mars exploration spacecraft.

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

A comparative study of quantitative immunohistochemistry and quantum dot immunohistochemistry for mutation carrier identification in Lynch syndrome.

PubMed

Barrow, Emma; Evans, D Gareth; McMahon, Ray; Hill, James; Byers, Richard

2011-03-01

Lynch Syndrome is caused by mutations in DNA mismatch repair (MMR) genes. Mutation carrier identification is facilitated by immunohistochemical detection of the MMR proteins MHL1 and MSH2 in tumour tissue and is desirable as colonoscopic screening reduces mortality. However, protein detection by conventional immunohistochemistry (IHC) is subjective, and quantitative techniques are required. Quantum dots (QDs) are novel fluorescent labels that enable quantitative multiplex staining. This study compared their use with quantitative 3,3'-diaminobenzidine (DAB) IHC for the diagnosis of Lynch Syndrome. Tumour sections from 36 mutation carriers and six controls were obtained. These were stained with DAB on an automated platform using antibodies against MLH1 and MSH2. Multiplex QD immunofluorescent staining of the sections was performed using antibodies against MLH1, MSH2 and smooth muscle actin (SMA). Multispectral analysis of the slides was performed. The staining intensity of DAB and QDs was measured in multiple colonic crypts, and the mean intensity scores calculated. Receiver operating characteristic (ROC) curves of staining performance for the identification of mutation carriers were evaluated. For quantitative DAB IHC, the area under the MLH1 ROC curve was 0.872 (95% CI 0.763 to 0.981), and the area under the MSH2 ROC curve was 0.832 (95% CI 0.704 to 0.960). For quantitative QD IHC, the area under the MLH1 ROC curve was 0.812 (95% CI 0.681 to 0.943), and the area under the MSH2 ROC curve was 0.598 (95% CI 0.418 to 0.777). Despite the advantage of QD staining to enable several markers to be measured simultaneously, it is of lower utility than DAB IHC for the identification of MMR mutation carriers. Automated DAB IHC staining and quantitative slide analysis may enable high-throughput IHC.

Quantitation of sweet steviol glycosides by means of a HILIC-MS/MS-SIDA approach.

PubMed

Well, Caroline; Frank, Oliver; Hofmann, Thomas

2013-11-27

Meeting the rising consumer demand for natural food ingredients, steviol glycosides, the sweet principle of Stevia rebaudiana Bertoni (Bertoni), have recently been approved as food additives in the European Union. As regulatory constraints require sensitive methods to analyze the sweet-tasting steviol glycosides in foods and beverages, a HILIC-MS/MS method was developed enabling the accurate and reliable quantitation of the major steviol glycosides stevioside, rebaudiosides A-F, steviolbioside, rubusoside, and dulcoside A by using the corresponding deuterated 16,17-dihydrosteviol glycosides as suitable internal standards. This quantitation not only enables the analysis of the individual steviol glycosides in foods and beverages but also can support the optimization of breeding and postharvest downstream processing of Stevia plants to produce preferentially sweet and least bitter tasting Stevia extracts.

Quantitative Imaging in Cancer Evolution and Ecology

PubMed Central

Grove, Olya; Gillies, Robert J.

2013-01-01

Cancer therapy, even when highly targeted, typically fails because of the remarkable capacity of malignant cells to evolve effective adaptations. These evolutionary dynamics are both a cause and a consequence of cancer system heterogeneity at many scales, ranging from genetic properties of individual cells to large-scale imaging features. Tumors of the same organ and cell type can have remarkably diverse appearances in different patients. Furthermore, even within a single tumor, marked variations in imaging features, such as necrosis or contrast enhancement, are common. Similar spatial variations recently have been reported in genetic profiles. Radiologic heterogeneity within tumors is usually governed by variations in blood flow, whereas genetic heterogeneity is typically ascribed to random mutations. However, evolution within tumors, as in all living systems, is subject to Darwinian principles; thus, it is governed by predictable and reproducible interactions between environmental selection forces and cell phenotype (not genotype). This link between regional variations in environmental properties and cellular adaptive strategies may permit clinical imaging to be used to assess and monitor intratumoral evolution in individual patients. This approach is enabled by new methods that extract, report, and analyze quantitative, reproducible, and mineable clinical imaging data. However, most current quantitative metrics lack spatialness, expressing quantitative radiologic features as a single value for a region of interest encompassing the whole tumor. In contrast, spatially explicit image analysis recognizes that tumors are heterogeneous but not well mixed and defines regionally distinct habitats, some of which appear to harbor tumor populations that are more aggressive and less treatable than others. By identifying regional variations in key environmental selection forces and evidence of cellular adaptation, clinical imaging can enable us to define intratumoral

Quantitative Oxygenation Venography from MRI Phase

PubMed Central

Fan, Audrey P.; Bilgic, Berkin; Gagnon, Louis; Witzel, Thomas; Bhat, Himanshu; Rosen, Bruce R.; Adalsteinsson, Elfar

2014-01-01

Purpose To demonstrate acquisition and processing methods for quantitative oxygenation venograms that map in vivo oxygen saturation (SvO2) along cerebral venous vasculature. Methods Regularized quantitative susceptibility mapping (QSM) is used to reconstruct susceptibility values and estimate SvO2 in veins. QSM with ℓ1 and ℓ2 regularization are compared in numerical simulations of vessel structures with known magnetic susceptibility. Dual-echo, flow-compensated phase images are collected in three healthy volunteers to create QSM images. Bright veins in the susceptibility maps are vectorized and used to form a three-dimensional vascular mesh, or venogram, along which to display SvO2 values from QSM. Results Quantitative oxygenation venograms that map SvO2 along brain vessels of arbitrary orientation and geometry are shown in vivo. SvO2 values in major cerebral veins lie within the normal physiological range reported by 15O positron emission tomography. SvO2 from QSM is consistent with previous MR susceptometry methods for vessel segments oriented parallel to the main magnetic field. In vessel simulations, ℓ1 regularization results in less than 10% SvO2 absolute error across all vessel tilt orientations and provides more accurate SvO2 estimation than ℓ2 regularization. Conclusion The proposed analysis of susceptibility images enables reliable mapping of quantitative SvO2 along venograms and may facilitate clinical use of venous oxygenation imaging. PMID:24006229

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

PubMed

Shewale, Jaiprakash G; Schneida, Elaine; Wilson, Jonathan; Walker, Jerilyn A; Batzer, Mark A; Sinha, Sudhir K

2007-03-01

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

A multiplexed system for quantitative comparisons of chromatin landscapes

PubMed Central

van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

2015-01-01

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

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.

Quantitation without Calibration: Response Profile as an Indicator of Target Amount.

PubMed

Debnath, Mrittika; Farace, Jessica M; Johnson, Kristopher D; Nesterova, Irina V

2018-06-21

Quantitative assessment of biomarkers is essential in numerous contexts from decision-making in clinical situations to food quality monitoring to interpretation of life-science research findings. However, appropriate quantitation techniques are not as widely addressed as detection methods. One of the major challenges in biomarker's quantitation is the need to have a calibration for correlating a measured signal to a target amount. The step complicates the methodologies and makes them less sustainable. In this work we address the issue via a new strategy: relying on position of response profile rather than on an absolute signal value for assessment of a target's amount. In order to enable the capability we develop a target-probe binding mechanism based on a negative cooperativity effect. A proof-of-concept example demonstrates that the model is suitable for quantitative analysis of nucleic acids over a wide concentration range. The general principles of the platform will be applicable toward a variety of biomarkers such as nucleic acids, proteins, peptides, and others.

Distance-based microfluidic quantitative detection methods for point-of-care testing.

PubMed

Tian, Tian; Li, Jiuxing; Song, Yanling; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong James

2016-04-07

Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings. Among various equipment-free approaches, distance-based visual quantitative detection methods rely on reading the visual signal length for corresponding target concentrations, thus eliminating the need for sophisticated instruments. The distance-based methods are low-cost, user-friendly and can be integrated into portable analytical devices. Moreover, such methods enable quantitative detection of various targets by the naked eye. In this review, we first introduce the concept and history of distance-based visual quantitative detection methods. Then, we summarize the main methods for translation of molecular signals to distance-based readout and discuss different microfluidic platforms (glass, PDMS, paper and thread) in terms of applications in biomedical diagnostics, food safety monitoring, and environmental analysis. Finally, the potential and future perspectives are discussed.

Identification of Phosphorylated Proteins on a Global Scale.

PubMed

Iliuk, Anton

2018-05-31

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

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.

Qualitative and quantitative interpretation of SEM image using digital image processing.

PubMed

Saladra, Dawid; Kopernik, Magdalena

2016-10-01

The aim of the this study is improvement of qualitative and quantitative analysis of scanning electron microscope micrographs by development of computer program, which enables automatic crack analysis of scanning electron microscopy (SEM) micrographs. Micromechanical tests of pneumatic ventricular assist devices result in a large number of micrographs. Therefore, the analysis must be automatic. Tests for athrombogenic titanium nitride/gold coatings deposited on polymeric substrates (Bionate II) are performed. These tests include microshear, microtension and fatigue analysis. Anisotropic surface defects observed in the SEM micrographs require support for qualitative and quantitative interpretation. Improvement of qualitative analysis of scanning electron microscope images was achieved by a set of computational tools that includes binarization, simplified expanding, expanding, simple image statistic thresholding, the filters Laplacian 1, and Laplacian 2, Otsu and reverse binarization. Several modifications of the known image processing techniques and combinations of the selected image processing techniques were applied. The introduced quantitative analysis of digital scanning electron microscope images enables computation of stereological parameters such as area, crack angle, crack length, and total crack length per unit area. This study also compares the functionality of the developed computer program of digital image processing with existing applications. The described pre- and postprocessing may be helpful in scanning electron microscopy and transmission electron microscopy surface investigations. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Measurements in quantitative research: how to select and report on research instruments.

PubMed

Hagan, Teresa L

2014-07-01

Measures exist to numerically represent degrees of attributes. Quantitative research is based on measurement and is conducted in a systematic, controlled manner. These measures enable researchers to perform statistical tests, analyze differences between groups, and determine the effectiveness of treatments. If something is not measurable, it cannot be tested.

EnableATIS strategy assessment.

DOT National Transportation Integrated Search

2014-02-01

Enabling Advanced Traveler Information Systems (EnableATIS) is the traveler information component of the Dynamic Mobility Application (DMA) program. The objective of the EnableATIS effort is to foster transformative traveler information applications ...

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

Probing myocardium biomechanics using quantitative optical coherence elastography

NASA Astrophysics Data System (ADS)

Wang, Shang; Lopez, Andrew L.; Morikawa, Yuka; Tao, Ge; Li, Jiasong; Larina, Irina V.; Martin, James F.; Larin, Kirill V.

2015-03-01

We present a quantitative optical coherence elastographic method for noncontact assessment of the myocardium elasticity. The method is based on shear wave imaging optical coherence tomography (SWI-OCT), where a focused air-puff system is used to induce localized tissue deformation through a low-pressure short-duration air stream and a phase-sensitive OCT system is utilized to monitor the propagation of the induced tissue displacement with nanoscale sensitivity. The 1-D scanning of M-mode OCT imaging and the application of optical phase retrieval and mapping techniques enable the reconstruction and visualization of 2-D depth-resolved shear wave propagation in tissue with ultra-high frame rate. The feasibility of this method in quantitative elasticity measurement is demonstrated on tissue-mimicking phantoms with the estimated Young's modulus compared with uniaxial compression tests. We also performed pilot experiments on ex vivo mouse cardiac muscle tissues with normal and genetically altered cardiomyocytes. Our results indicate this noncontact quantitative optical coherence elastographic method can be a useful tool for the cardiac muscle research and studies.

Quantitation of Staphylococcus aureus in Seawater Using CHROMagar™ SA

PubMed Central

Pombo, David; Hui, Jennifer; Kurano, Michelle; Bankowski, Matthew J; Seifried, Steven E

2010-01-01

A microbiological algorithm has been developed to analyze beach water samples for the determination of viable colony forming units (CFU) of Staphylococcus aureus (S. aureus). Membrane filtration enumeration of S. aureus from recreational beach waters using the chromogenic media CHROMagar™SA alone yields a positive predictive value (PPV) of 70%. Presumptive CHROMagar™SA colonies were confirmed as S. aureus by 24-hour tube coagulase test. Combined, these two tests yield a PPV of 100%. This algorithm enables accurate quantitation of S. aureus in seawater in 72 hours and could support risk-prediction processes for recreational waters. A more rapid protocol, utilizing a 4-hour tube coagulase confirmatory test, enables a 48-hour turnaround time with a modest false negative rate of less than 10%. PMID:20222490

Toward genome-enabled mycology.

PubMed

Hibbett, David S; Stajich, Jason E; Spatafora, Joseph W

2013-01-01

Genome-enabled mycology is a rapidly expanding field that is characterized by the pervasive use of genome-scale data and associated computational tools in all aspects of fungal biology. Genome-enabled mycology is integrative and often requires teams of researchers with diverse skills in organismal mycology, bioinformatics and molecular biology. This issue of Mycologia presents the first complete fungal genomes in the history of the journal, reflecting the ongoing transformation of mycology into a genome-enabled science. Here, we consider the prospects for genome-enabled mycology and the technical and social challenges that will need to be overcome to grow the database of complete fungal genomes and enable all fungal biologists to make use of the new data.

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

Automated quantitative cytological analysis using portable microfluidic microscopy.

PubMed

Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

2016-06-01

In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and Quantitation of Asparagine and Citrulline Using High-Performance Liquid Chromatography (HPLC)

PubMed Central

Bai, Cheng; Reilly, Charles C.; Wood, Bruce W.

2007-01-01

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (μMol ml−1/μMol ml−1)], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides. PMID:19662174

Identification and quantitation of asparagine and citrulline using high-performance liquid chromatography (HPLC).

PubMed

Bai, Cheng; Reilly, Charles C; Wood, Bruce W

2007-03-28

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (microMol ml(-1)/microMol ml(-1))], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.

High-resolution quantitative determination of dielectric function by using scattering scanning near-field optical microscopy

PubMed Central

Tranca, D. E.; Stanciu, S. G.; Hristu, R.; Stoichita, C.; Tofail, S. A. M.; Stanciu, G. A.

2015-01-01

A new method for high-resolution quantitative measurement of the dielectric function by using scattering scanning near-field optical microscopy (s-SNOM) is presented. The method is based on a calibration procedure that uses the s-SNOM oscillating dipole model of the probe-sample interaction and quantitative s-SNOM measurements. The nanoscale capabilities of the method have the potential to enable novel applications in various fields such as nano-electronics, nano-photonics, biology or medicine. PMID:26138665

Defining and Enabling Resiliency of Electric Distribution Systems With Multiple Microgrids

DOE PAGES

Chanda, Sayonsom; Srivastava, Anurag K.

2016-05-02

This paper presents a method for quantifying and enabling the resiliency of a power distribution system (PDS) using analytical hierarchical process and percolation theory. Using this metric, quantitative analysis can be done to analyze the impact of possible control decisions to pro-actively enable the resilient operation of distribution system with multiple microgrids and other resources. Developed resiliency metric can also be used in short term distribution system planning. The benefits of being able to quantify resiliency can help distribution system planning engineers and operators to justify control actions, compare different reconfiguration algorithms, develop proactive control actions to avert power systemmore » outage due to impending catastrophic weather situations or other adverse events. Validation of the proposed method is done using modified CERTS microgrids and a modified industrial distribution system. Furthermore, simulation results show topological and composite metric considering power system characteristics to quantify the resiliency of a distribution system with the proposed methodology, and improvements in resiliency using two-stage reconfiguration algorithm and multiple microgrids.« less

Automated Planning Enables Complex Protocols on Liquid-Handling Robots.

PubMed

Whitehead, Ellis; Rudolf, Fabian; Kaltenbach, Hans-Michael; Stelling, Jörg

2018-03-16

Robotic automation in synthetic biology is especially relevant for liquid handling to facilitate complex experiments. However, research tasks that are not highly standardized are still rarely automated in practice. Two main reasons for this are the substantial investments required to translate molecular biological protocols into robot programs, and the fact that the resulting programs are often too specific to be easily reused and shared. Recent developments of standardized protocols and dedicated programming languages for liquid-handling operations addressed some aspects of ease-of-use and portability of protocols. However, either they focus on simplicity, at the expense of enabling complex protocols, or they entail detailed programming, with corresponding skills and efforts required from the users. To reconcile these trade-offs, we developed Roboliq, a software system that uses artificial intelligence (AI) methods to integrate (i) generic formal, yet intuitive, protocol descriptions, (ii) complete, but usually hidden, programming capabilities, and (iii) user-system interactions to automatically generate executable, optimized robot programs. Roboliq also enables high-level specifications of complex tasks with conditional execution. To demonstrate the system's benefits for experiments that are difficult to perform manually because of their complexity, duration, or time-critical nature, we present three proof-of-principle applications for the reproducible, quantitative characterization of GFP variants.

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.

Quantitative modeling of the reaction/diffusion kinetics of two-chemistry photopolymers

NASA Astrophysics Data System (ADS)

Kowalski, Benjamin Andrew

Optically driven diffusion in photopolymers is an appealing material platform for a broad range of applications, in which the recorded refractive index patterns serve either as images (e.g. data storage, display holography) or as optical elements (e.g. custom GRIN components, integrated optical devices). A quantitative understanding of the reaction/diffusion kinetics is difficult to obtain directly, but is nevertheless necessary in order to fully exploit the wide array of design freedoms in these materials. A general strategy for characterizing these kinetics is proposed, in which key processes are decoupled and independently measured. This strategy enables prediction of a material's potential refractive index change, solely on the basis of its chemical components. The degree to which a material does not reach this potential reveals the fraction of monomer that has participated in unwanted reactions, reducing spatial resolution and dynamic range. This approach is demonstrated for a model material similar to commercial media, achieving quantitative predictions of index response over three orders of exposure dose (~1 to ~103 mJ cm-2) and three orders of feature size (0.35 to 500 microns). The resulting insights enable guided, rational design of new material formulations with demonstrated performance improvement.

Quantitative, equal carbon response HSQC experiment, QEC-HSQC

NASA Astrophysics Data System (ADS)

Mäkelä, Valtteri; Helminen, Jussi; Kilpeläinen, Ilkka; Heikkinen, Sami

2016-10-01

Quantitative NMR has become increasingly useful and popular in recent years, with many new and emerging applications in metabolomics, quality control, reaction monitoring and other types of mixture analysis. While sensitive and simple to acquire, the low resolving power of 1D 1H NMR spectra can be a limiting factor when analyzing complex mixtures. This drawback can be solved by observing a different type of nuclei offering improved resolution or with multidimensional experiments, such as HSQC. In this paper, we present a novel Quantitative, Equal Carbon HSQC (QEC-HSQC) experiment providing an equal response across different type of carbons regardless of the number of attached protons, in addition to an uniform response over a wide range of 1JCH couplings. This enables rapid quantification and integration over multiple signals without the need for complete resonance assignments and simplifies the integration of overlapping signals.

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.

Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR.

PubMed

Shuga, Joe; Zeng, Yong; Novak, Richard; Lan, Qing; Tang, Xiaojiang; Rothman, Nathaniel; Vermeulen, Roel; Li, Laiyu; Hubbard, Alan; Zhang, Luoping; Mathies, Richard A; Smith, Martyn T

2013-09-01

Cancers are heterogeneous and genetically unstable. New methods are needed that provide the sensitivity and specificity to query single cells at the genetic loci that drive cancer progression, thereby enabling researchers to study the progression of individual tumors. Here, we report the development and application of a bead-based hemi-nested microfluidic droplet digital PCR (dPCR) technology to achieve 'quantitative' measurement and single-molecule sequencing of somatically acquired carcinogenic translocations at extremely low levels (<10(-6)) in healthy subjects. We use this technique in our healthy study population to determine the overall concentration of the t(14;18) translocation, which is strongly associated with follicular lymphoma. The nested dPCR approach improves the detection limit to 1×10(-7) or lower while maintaining the analysis efficiency and specificity. Further, the bead-based dPCR enabled us to isolate and quantify the relative amounts of the various clonal forms of t(14;18) translocation in these subjects, and the single-molecule sensitivity and resolution of dPCR led to the discovery of new clonal forms of t(14;18) that were otherwise masked by the conventional quantitative PCR measurements. In this manner, we created a quantitative map for this carcinogenic mutation in this healthy population and identified the positions on chromosomes 14 and 18 where the vast majority of these t(14;18) events occur.

Quantitative analysis of periodontal pathogens by ELISA and real-time polymerase chain reaction.

PubMed

Hamlet, Stephen M

2010-01-01

The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

DMD-based quantitative phase microscopy and optical diffraction tomography

NASA Astrophysics Data System (ADS)

Zhou, Renjie

2018-02-01

Digital micromirror devices (DMDs), which offer high speed and high degree of freedoms in steering light illuminations, have been increasingly applied to optical microscopy systems in recent years. Lately, we introduced DMDs into digital holography to enable new imaging modalities and break existing imaging limitations. In this paper, we will first present our progress in using DMDs for demonstrating laser-illumination Fourier ptychographic microscopy (FPM) with shotnoise limited detection. After that, we will present a novel common-path quantitative phase microscopy (QPM) system based on using a DMD. Building on those early developments, a DMD-based high speed optical diffraction tomography (ODT) system has been recently demonstrated, and the results will also be presented. This ODT system is able to achieve video-rate 3D refractive-index imaging, which can potentially enable observations of high-speed 3D sample structural changes.

Quantitative characterization of genetic parts and circuits for plant synthetic biology.

PubMed

Schaumberg, Katherine A; Antunes, Mauricio S; Kassaw, Tessema K; Xu, Wenlong; Zalewski, Christopher S; Medford, June I; Prasad, Ashok

2016-01-01

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

Bioprocess automation on a Mini Pilot Plant enables fast quantitative microbial phenotyping.

PubMed

Unthan, Simon; Radek, Andreas; Wiechert, Wolfgang; Oldiges, Marco; Noack, Stephan

2015-03-11

The throughput of cultivation experiments in bioprocess development has drastically increased in recent years due to the availability of sophisticated microliter scale cultivation devices. However, as these devices still require time-consuming manual work, the bottleneck was merely shifted to media preparation, inoculation and finally the analyses of cultivation samples. A first step towards solving these issues was undertaken in our former study by embedding a BioLector in a robotic workstation. This workstation already allowed for the optimization of heterologous protein production processes, but remained limited when aiming for the characterization of small molecule producer strains. In this work, we extended our workstation to a versatile Mini Pilot Plant (MPP) by integrating further robotic workflows and microtiter plate assays that now enable a fast and accurate phenotyping of a broad range of microbial production hosts. A fully automated harvest procedure was established, which repeatedly samples up to 48 wells from BioLector cultivations in response to individually defined trigger conditions. The samples are automatically clarified by centrifugation and finally frozen for subsequent analyses. Sensitive metabolite assays in 384-well plate scale were integrated on the MPP for the direct determination of substrate uptake (specifically D-glucose and D-xylose) and product formation (specifically amino acids). In a first application, we characterized a set of Corynebacterium glutamicum L-lysine producer strains and could rapidly identify a unique strain showing increased L-lysine titers, which was subsequently confirmed in lab-scale bioreactor experiments. In a second study, we analyzed the substrate uptake kinetics of a previously constructed D-xylose-converting C. glutamicum strain during cultivation on mixed carbon sources in a fully automated experiment. The presented MPP is designed to face the challenges typically encountered during early-stage bioprocess

Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Hardie, Darryl B; Borchers, Christoph H

2014-05-01

Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

Grid workflow validation using ontology-based tacit knowledge: A case study for quantitative remote sensing applications

NASA Astrophysics Data System (ADS)

Liu, Jia; Liu, Longli; Xue, Yong; Dong, Jing; Hu, Yingcui; Hill, Richard; Guang, Jie; Li, Chi

2017-01-01

Workflow for remote sensing quantitative retrieval is the ;bridge; between Grid services and Grid-enabled application of remote sensing quantitative retrieval. Workflow averts low-level implementation details of the Grid and hence enables users to focus on higher levels of application. The workflow for remote sensing quantitative retrieval plays an important role in remote sensing Grid and Cloud computing services, which can support the modelling, construction and implementation of large-scale complicated applications of remote sensing science. The validation of workflow is important in order to support the large-scale sophisticated scientific computation processes with enhanced performance and to minimize potential waste of time and resources. To research the semantic correctness of user-defined workflows, in this paper, we propose a workflow validation method based on tacit knowledge research in the remote sensing domain. We first discuss the remote sensing model and metadata. Through detailed analysis, we then discuss the method of extracting the domain tacit knowledge and expressing the knowledge with ontology. Additionally, we construct the domain ontology with Protégé. Through our experimental study, we verify the validity of this method in two ways, namely data source consistency error validation and parameters matching error validation.

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.

Quantitative Image Restoration in Bright Field Optical Microscopy.

PubMed

Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

2017-11-07

Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Health promotion and sustainability programmes in Australia: barriers and enablers to evaluation.

PubMed

Patrick, Rebecca; Kingsley, Jonathan

2017-08-01

In an era characterised by the adverse impacts of climate change and environmental degradation, health promotion programmes are beginning to actively link human health with environmental sustainability imperatives. This paper draws on a study of health promotion and sustainability programmes in Australia, providing insights to evaluation approaches being used and barriers and enablers to these evaluations. The study was based on a multi-strategy research involving both quantitative and qualitative methods. Health promotion practitioners explained through surveys and semi-structured interviews that they focused on five overarching health and sustainability programme types (healthy and sustainable food, active transport, energy efficiency, contact with nature, and capacity building). Various evaluation methods and indicators (health, social, environmental, economic and demographic) were identified as being valuable for monitoring and evaluating health and sustainability programmes. Findings identified several evaluation enablers such as successful community engagement, knowledge of health and sustainability issues and programme champions, whereas barriers included resource constraints and competing interests. This paper highlights the need for ecological models and evaluation tools to support the design and monitoring of health promotion and sustainability programmes.

Quantitative analysis of pork and chicken products by droplet digital PCR.

PubMed

Cai, Yicun; Li, Xiang; Lv, Rong; Yang, Jielin; Li, Jian; He, Yuping; Pan, Liangwen

2014-01-01

In this project, a highly precise quantitative method based on the digital polymerase chain reaction (dPCR) technique was developed to determine the weight of pork and chicken in meat products. Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of species-specific DNAs in meat products. However, it is limited in amplification efficiency and relies on standard curves based Ct values, detecting and quantifying low copy number target DNA, as in some complex mixture meat products. By using the dPCR method, we find the relationships between the raw meat weight and DNA weight and between the DNA weight and DNA copy number were both close to linear. This enabled us to establish formulae to calculate the raw meat weight based on the DNA copy number. The accuracy and applicability of this method were tested and verified using samples of pork and chicken powder mixed in known proportions. Quantitative analysis indicated that dPCR is highly precise in quantifying pork and chicken in meat products and therefore has the potential to be used in routine analysis by government regulators and quality control departments of commercial food and feed enterprises.

Developing a national dental education research strategy: priorities, barriers and enablers

PubMed Central

Barton, Karen L; Dennis, Ashley A; Rees, Charlotte E

2017-01-01

Objectives This study aimed to identify national dental education research (DER) priorities for the next 3–5 years and to identify barriers and enablers to DER. Setting Scotland. Participants In this two-stage online questionnaire study, we collected data with multiple dental professions (eg, dentistry, dental nursing and dental hygiene) and stakeholder groups (eg, learners, clinicians, educators, managers, researchers and academics). Eighty-five participants completed the Stage 1 qualitative questionnaire and 649 participants the Stage 2 quantitative questionnaire. Results Eight themes were identified at Stage 1. Of the 24 DER priorities identified, the top three were: role of assessments in identifying competence; undergraduate curriculum prepares for practice and promoting teamwork. Following exploratory factor analysis, the 24 items loaded onto four factors: teamwork and professionalism, measuring and enhancing performance, dental workforce issues and curriculum integration and innovation. Barriers and enablers existed at multiple levels: individual, interpersonal, institutional structures and cultures and technology. Conclusions This priority setting exercise provides a necessary first step to developing a national DER strategy capturing multiple perspectives. Promoting DER requires improved resourcing alongside efforts to overcome peer stigma and lack of valuing and motivation. PMID:28360237

Usefulness of quantitative susceptibility mapping for the diagnosis of Parkinson disease.

PubMed

Murakami, Y; Kakeda, S; Watanabe, K; Ueda, I; Ogasawara, A; Moriya, J; Ide, S; Futatsuya, K; Sato, T; Okada, K; Uozumi, T; Tsuji, S; Liu, T; Wang, Y; Korogi, Y

2015-06-01

Quantitative susceptibility mapping allows overcoming several nonlocal restrictions of susceptibility-weighted and phase imaging and enables quantification of magnetic susceptibility. We compared the diagnostic accuracy of quantitative susceptibility mapping and R2* (1/T2*) mapping to discriminate between patients with Parkinson disease and controls. For 21 patients with Parkinson disease and 21 age- and sex-matched controls, 2 radiologists measured the quantitative susceptibility mapping values and R2* values in 6 brain structures (the thalamus, putamen, caudate nucleus, pallidum, substantia nigra, and red nucleus). The quantitative susceptibility mapping values and R2* values of the substantia nigra were significantly higher in patients with Parkinson disease (P < .01); measurements in other brain regions did not differ significantly between patients and controls. For the discrimination of patients with Parkinson disease from controls, receiver operating characteristic analysis suggested that the optimal cutoff values for the substantia nigra, based on the Youden Index, were >0.210 for quantitative susceptibility mapping and >28.8 for R2*. The sensitivity, specificity, and accuracy of quantitative susceptibility mapping were 90% (19 of 21), 86% (18 of 21), and 88% (37 of 42), respectively; for R2* mapping, they were 81% (17 of 21), 52% (11 of 21), and 67% (28 of 42). Pair-wise comparisons showed that the areas under the receiver operating characteristic curves were significantly larger for quantitative susceptibility mapping than for R2* mapping (0.91 versus 0.69, P < .05). Quantitative susceptibility mapping showed higher diagnostic performance than R2* mapping for the discrimination between patients with Parkinson disease and controls. © 2015 by American Journal of Neuroradiology.

Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species.

PubMed

Veldhoen, Nik; Propper, Catherine R; Helbing, Caren C

2014-03-01

Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the "thyroid assays across indicator and sentinel species" (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available. Copyright © 2014 Elsevier B.V. All rights reserved.

Web-based interactive visualization in a Grid-enabled neuroimaging application using HTML5.

PubMed

Siewert, René; Specovius, Svenja; Wu, Jie; Krefting, Dagmar

2012-01-01

Interactive visualization and correction of intermediate results are required in many medical image analysis pipelines. To allow certain interaction in the remote execution of compute- and data-intensive applications, new features of HTML5 are used. They allow for transparent integration of user interaction into Grid- or Cloud-enabled scientific workflows. Both 2D and 3D visualization and data manipulation can be performed through a scientific gateway without the need to install specific software or web browser plugins. The possibilities of web-based visualization are presented along the FreeSurfer-pipeline, a popular compute- and data-intensive software tool for quantitative neuroimaging.

Generation of High-Quality SWATH® Acquisition Data for Label-free Quantitative Proteomics Studies Using TripleTOF® Mass Spectrometers

PubMed Central

Schilling, Birgit; Gibson, Bradford W.; Hunter, Christie L.

2017-01-01

Data-independent acquisition is a powerful mass spectrometry technique that enables comprehensive MS and MS/MS analysis of all detectable species, providing an information rich data file that can be mined deeply. Here, we describe how to acquire high-quality SWATH® Acquisition data to be used for large quantitative proteomic studies. We specifically focus on using variable sized Q1 windows for acquisition of MS/MS data for generating higher specificity quantitative data. PMID:28188533

Quantitative phase microscopy for cellular dynamics based on transport of intensity equation.

PubMed

Li, Ying; Di, Jianglei; Ma, Chaojie; Zhang, Jiwei; Zhong, Jinzhan; Wang, Kaiqiang; Xi, Teli; Zhao, Jianlin

2018-01-08

We demonstrate a simple method for quantitative phase imaging of tiny transparent objects such as living cells based on the transport of intensity equation. The experiments are performed using an inverted bright field microscope upgraded with a flipping imaging module, which enables to simultaneously create two laterally separated images with unequal defocus distances. This add-on module does not include any lenses or gratings and is cost-effective and easy-to-alignment. The validity of this method is confirmed by the measurement of microlens array and human osteoblastic cells in culture, indicating its potential in the applications of dynamically measuring living cells and other transparent specimens in a quantitative, non-invasive and label-free manner.

Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos.

PubMed

Zhao, Ziqing W; White, Melanie D; Bissiere, Stephanie; Levi, Valeria; Plachta, Nicolas

2016-12-23

Probing dynamic processes occurring within the cell nucleus at the quantitative level has long been a challenge in mammalian biology. Advances in bio-imaging techniques over the past decade have enabled us to directly visualize nuclear processes in situ with unprecedented spatial and temporal resolution and single-molecule sensitivity. Here, using transcription as our primary focus, we survey recent imaging studies that specifically emphasize the quantitative understanding of nuclear dynamics in both time and space. These analyses not only inform on previously hidden physical parameters and mechanistic details, but also reveal a hierarchical organizational landscape for coordinating a wide range of transcriptional processes shared by mammalian systems of varying complexity, from single cells to whole embryos.

Development of an ultra-sensitive Simoa assay to enable GDF11 detection: a comparison across bioanalytical platforms.

PubMed

Myzithras, Maria; Li, Hua; Bigwarfe, Tammy; Waltz, Erica; Gupta, Priyanka; Low, Sarah; Hayes, David B; MacDonnell, Scott; Ahlberg, Jennifer; Franti, Michael; Roberts, Simon

2016-03-01

Four bioanalytical platforms were evaluated to optimize sensitivity and enable detection of recombinant human GDF11 in biological matrices; ELISA, Meso Scale Discovery, Gyrolab xP Workstation and Simoa HD-1. Results & methodology: After completion of custom assay development, the single-molecule ELISA (Simoa) achieved the greatest sensitivity with a lower limit of quantitation of 0.1 ng/ml, an improvement of 100-fold over the next sensitive platform (MSD). This improvement was essential to enable detection of GDF11 in biological samples, and without the technology the sensitivity achieved on the other platforms would not have been sufficient. Other factors such as ease of use, cost, assay time and automation capability can also be considered when developing custom immunoassays, based on the requirements of the bioanalyst.

End-to-end deep neural network for optical inversion in quantitative photoacoustic imaging.

PubMed

Cai, Chuangjian; Deng, Kexin; Ma, Cheng; Luo, Jianwen

2018-06-15

An end-to-end deep neural network, ResU-net, is developed for quantitative photoacoustic imaging. A residual learning framework is used to facilitate optimization and to gain better accuracy from considerably increased network depth. The contracting and expanding paths enable ResU-net to extract comprehensive context information from multispectral initial pressure images and, subsequently, to infer a quantitative image of chromophore concentration or oxygen saturation (sO 2 ). According to our numerical experiments, the estimations of sO 2 and indocyanine green concentration are accurate and robust against variations in both optical property and object geometry. An extremely short reconstruction time of 22 ms is achieved.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

PubMed

He, Dan; Xie, Xiao; Yang, Fan; Zhang, Heng; Su, Haomiao; Ge, Yun; Song, Haiping; Chen, Peng R

2017-11-13

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Enabling comparative effectiveness research with informatics: show me the data!

PubMed

Safdar, Nabile M; Siegel, Eliot; Erickson, Bradley J; Nagy, Paul

2011-09-01

Both outcomes researchers and informaticians are concerned with information and data. As such, some of the central challenges to conducting successful comparative effectiveness research can be addressed with informatics solutions. Specific informatics solutions which address how data in comparative effectiveness research are enriched, stored, shared, and analyzed are reviewed. Imaging data can be made more quantitative, uniform, and structured for researchers through the use of lexicons and structured reporting. Secure and scalable storage of research data is enabled through data warehouses and cloud services. There are a number of national efforts to help researchers share research data and analysis tools. There is a diverse arsenal of informatics tools designed to meet the needs of comparative effective researchers. Copyright © 2011 AUR. Published by Elsevier Inc. All rights reserved.

Web-enabling technologies for the factory floor: a web-enabling strategy for emanufacturing

NASA Astrophysics Data System (ADS)

Velez, Ricardo; Lastra, Jose L. M.; Tuokko, Reijo O.

2001-10-01

This paper is intended to address the different technologies available for Web-enabling of the factory floor. It will give an overview of the importance of Web-enabling of the factory floor, in the application of the concepts of flexible and intelligent manufacturing, in conjunction with e-commerce. As a last section, it will try to define a Web-enabling strategy for the application in eManufacturing. This is made under the scope of the electronics manufacturing industry, so every application, technology or related matter is presented under such scope.

Quantitative Secondary Electron Detector (QSED)

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

Nayak, Subu; Joy, David C.

2013-12-31

Research is proposed to investigate the feasibility of applying recent advances in semiconductor technology to fabricate direct digital Quantitative Secondary Electron Detectors (QSED) for scanning electron microscopes (SEMs). If successful, commercial versions of the QSED would transform the SEM into a quantitative, metrological system with enhanced capabilities that, in turn, would broaden research horizons across industries. This project will be conducted in collaboration with Dr. David C Joy at the University of Tennessee, who has demonstrated limited (to the 1keV range) digital collection of the energy from backscattered signals in a SEM using a modified silicon drift detector. Several detectormore » configurations will be fabricated and tested for sensitivities, background noise reduction, DC offset elimination, and metrological capabilities (linearity, accuracy, etc.) against a set of commercially important performance criteria to ascertain concept feasibility. Once feasibility is proven, the solid state digital device array and its switching frequency will be scaled-up, in Phase II, to improve temporal resolution. If successful, this work will produce a crucial advancement in electron microscopy with wide-ranging applications. The following are key advantages anticipated from direct digital QSED: 1. High signal-to-noise ratio will improve SEM resolution in nano-scale, which is critical for dimensional metrology in any application. 2. Quantitative measurement will enhance process control and design validation in semiconductors, photo-voltaics, bio-medical devices and catalysts; and will improve accuracy in predicting the reliability and the lifecycle of materials across industries. 3. Video and dynamic-imaging capabilities will advance study in nano-scale phenomena in a variety of industries, including pharmaceutical and semiconductor materials. 4. Lower cost will make high-performing electron microscopes affordable to more researchers. 5. Compact size and

Physician Enabling Skills Questionnaire

PubMed Central

Hudon, Catherine; Lambert, Mireille; Almirall, José

2015-01-01

Abstract Objective To evaluate the reliability and validity of the newly developed Physician Enabling Skills Questionnaire (PESQ) by assessing its internal consistency, test-retest reliability, concurrent validity with patient-centred care, and predictive validity with patient activation and patient enablement. Design Validation study. Setting Saguenay, Que. Participants One hundred patients with at least 1 chronic disease who presented in a waiting room of a regional health centre family medicine unit. Main outcome measures Family physicians’ enabling skills, measured with the PESQ at 2 points in time (ie, while in the waiting room at the family medicine unit and 2 weeks later through a mail survey); patient-centred care, assessed with the Patient Perception of Patient-Centredness instrument; patient activation, assessed with the Patient Activation Measure; and patient enablement, assessed with the Patient Enablement Instrument. Results The internal consistency of the 6 subscales of the PESQ was adequate (Cronbach α = .69 to .92). The test-retest reliability was very good (r = 0.90; 95% CI 0.84 to 0.93). Concurrent validity with the Patient Perception of Patient-Centredness instrument was good (r = −0.67; 95% CI −0.78 to −0.53; P < .001). The PESQ accounts for 11% of the total variance with the Patient Activation Measure (r2 = 0.11; P = .002) and 19% of the variance with the Patient Enablement Instrument (r2 = 0.19; P < .001). Conclusion The newly developed PESQ presents good psychometric properties, allowing for its use in practice and research. PMID:26889507

Outcomes from Enabling Courses.

ERIC Educational Resources Information Center

Phan, Oanh; Ball, Katrina

The outcomes of enabling courses offered in Australia's vocational education and training (VET) sector were examined. "Enabling course" was defined as lower-level preparatory and prevocational courses covering a wide range of areas, including remedial education, bridging courses, precertificate courses, and general employment preparation…

Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera L. ssp. sativa) berry: a quantitative trait locus to quantitative trait nucleotide integrated study.

PubMed

Fournier-Level, Alexandre; Le Cunff, Loïc; Gomez, Camila; Doligez, Agnès; Ageorges, Agnès; Roux, Catherine; Bertrand, Yves; Souquet, Jean-Marc; Cheynier, Véronique; This, Patrice

2009-11-01

the continuous variation in anthocyanin content in grape was explained mainly by a single gene cluster of three VvMybA genes. The use of natural diversity helped to reduce one QTL to a set of five quantitative trait nucleotides and gave a clear picture of how isogenes combined their effects to shape grape color. Such analysis also illustrates how isogenes combine their effect to shape a complex quantitative trait and enables the definition of markers directly targeted for upcoming breeding programs.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Flexible opto-electronics enabled microfluidics systems with cloud connectivity for point-of-care micronutrient analysis.

PubMed

Lee, Stephen; Aranyosi, A J; Wong, Michelle D; Hong, Ji Hyung; Lowe, Jared; Chan, Carol; Garlock, David; Shaw, Scott; Beattie, Patrick D; Kratochvil, Zachary; Kubasti, Nick; Seagers, Kirsten; Ghaffari, Roozbeh; Swanson, Christina D

2016-04-15

In developing countries, the deployment of medical diagnostic technologies remains a challenge because of infrastructural limitations (e.g. refrigeration, electricity), and paucity of health professionals, distribution centers and transportation systems. Here we demonstrate the technical development and clinical testing of a novel electronics enabled microfluidic paper-based analytical device (EE-μPAD) for quantitative measurement of micronutrient concentrations in decentralized, resource-limited settings. The system performs immune-detection using paper-based microfluidics, instrumented with flexible electronics and optoelectronic sensors in a mechanically robust, ultrathin format comparable in size to a credit card. Autonomous self-calibration, plasma separation, flow monitoring, timing and data storage enable multiple devices to be run simultaneously. Measurements are wirelessly transferred to a mobile phone application that geo-tags the data and transmits it to a remote server for real time tracking of micronutrient deficiencies. Clinical tests of micronutrient levels from whole blood samples (n=95) show comparable sensitivity and specificity to ELISA-based tests. These results demonstrate instantaneous acquisition and global aggregation of diagnostics data using a fully integrated point of care system that will enable rapid and distributed surveillance of disease prevalence and geographical progression. Copyright © 2015 Elsevier B.V. All rights reserved.

Technology Enabled Learning. Symposium.

ERIC Educational Resources Information Center

2002

This document contains three papers on technology-enabled learning and human resource development. Among results found in "Current State of Technology-enabled Learning Programs in Select Federal Government Organizations: a Case Study of Ten Organizations" (Letitia A. Combs) are the following: the dominant delivery method is traditional…

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.

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

Methods to enable the design of bioactive small molecules targeting RNA.

PubMed

Disney, Matthew D; Yildirim, Ilyas; Childs-Disney, Jessica L

2014-02-21

RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including structure-activity relationships through sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome.

On the analysis of complex biological supply chains: From Process Systems Engineering to Quantitative Systems Pharmacology.

PubMed

Rao, Rohit T; Scherholz, Megerle L; Hartmanshenn, Clara; Bae, Seul-A; Androulakis, Ioannis P

2017-12-05

The use of models in biology has become particularly relevant as it enables investigators to develop a mechanistic framework for understanding the operating principles of living systems as well as in quantitatively predicting their response to both pathological perturbations and pharmacological interventions. This application has resulted in a synergistic convergence of systems biology and pharmacokinetic-pharmacodynamic modeling techniques that has led to the emergence of quantitative systems pharmacology (QSP). In this review, we discuss how the foundational principles of chemical process systems engineering inform the progressive development of more physiologically-based systems biology models.

Alzheimer disease: Quantitative analysis of I-123-iodoamphetamine SPECT brain imaging

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

Hellman, R.S.; Tikofsky, R.S.; Collier, B.D.

1989-07-01

To enable a more quantitative diagnosis of senile dementia of the Alzheimer type (SDAT), the authors developed and tested a semiautomated method to define regions of interest (ROIs) to be used in quantitating results from single photon emission computed tomography (SPECT) of regional cerebral blood flow performed with N-isopropyl iodine-123-iodoamphetamine. SPECT/IMP imaging was performed in ten patients with probable SDAT and seven healthy subjects. Multiple ROIs were manually and semiautomatically generated, and uptake was quantitated for each ROI. Mean cortical activity was estimated as the average of the mean activity in 24 semiautomatically generated ROIs; mean cerebellar activity was determinedmore » from the mean activity in separate ROIs. A ratio of parietal to cerebellar activity less than 0.60 and a ratio of parietal to mean cortical activity less than 0.90 allowed correct categorization of nine of ten and eight of ten patients, respectively, with SDAT and all control subjects. The degree of diminished mental status observed in patients with SDAT correlated with both global and regional changes in IMP uptake.« less

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

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

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

2016-01-01

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. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

Quantitative electrophysiological monitoring of anti-histamine drug effects on live cells via reusable sensor platforms.

PubMed

Pham Ba, Viet Anh; Cho, Dong-Guk; Kim, Daesan; Yoo, Haneul; Ta, Van-Thao; Hong, Seunghun

2017-08-15

We demonstrated the quantitative electrophysiological monitoring of histamine and anti-histamine drug effects on live cells via reusable sensor platforms based on carbon nanotube transistors. This method enabled us to monitor the real-time electrophysiological responses of a single HeLa cell to histamine with different concentrations. The measured electrophysiological responses were attributed to the activity of histamine type 1 receptors on a HeLa cell membrane by histamine. Furthermore, the effects of anti-histamine drugs such as cetirizine or chlorphenamine on the electrophysiological activities of HeLa cells were also evaluated quantitatively. Significantly, we utilized only a single device to monitor the responses of multiple HeLa cells to each drug, which allowed us to quantitatively analyze the antihistamine drug effects on live cells without errors from the device-to-device variation in device characteristics. Such quantitative evaluation capability of our method would promise versatile applications such as drug screening and nanoscale bio sensor researches. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

NASA Astrophysics Data System (ADS)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

Quantitative structure parameters from the NMR spectroscopy of quadrupolar nuclei

DOE PAGES

Perras, Frederic A.

2015-12-15

Here, nuclear magnetic resonance (NMR) spectroscopy is one of the most important characterization tools in chemistry, however, 3/4 of the NMR active nuclei are underutilized due to their quadrupolar nature. This short review centers on the development of methods that use solid-state NMR of quadrupolar nuclei for obtaining quantitative structural information. Namely, techniques using dipolar recoupling as well as the resolution afforded by double-rotation are presented for the measurement of spin–spin coupling between quadrupoles, enabling the measurement of internuclear distances and connectivities.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

PubMed

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Developing a national dental education research strategy: priorities, barriers and enablers.

PubMed

Ajjawi, Rola; Barton, Karen L; Dennis, Ashley A; Rees, Charlotte E

2017-03-29

This study aimed to identify national dental education research (DER) priorities for the next 3-5 years and to identify barriers and enablers to DER. Scotland. In this two-stage online questionnaire study, we collected data with multiple dental professions (eg, dentistry, dental nursing and dental hygiene) and stakeholder groups (eg, learners, clinicians, educators, managers, researchers and academics). Eighty-five participants completed the Stage 1 qualitative questionnaire and 649 participants the Stage 2 quantitative questionnaire. Eight themes were identified at Stage 1. Of the 24 DER priorities identified, the top three were: role of assessments in identifying competence; undergraduate curriculum prepares for practice and promoting teamwork. Following exploratory factor analysis, the 24 items loaded onto four factors: teamwork and professionalism, measuring and enhancing performance, dental workforce issues and curriculum integration and innovation. Barriers and enablers existed at multiple levels: individual, interpersonal, institutional structures and cultures and technology. This priority setting exercise provides a necessary first step to developing a national DER strategy capturing multiple perspectives. Promoting DER requires improved resourcing alongside efforts to overcome peer stigma and lack of valuing and motivation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

A strategy to apply quantitative epistasis analysis on developmental traits.

PubMed

Labocha, Marta K; Yuan, Wang; Aleman-Meza, Boanerges; Zhong, Weiwei

2017-05-15

Genetic interactions are keys to understand complex traits and evolution. Epistasis analysis is an effective method to map genetic interactions. Large-scale quantitative epistasis analysis has been well established for single cells. However, there is a substantial lack of such studies in multicellular organisms and their complex phenotypes such as development. Here we present a method to extend quantitative epistasis analysis to developmental traits. In the nematode Caenorhabditis elegans, we applied RNA interference on mutants to inactivate two genes, used an imaging system to quantitatively measure phenotypes, and developed a set of statistical methods to extract genetic interactions from phenotypic measurement. Using two different C. elegans developmental phenotypes, body length and sex ratio, as examples, we showed that this method could accommodate various metazoan phenotypes with performances comparable to those methods in single cell growth studies. Comparing with qualitative observations, this method of quantitative epistasis enabled detection of new interactions involving subtle phenotypes. For example, several sex-ratio genes were found to interact with brc-1 and brd-1, the orthologs of the human breast cancer genes BRCA1 and BARD1, respectively. We confirmed the brc-1 interactions with the following genes in DNA damage response: C34F6.1, him-3 (ortholog of HORMAD1, HORMAD2), sdc-1, and set-2 (ortholog of SETD1A, SETD1B, KMT2C, KMT2D), validating the effectiveness of our method in detecting genetic interactions. We developed a reliable, high-throughput method for quantitative epistasis analysis of developmental phenotypes.

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

PubMed Central

Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

2015-01-01

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

Global Quantitative Modeling of Chromatin Factor Interactions

PubMed Central

Zhou, Jian; Troyanskaya, Olga G.

2014-01-01

Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

Imaging enabled platforms for development of therapeutics

NASA Astrophysics Data System (ADS)

Celli, Jonathan; Rizvi, Imran; Blanden, Adam R.; Evans, Conor L.; Abu-Yousif, Adnan O.; Spring, Bryan Q.; Muzikansky, Alona; Pogue, Brian W.; Finkelstein, Dianne M.; Hasan, Tayyaba

2011-03-01

Advances in imaging and spectroscopic technologies have enabled the optimization of many therapeutic modalities in cancer and noncancer pathologies either by earlier disease detection or by allowing therapy monitoring. Amongst the therapeutic options benefiting from developments in imaging technologies, photodynamic therapy (PDT) is exceptional. PDT is a photochemistry-based therapeutic approach where a light-sensitive molecule (photosensitizer) is activated with light of appropriate energy (wavelength) to produce reactive molecular species such as free radicals and singlet oxygen. These molecular entities then react with biological targets such as DNA, membranes and other cellular components to impair their function and lead to eventual cell and tissue death. Development of PDT-based imaging also provides a platform for rapid screening of new therapeutics in novel in vitro models prior to expensive and labor-intensive animal studies. In this study we demonstrate how an imaging platform can be used for strategizing a novel combination treatment strategy for multifocal ovarian cancer. Using an in vitro 3D model for micrometastatic ovarian cancer in conjunction with quantitative imaging we examine dose and scheduling strategies for PDT in combination with carboplatin, a chemotherapeutic agent presently in clinical use for management of this deadly form of cancer.

Developing High-Frequency Quantitative Ultrasound Techniques to Characterize Three-Dimensional Engineered Tissues

NASA Astrophysics Data System (ADS)

Mercado, Karla Patricia E.

Tissue engineering holds great promise for the repair or replacement of native tissues and organs. Further advancements in the fabrication of functional engineered tissues are partly dependent on developing new and improved technologies to monitor the properties of engineered tissues volumetrically, quantitatively, noninvasively, and nondestructively over time. Currently, engineered tissues are evaluated during fabrication using histology, biochemical assays, and direct mechanical tests. However, these techniques destroy tissue samples and, therefore, lack the capability for real-time, longitudinal monitoring. The research reported in this thesis developed nondestructive, noninvasive approaches to characterize the structural, biological, and mechanical properties of 3-D engineered tissues using high-frequency quantitative ultrasound and elastography technologies. A quantitative ultrasound technique, using a system-independent parameter known as the integrated backscatter coefficient (IBC), was employed to visualize and quantify structural properties of engineered tissues. Specifically, the IBC was demonstrated to estimate cell concentration and quantitatively detect differences in the microstructure of 3-D collagen hydrogels. Additionally, the feasibility of an ultrasound elastography technique called Single Tracking Location Acoustic Radiation Force Impulse (STL-ARFI) imaging was demonstrated for estimating the shear moduli of 3-D engineered tissues. High-frequency ultrasound techniques can be easily integrated into sterile environments necessary for tissue engineering. Furthermore, these high-frequency quantitative ultrasound techniques can enable noninvasive, volumetric characterization of the structural, biological, and mechanical properties of engineered tissues during fabrication and post-implantation.

Grid-Enabled Quantitative Analysis of Breast Cancer

DTIC Science & Technology

2009-10-01

large-scale, multi-modality computerized image analysis . The central hypothesis of this research is that large-scale image analysis for breast cancer...pilot study to utilize large scale parallel Grid computing to harness the nationwide cluster infrastructure for optimization of medical image ... analysis parameters. Additionally, we investigated the use of cutting edge dataanalysis/ mining techniques as applied to Ultrasound, FFDM, and DCE-MRI Breast

Grid-Enabled Quantitative Analysis of Breast Cancer

DTIC Science & Technology

2010-10-01

large-scale, multi-modality computerized image analysis . The central hypothesis of this research is that large-scale image analysis for breast cancer...research, we designed a pilot study utilizing large scale parallel Grid computing harnessing nationwide infrastructure for medical image analysis . Also

Broadband quantitative NQR for authentication of vitamins and dietary supplements

NASA Astrophysics Data System (ADS)

Chen, Cheng; Zhang, Fengchao; Bhunia, Swarup; Mandal, Soumyajit

2017-05-01

We describe hardware, pulse sequences, and algorithms for nuclear quadrupole resonance (NQR) spectroscopy of medicines and dietary supplements. Medicine and food safety is a pressing problem that has drawn more and more attention. NQR is an ideal technique for authenticating these substances because it is a non-invasive method for chemical identification. We have recently developed a broadband NQR front-end that can excite and detect 14N NQR signals over a wide frequency range; its operating frequency can be rapidly set by software, while sensitivity is comparable to conventional narrowband front-ends over the entire range. This front-end improves the accuracy of authentication by enabling multiple-frequency experiments. We have also developed calibration and signal processing techniques to convert measured NQR signal amplitudes into nuclear spin densities, thus enabling its use as a quantitative technique. Experimental results from several samples are used to illustrate the proposed methods.

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, ...

Towards real-time quantitative optical imaging for surgery

NASA Astrophysics Data System (ADS)

Gioux, Sylvain

2017-07-01

There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively leading to a large number of failures, patient morbidity and increased healthcare cost. Because near-infrared (NIR) optical imaging is safe, does not require contact, and can provide relatively deep information (several mm), it offers unparalleled capabilities for providing image guidance during surgery. In this work, we introduce a novel concept that enables the quantitative imaging of endogenous molecular information over large fields-of-view. Because this concept can be implemented in real-time, it is amenable to provide video-rate endogenous information during surgery.

Quantitative Thermochronology

NASA Astrophysics Data System (ADS)

Braun, Jean; van der Beek, Peter; Batt, Geoffrey

2006-05-01

Thermochronology, the study of the thermal history of rocks, enables us to quantify the nature and timing of tectonic processes. Quantitative Thermochronology is a robust review of isotopic ages, and presents a range of numerical modeling techniques to allow the physical implications of isotopic age data to be explored. The authors provide analytical, semi-analytical, and numerical solutions to the heat transfer equation in a range of tectonic settings and under varying boundary conditions. They then illustrate their modeling approach built around a large number of case studies. The benefits of different thermochronological techniques are also described. Computer programs on an accompanying website at www.cambridge.org/9780521830577 are introduced through the text and provide a means of solving the heat transport equation in the deforming Earth to predict the ages of rocks and compare them directly to geological and geochronological data. Several short tutorials, with hints and solutions, are also included. Numerous case studies help geologists to interpret age data and relate it to Earth processes Essential background material to aid understanding and using thermochronological data Provides a thorough treatise on numerical modeling of heat transport in the Earth's crust Supported by a website hosting relevant computer programs and colour slides of figures from the book for use in teaching

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Differential diagnosis of breast cancer using quantitative, label-free and molecular vibrational imaging

PubMed Central

Yang, Yaliang; Li, Fuhai; Gao, Liang; Wang, Zhiyong; Thrall, Michael J.; Shen, Steven S.; Wong, Kelvin K.; Wong, Stephen T. C.

2011-01-01

We present a label-free, chemically-selective, quantitative imaging strategy to identify breast cancer and differentiate its subtypes using coherent anti-Stokes Raman scattering (CARS) microscopy. Human normal breast tissue, benign proliferative, as well as in situ and invasive carcinomas, were imaged ex vivo. Simply by visualizing cellular and tissue features appearing on CARS images, cancerous lesions can be readily separated from normal tissue and benign proliferative lesion. To further distinguish cancer subtypes, quantitative disease-related features, describing the geometry and distribution of cancer cell nuclei, were extracted and applied to a computerized classification system. The results show that in situ carcinoma was successfully distinguished from invasive carcinoma, while invasive ductal carcinoma (IDC) and invasive lobular carcinoma were also distinguished from each other. Furthermore, 80% of intermediate-grade IDC and 85% of high-grade IDC were correctly distinguished from each other. The proposed quantitative CARS imaging method has the potential to enable rapid diagnosis of breast cancer. PMID:21833355

Quantitative Investigation of the Role of Intra-/Intercellular Dynamics in Bacterial Quorum Sensing.

PubMed

Leaman, Eric J; Geuther, Brian Q; Behkam, Bahareh

2018-04-20

Bacteria utilize diffusible signals to regulate population density-dependent coordinated gene expression in a process called quorum sensing (QS). While the intracellular regulatory mechanisms of QS are well-understood, the effect of spatiotemporal changes in the population configuration on the sensitivity and robustness of the QS response remains largely unexplored. Using a microfluidic device, we quantitatively characterized the emergent behavior of a population of swimming E. coli bacteria engineered with the lux QS system and a GFP reporter. We show that the QS activation time follows a power law with respect to bacterial population density, but this trend is disrupted significantly by microscale variations in population configuration and genetic circuit noise. We then developed a computational model that integrates population dynamics with genetic circuit dynamics to enable accurate (less than 7% error) quantitation of the bacterial QS activation time. Through modeling and experimental analyses, we show that changes in spatial configuration of swimming bacteria can drastically alter the QS activation time, by up to 22%. The integrative model developed herein also enables examination of the performance robustness of synthetic circuits with respect to growth rate, circuit sensitivity, and the population's initial size and spatial structure. Our framework facilitates quantitative tuning of microbial systems performance through rational engineering of synthetic ribosomal binding sites. We have demonstrated this through modulation of QS activation time over an order of magnitude. Altogether, we conclude that predictive engineering of QS-based bacterial systems requires not only the precise temporal modulation of gene expression (intracellular dynamics) but also accounting for the spatiotemporal changes in population configuration (intercellular dynamics).

Dynamic mobility applications policy analysis : policy and institutional issues for enabling advanced traveler information services (EnableATIS).

DOT National Transportation Integrated Search

2014-10-01

This report documents policy considerations for Enabling Advanced Traveler Information Services, or EnableATIS. EnableATIS is the traveler information element of the Dynamic Mobility Applications program, and it provides a framework to develop multi-...

Methods to enable the design of bioactive small molecules targeting RNA

PubMed Central

Disney, Matthew D.; Yildirim, Ilyas; Childs-Disney, Jessica L.

2014-01-01

RNA is an immensely important target for small molecule therapeutics or chemical probes of function. However, methods that identify, annotate, and optimize RNA-small molecule interactions that could enable the design of compounds that modulate RNA function are in their infancies. This review describes recent approaches that have been developed to understand and optimize RNA motif-small molecule interactions, including Structure-Activity Relationships Through Sequencing (StARTS), quantitative structure-activity relationships (QSAR), chemical similarity searching, structure-based design and docking, and molecular dynamics (MD) simulations. Case studies described include the design of small molecules targeting RNA expansions, the bacterial A-site, viral RNAs, and telomerase RNA. These approaches can be combined to afford a synergistic method to exploit the myriad of RNA targets in the transcriptome. PMID:24357181

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).

Affinity Proteomics for Fast, Sensitive, Quantitative Analysis of Proteins in Plasma.

PubMed

O'Grady, John P; Meyer, Kevin W; Poe, Derrick N

2017-01-01

The improving efficacy of many biological therapeutics and identification of low-level biomarkers are driving the analytical proteomics community to deal with extremely high levels of sample complexity relative to their analytes. Many protein quantitation and biomarker validation procedures utilize an immunoaffinity enrichment step to purify the sample and maximize the sensitivity of the corresponding liquid chromatography tandem mass spectrometry measurements. In order to generate surrogate peptides with better mass spectrometric properties, protein enrichment is followed by a proteolytic cleavage step. This is often a time-consuming multistep process. Presented here is a workflow which enables rapid protein enrichment and proteolytic cleavage to be performed in a single, easy-to-use reactor. Using this strategy Klotho, a low-abundance biomarker found in plasma, can be accurately quantitated using a protocol that takes under 5 h from start to finish.

Micro/nano-computed tomography technology for quantitative dynamic, multi-scale imaging of morphogenesis.

PubMed

Gregg, Chelsea L; Recknagel, Andrew K; Butcher, Jonathan T

2015-01-01

Tissue morphogenesis and embryonic development are dynamic events challenging to quantify, especially considering the intricate events that happen simultaneously in different locations and time. Micro- and more recently nano-computed tomography (micro/nanoCT) has been used for the past 15 years to characterize large 3D fields of tortuous geometries at high spatial resolution. We and others have advanced micro/nanoCT imaging strategies for quantifying tissue- and organ-level fate changes throughout morphogenesis. Exogenous soft tissue contrast media enables visualization of vascular lumens and tissues via extravasation. Furthermore, the emergence of antigen-specific tissue contrast enables direct quantitative visualization of protein and mRNA expression. Micro-CT X-ray doses appear to be non-embryotoxic, enabling longitudinal imaging studies in live embryos. In this chapter we present established soft tissue contrast protocols for obtaining high-quality micro/nanoCT images and the image processing techniques useful for quantifying anatomical and physiological information from the data sets.

A quantitative literature-curated gold standard for kinase-substrate pairs

PubMed Central

2011-01-01

We describe the Yeast Kinase Interaction Database (KID, http://www.moseslab.csb.utoronto.ca/KID/), which contains high- and low-throughput data relevant to phosphorylation events. KID includes 6,225 low-throughput and 21,990 high-throughput interactions, from greater than 35,000 experiments. By quantitatively integrating these data, we identified 517 high-confidence kinase-substrate pairs that we consider a gold standard. We show that this gold standard can be used to assess published high-throughput datasets, suggesting that it will enable similar rigorous assessments in the future. PMID:21492431

Enabling Soldiers with Robots

DTIC Science & Technology

2012-04-17

difficult to imagine that lethal robots would find themselves among the list of particularly inhumane weapons. Albert Einstein whose research was...REPORT DATE (DD-MM-YYYY) 17-04-2012 2. REPORT TYPE Strategy Research Project 3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Enabling...Enabling Soldiers with Robots FORMAT: Strategy Research Project DATE : 17 April 2012 WORD COUNT: 5202 PAGES: 26 KEY TERMS: Ethics, Doctrine

Quantitative Evaluation of the Use of Actigraphy for Neurological and Psychiatric Disorders

PubMed Central

Song, Yu; Kwak, Shin; Yoshida, Sohei; Yamamoto, Yoshiharu

2014-01-01

Quantitative and objective evaluation of disease severity and/or drug effect is necessary in clinical practice. Wearable accelerometers such as an actigraph enable long-term recording of a patient's movement during activities and they can be used for quantitative assessment of symptoms due to various diseases. We reviewed some applications of actigraphy with analytical methods that are sufficiently sensitive and reliable to determine the severity of diseases and disorders such as motor and nonmotor disorders like Parkinson's disease, sleep disorders, depression, behavioral and psychological symptoms of dementia (BPSD) for vascular dementia (VD), seasonal affective disorder (SAD), and stroke, as well as the effects of drugs used to treat them. We believe it is possible to develop analytical methods to assess more neurological or psychopathic disorders using actigraphy records. PMID:25214709

Visualization and Quantitative Analysis of Crack-Tip Plastic Zone in Pure Nickel

NASA Astrophysics Data System (ADS)

Kelton, Randall; Sola, Jalal Fathi; Meletis, Efstathios I.; Huang, Haiying

2018-05-01

Changes in surface morphology have long been thought to be associated with crack propagation in metallic materials. We have studied areal surface texture changes around crack tips in an attempt to understand the correlations between surface texture changes and crack growth behavior. Detailed profiling of the fatigue sample surface was carried out at short fatigue intervals. An image processing algorithm was developed to calculate the surface texture changes. Quantitative analysis of the crack-tip plastic zone, crack-arrested sites near triple points, and large surface texture changes associated with crack release from arrested locations was carried out. The results indicate that surface texture imaging enables visualization of the development of plastic deformation around a crack tip. Quantitative analysis of the surface texture changes reveals the effects of local microstructures on the crack growth behavior.

Coupling Reagent for UV/vis Absorbing Azobenzene-Based Quantitative Analysis of the Extent of Functional Group Immobilization on Silica.

PubMed

Choi, Ra-Young; Lee, Chang-Hee; Jun, Chul-Ho

2018-05-18

A methallylsilane coupling reagent, containing both a N-hydroxysuccinimidyl(NHS)-ester group and a UV/vis absorbing azobenzene linker undergoes acid-catalyzed immobilization on silica. Analysis of the UV/vis absorption band associated with the azobenzene group in the adduct enables facile quantitative determination of the extent of loading of the NHS groups. Reaction of NHS-groups on the silica surface with amine groups of GOx and rhodamine can be employed to generate enzyme or dye-immobilized silica for quantitative analysis.

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

Quantitation of permethylated N-glycans through multiple-reaction monitoring (MRM) LC-MS/MS.

PubMed

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

PubMed Central

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-01-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan structures was determined to be 30% while it was found to be 35% for either fucosylated or sialylated structures The optimum CE for mannose and complex type N-glycan structures was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan structures in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these structures was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitudes. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan structures enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples. PMID:25698222

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

NASA Astrophysics Data System (ADS)

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Selecting the most appropriate inferential statistical test for your quantitative research study.

PubMed

Bettany-Saltikov, Josette; Whittaker, Victoria Jane

2014-06-01

To discuss the issues and processes relating to the selection of the most appropriate statistical test. A review of the basic research concepts together with a number of clinical scenarios is used to illustrate this. Quantitative nursing research generally features the use of empirical data which necessitates the selection of both descriptive and statistical tests. Different types of research questions can be answered by different types of research designs, which in turn need to be matched to a specific statistical test(s). Discursive paper. This paper discusses the issues relating to the selection of the most appropriate statistical test and makes some recommendations as to how these might be dealt with. When conducting empirical quantitative studies, a number of key issues need to be considered. Considerations for selecting the most appropriate statistical tests are discussed and flow charts provided to facilitate this process. When nursing clinicians and researchers conduct quantitative research studies, it is crucial that the most appropriate statistical test is selected to enable valid conclusions to be made. © 2013 John Wiley & Sons Ltd.

Assessing signal-to-noise in quantitative proteomics: multivariate statistical analysis in DIGE experiments.

PubMed

Friedman, David B

2012-01-01

All quantitative proteomics experiments measure variation between samples. When performing large-scale experiments that involve multiple conditions or treatments, the experimental design should include the appropriate number of individual biological replicates from each condition to enable the distinction between a relevant biological signal from technical noise. Multivariate statistical analyses, such as principal component analysis (PCA), provide a global perspective on experimental variation, thereby enabling the assessment of whether the variation describes the expected biological signal or the unanticipated technical/biological noise inherent in the system. Examples will be shown from high-resolution multivariable DIGE experiments where PCA was instrumental in demonstrating biologically significant variation as well as sample outliers, fouled samples, and overriding technical variation that would not be readily observed using standard univariate tests.

Quantitative 3D investigation of Neuronal network in mouse spinal cord model

NASA Astrophysics Data System (ADS)

Bukreeva, I.; Campi, G.; Fratini, M.; Spanò, R.; Bucci, D.; Battaglia, G.; Giove, F.; Bravin, A.; Uccelli, A.; Venturi, C.; Mastrogiacomo, M.; Cedola, A.

2017-01-01

The investigation of the neuronal network in mouse spinal cord models represents the basis for the research on neurodegenerative diseases. In this framework, the quantitative analysis of the single elements in different districts is a crucial task. However, conventional 3D imaging techniques do not have enough spatial resolution and contrast to allow for a quantitative investigation of the neuronal network. Exploiting the high coherence and the high flux of synchrotron sources, X-ray Phase-Contrast multiscale-Tomography allows for the 3D investigation of the neuronal microanatomy without any aggressive sample preparation or sectioning. We investigated healthy-mouse neuronal architecture by imaging the 3D distribution of the neuronal-network with a spatial resolution of 640 nm. The high quality of the obtained images enables a quantitative study of the neuronal structure on a subject-by-subject basis. We developed and applied a spatial statistical analysis on the motor neurons to obtain quantitative information on their 3D arrangement in the healthy-mice spinal cord. Then, we compared the obtained results with a mouse model of multiple sclerosis. Our approach paves the way to the creation of a “database” for the characterization of the neuronal network main features for a comparative investigation of neurodegenerative diseases and therapies.

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

PubMed

Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

2015-04-01

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantitative nondestructive evaluation: Requirements for tomorrow's reliability

NASA Technical Reports Server (NTRS)

Heyman, Joseph S.

1991-01-01

Quantitative Nondestructive Evaluation (QNDE) is the technology of measurement, analysis, and prediction of the state of material/structural systems for safety, reliability, and mission assurance. QNDE has impact on everyday life from the cars we drive, the planes we fly, the buildings we work or live in, literally to the infrastructure of our world. Here, researchers highlight some of the new sciences and technologies that are part of a safer, cost effective tomorrow. Specific technologies that are discussed are thermal QNDE of aircraft structural integrity, ultrasonic QNDE for materials characterization, and technology spinoffs from aerospace to the medical sector. In each case, examples are given of how new requirements result in enabling measurement technologies, which in turn change the boundaries of design/practice.

A quantitative assay for mitochondrial fusion using Renilla luciferase complementation

PubMed Central

Huang, Huiyan; Choi, Seok-Yong; Frohman, Michael A.

2010-01-01

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression. PMID:20488258

A quantitative assay for mitochondrial fusion using Renilla luciferase complementation.

PubMed

Huang, Huiyan; Choi, Seok-Yong; Frohman, Michael A

2010-08-01

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression. (c) 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.

Dominant Epistasis Between Two Quantitative Trait Loci Governing Sporulation Efficiency in Yeast Saccharomyces cerevisiae

PubMed Central

Bergman, Juraj; Mitrikeski, Petar T.

2015-01-01

Summary Sporulation efficiency in the yeast Saccharomyces cerevisiae is a well-established model for studying quantitative traits. A variety of genes and nucleotides causing different sporulation efficiencies in laboratory, as well as in wild strains, has already been extensively characterised (mainly by reciprocal hemizygosity analysis and nucleotide exchange methods). We applied a different strategy in order to analyze the variation in sporulation efficiency of laboratory yeast strains. Coupling classical quantitative genetic analysis with simulations of phenotypic distributions (a method we call phenotype modelling) enabled us to obtain a detailed picture of the quantitative trait loci (QTLs) relationships underlying the phenotypic variation of this trait. Using this approach, we were able to uncover a dominant epistatic inheritance of loci governing the phenotype. Moreover, a molecular analysis of known causative quantitative trait genes and nucleotides allowed for the detection of novel alleles, potentially responsible for the observed phenotypic variation. Based on the molecular data, we hypothesise that the observed dominant epistatic relationship could be caused by the interaction of multiple quantitative trait nucleotides distributed across a 60--kb QTL region located on chromosome XIV and the RME1 locus on chromosome VII. Furthermore, we propose a model of molecular pathways which possibly underlie the phenotypic variation of this trait. PMID:27904371

Morphology enabled dipole inversion (MEDI) from a single-angle acquisition: comparison with COSMOS in human brain imaging.

PubMed

Liu, Tian; Liu, Jing; de Rochefort, Ludovic; Spincemaille, Pascal; Khalidov, Ildar; Ledoux, James Robert; Wang, Yi

2011-09-01

Magnetic susceptibility varies among brain structures and provides insights into the chemical and molecular composition of brain tissues. However, the determination of an arbitrary susceptibility distribution from the measured MR signal phase is a challenging, ill-conditioned inverse problem. Although a previous method named calculation of susceptibility through multiple orientation sampling (COSMOS) has solved this inverse problem both theoretically and experimentally using multiple angle acquisitions, it is often impractical to carry out on human subjects. Recently, the feasibility of calculating the brain susceptibility distribution from a single-angle acquisition was demonstrated using morphology enabled dipole inversion (MEDI). In this study, we further improved the original MEDI method by sparsifying the edges in the quantitative susceptibility map that do not have a corresponding edge in the magnitude image. Quantitative susceptibility maps generated by the improved MEDI were compared qualitatively and quantitatively with those generated by calculation of susceptibility through multiple orientation sampling. The results show a high degree of agreement between MEDI and calculation of susceptibility through multiple orientation sampling, and the practicality of MEDI allows many potential clinical applications. Copyright © 2011 Wiley-Liss, Inc.

Quantitative Image Informatics for Cancer Research (QIICR) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

Imaging has enormous untapped potential to improve cancer research through software to extract and process morphometric and functional biomarkers. In the era of non-cytotoxic treatment agents, multi- modality image-guided ablative therapies and rapidly evolving computational resources, quantitative imaging software can be transformative in enabling minimally invasive, objective and reproducible evaluation of cancer treatment response. Post-processing algorithms are integral to high-throughput analysis and fine- grained differentiation of multiple molecular targets.

Quantitative imaging biomarker ontology (QIBO) for knowledge representation of biomedical imaging biomarkers.

PubMed

Buckler, Andrew J; Liu, Tiffany Ting; Savig, Erica; Suzek, Baris E; Ouellette, M; Danagoulian, J; Wernsing, G; Rubin, Daniel L; Paik, David

2013-08-01

A widening array of novel imaging biomarkers is being developed using ever more powerful clinical and preclinical imaging modalities. These biomarkers have demonstrated effectiveness in quantifying biological processes as they occur in vivo and in the early prediction of therapeutic outcomes. However, quantitative imaging biomarker data and knowledge are not standardized, representing a critical barrier to accumulating medical knowledge based on quantitative imaging data. We use an ontology to represent, integrate, and harmonize heterogeneous knowledge across the domain of imaging biomarkers. This advances the goal of developing applications to (1) improve precision and recall of storage and retrieval of quantitative imaging-related data using standardized terminology; (2) streamline the discovery and development of novel imaging biomarkers by normalizing knowledge across heterogeneous resources; (3) effectively annotate imaging experiments thus aiding comprehension, re-use, and reproducibility; and (4) provide validation frameworks through rigorous specification as a basis for testable hypotheses and compliance tests. We have developed the Quantitative Imaging Biomarker Ontology (QIBO), which currently consists of 488 terms spanning the following upper classes: experimental subject, biological intervention, imaging agent, imaging instrument, image post-processing algorithm, biological target, indicated biology, and biomarker application. We have demonstrated that QIBO can be used to annotate imaging experiments with standardized terms in the ontology and to generate hypotheses for novel imaging biomarker-disease associations. Our results established the utility of QIBO in enabling integrated analysis of quantitative imaging data.

Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes

PubMed Central

Di Napoli, Claudia; Pope, Iestyn; Masia, Francesco; Watson, Peter; Langbein, Wolfgang; Borri, Paola

2014-01-01

In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200–2000/cm) and in the CH stretch region (2600–3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods. PMID:24877002

Quantitative adhesion characterization of antireflective coatings in multijunction photovoltaics

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

Brock, Ryan; Rewari, Raunaq; Novoa, Fernando D.

We discuss the development of a new composite dual cantilever beam (cDCB) thin-film adhesion testing method, which enables the quantitative measurement of adhesion on the thin and fragile substrates used in multijunction photovoltaics. In particular, we address the adhesion of several 2- and 3-layer antireflective coating systems on multijunction cells. By varying interface chemistry and morphology through processing, we demonstrate the marked effects on adhesion and help to develop an understanding of how high adhesion can be achieved, as adhesion values ranging from 0.5 J/m2 to 10 J/m2 were measured. Damp heat (85 degrees C/85% RH) was used to invokemore » degradation of interfacial adhesion. We demonstrate that even with germanium substrates that fracture relatively easily, quantitative measurements of adhesion can be made at high test yield. The cDCB test is discussed as an important new methodology, which can be broadly applied to any system that makes use of thin, brittle, or otherwise fragile substrates.« less

Toward Accurate and Quantitative Comparative Metagenomics

PubMed Central

Nayfach, Stephen; Pollard, Katherine S.

2016-01-01

Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. PMID:27565341

Quantitative characterization of surface topography using spectral analysis

NASA Astrophysics Data System (ADS)

Jacobs, Tevis D. B.; Junge, Till; Pastewka, Lars

2017-03-01

Roughness determines many functional properties of surfaces, such as adhesion, friction, and (thermal and electrical) contact conductance. Recent analytical models and simulations enable quantitative prediction of these properties from knowledge of the power spectral density (PSD) of the surface topography. The utility of the PSD is that it contains statistical information that is unbiased by the particular scan size and pixel resolution chosen by the researcher. In this article, we first review the mathematical definition of the PSD, including the one- and two-dimensional cases, and common variations of each. We then discuss strategies for reconstructing an accurate PSD of a surface using topography measurements at different size scales. Finally, we discuss detecting and mitigating artifacts at the smallest scales, and computing upper/lower bounds on functional properties obtained from models. We accompany our discussion with virtual measurements on computer-generated surfaces. This discussion summarizes how to analyze topography measurements to reconstruct a reliable PSD. Analytical models demonstrate the potential for tuning functional properties by rationally tailoring surface topography—however, this potential can only be achieved through the accurate, quantitative reconstruction of the PSDs of real-world surfaces.

Quantitative Evaluation of Performance during Robot-assisted Treatment.

PubMed

Peri, E; Biffi, E; Maghini, C; Servodio Iammarrone, F; Gagliardi, C; Germiniasi, C; Pedrocchi, A; Turconi, A C; Reni, G

2016-01-01

This article is part of the Focus Theme of Methods of Information in Medicine on "Methodologies, Models and Algorithms for Patients Rehabilitation". The great potential of robots in extracting quantitative and meaningful data is not always exploited in clinical practice. The aim of the present work is to describe a simple parameter to assess the performance of subjects during upper limb robotic training exploiting data automatically recorded by the robot, with no additional effort for patients and clinicians. Fourteen children affected by cerebral palsy (CP) performed a training with Armeo®Spring. Each session was evaluated with P, a simple parameter that depends on the overall performance recorded, and median and interquartile values were computed to perform a group analysis. Median (interquartile) values of P significantly increased from 0.27 (0.21) at T0 to 0.55 (0.27) at T1 . This improvement was functionally validated by a significant increase of the Melbourne Assessment of Unilateral Upper Limb Function. The parameter described here was able to show variations in performance over time and enabled a quantitative evaluation of motion abilities in a way that is reliable with respect to a well-known clinical scale.

Barriers to and enablers of diabetic retinopathy screening attendance: a systematic review of published and grey literature.

PubMed

Graham-Rowe, E; Lorencatto, F; Lawrenson, J G; Burr, J M; Grimshaw, J M; Ivers, N M; Presseau, J; Vale, L; Peto, T; Bunce, C; Francis, J J

2018-05-23

To identify and synthesize studies reporting modifiable barriers/enablers associated with retinopathy screening attendance in people with Type 1 or Type 2 diabetes, and to identify those most likely to influence attendance. We searched MEDLINE, EMBASE, PsycINFO, Cochrane Library and the 'grey literature' for quantitative and qualitative studies to February 2017. Data (i.e. participant quotations, interpretive summaries, survey results) reporting barriers/enablers were extracted and deductively coded into domains from the Theoretical Domains Framework; with domains representing categories of theoretical barriers/enablers proposed to mediate behaviour change. Inductive thematic analysis was conducted within domains to describe the role each domain plays in facilitating or hindering screening attendance. Domains that were more frequently coded and for which more themes were generated were judged more likely to influence attendance. Sixty-nine primary studies were included. We identified six theoretical domains ['environmental context and resources' (75% of included studies), 'social influences' (51%), 'knowledge' (50%), 'memory, attention, decision processes' (50%), 'beliefs about consequences' (38%) and 'emotions' (33%)] as the key mediators of diabetic retinopathy screening attendance. Examples of barriers populating these domains included inaccurate diabetic registers and confusion between routine eye care and retinopathy screening. Recommendations by healthcare professionals and community-level media coverage acted as enablers. Across a variety of contexts, we found common barriers to and enablers of retinopathy screening that could be targeted in interventions aiming to increase screening attendance. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Lipid Informed Quantitation and Identification

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

Kevin Crowell, PNNL

2014-07-21

LIQUID (Lipid Informed Quantitation and Identification) is a software program that has been developed to enable users to conduct both informed and high-throughput global liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based lipidomics analysis. This newly designed desktop application can quickly identify and quantify lipids from LC-MS/MS datasets while providing a friendly graphical user interface for users to fully explore the data. Informed data analysis simply involves the user specifying an electrospray ionization mode, lipid common name (i.e. PE(16:0/18:2)), and associated charge carrier. A stemplot of the isotopic profile and a line plot of the extracted ion chromatogram are also provided to showmore » the MS-level evidence of the identified lipid. In addition to plots, other information such as intensity, mass measurement error, and elution time are also provided. Typically, a global analysis for 15,000 lipid targets« less

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

PubMed

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Quantitative phylogenetic assessment of microbial communities in diverse environments.

PubMed

von Mering, C; Hugenholtz, P; Raes, J; Tringe, S G; Doerks, T; Jensen, L J; Ward, N; Bork, P

2007-02-23

The taxonomic composition of environmental communities is an important indicator of their ecology and function. We used a set of protein-coding marker genes, extracted from large-scale environmental shotgun sequencing data, to provide a more direct, quantitative, and accurate picture of community composition than that provided by traditional ribosomal RNA-based approaches depending on the polymerase chain reaction. Mapping marker genes from four diverse environmental data sets onto a reference species phylogeny shows that certain communities evolve faster than others. The method also enables determination of preferred habitats for entire microbial clades and provides evidence that such habitat preferences are often remarkably stable over time.

Wires in the soup: quantitative models of cell signaling

PubMed Central

Cheong, Raymond; Levchenko, Andre

2014-01-01

Living cells are capable of extracting information from their environments and mounting appropriate responses to a variety of associated challenges. The underlying signal transduction networks enabling this can be quite complex, necessitating for their unraveling by sophisticated computational modeling coupled with precise experimentation. Although we are still at the beginning of this process, some recent examples of integrative analysis of cell signaling are very encouraging. This review highlights the case of the NF-κB pathway in order to illustrate how a quantitative model of a signaling pathway can be gradually constructed through continuous experimental validation, and what lessons one might learn from such exercises. PMID:18291655

SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin

PubMed Central

Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

2017-01-01

Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin. PMID:28402964

SET mediates TCE-induced liver cell apoptosis through dephosphorylation and upregulation of nucleolin.

PubMed

Ren, Xiaohu; Huang, Xinfeng; Yang, Xifei; Liu, Yungang; Liu, Wei; Huang, Haiyan; Wu, Desheng; Zou, Fei; Liu, Jianjun

2017-06-20

Trichloroethylene (TCE) is an occupational and environmental chemical that can cause severe hepatotoxicity. While our previous studies showed that the phosphatase inhibitor SET is a key mediator of TCE-induced liver cell apoptosis, the molecular mechanisms remain elusive. Using quantitative phosphoproteomic analysis, we report here that nucleolin is a SET-regulated phosphoprotein in human liver HL-7702 cells. Functional analysis suggested that SET promoted dephosphorylation of nucleolin, decreased its binding to its transcriptional activator, c-myc, and upregulated nucleolin expression in TCE-treated cells. Importantly, TCE-induced hepatocyte apoptosis was significantly attenuated when nucleolin was downregulated with specific siRNAs. These findings indicate that TCE may induce hepatocyte apoptosis via SET-mediated dephosphorylation and overexpression of nucleolin.

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.

Evaluation of empirical rule of linearly correlated peptide selection (ERLPS) for proteotypic peptide-based quantitative proteomics.

PubMed

Liu, Kehui; Zhang, Jiyang; Fu, Bin; Xie, Hongwei; Wang, Yingchun; Qian, Xiaohong

2014-07-01

Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide-based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an "empirical rule for linearly correlated peptide selection (ERLPS)" in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label-free to O18/O16-labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide-based quantitative proteomics over a large dynamic range. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2017-01-01

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

The Development of Mathematical Knowledge for Teaching for Quantitative Reasoning Using Video-Based Instruction

NASA Astrophysics Data System (ADS)

Walters, Charles David

Quantitative reasoning (P. W. Thompson, 1990, 1994) is a powerful mathematical tool that enables students to engage in rich problem solving across the curriculum. One way to support students' quantitative reasoning is to develop prospective secondary teachers' (PSTs) mathematical knowledge for teaching (MKT; Ball, Thames, & Phelps, 2008) related to quantitative reasoning. However, this may prove challenging, as prior to entering the classroom, PSTs often have few opportunities to develop MKT by examining and reflecting on students' thinking. Videos offer one avenue through which such opportunities are possible. In this study, I report on the design of a mini-course for PSTs that featured a series of videos created as part of a proof-of-concept NSF-funded project. These MathTalk videos highlight the ways in which the quantitative reasoning of two high school students developed over time. Using a mixed approach to grounded theory, I analyzed pre- and postinterviews using an extant coding scheme based on the Silverman and Thompson (2008) framework for the development of MKT. This analysis revealed a shift in participants' affect as well as three distinct shifts in their MKT around quantitative reasoning with distances, including shifts in: (a) quantitative reasoning; (b) point of view (decentering); and (c) orientation toward problem solving. Using the four-part focusing framework (Lobato, Hohensee, & Rhodehamel, 2013), I analyzed classroom data to account for how participants' noticing was linked with the shifts in MKT. Notably, their increased noticing of aspects of MKT around quantitative reasoning with distances, which features prominently in the MathTalk videos, seemed to contribute to the emergence of the shifts in MKT. Results from this study link elements of the learning environment to the development of specific facets of MKT around quantitative reasoning with distances. These connections suggest that vicarious experiences with two students' quantitative

Development of an exposure measurement database on five lung carcinogens (ExpoSYN) for quantitative retrospective occupational exposure assessment.

PubMed

Peters, Susan; Vermeulen, Roel; Olsson, Ann; Van Gelder, Rainer; Kendzia, Benjamin; Vincent, Raymond; Savary, Barbara; Williams, Nick; Woldbæk, Torill; Lavoué, Jérôme; Cavallo, Domenico; Cattaneo, Andrea; Mirabelli, Dario; Plato, Nils; Dahmann, Dirk; Fevotte, Joelle; Pesch, Beate; Brüning, Thomas; Straif, Kurt; Kromhout, Hans

2012-01-01

SYNERGY is a large pooled analysis of case-control studies on the joint effects of occupational carcinogens and smoking in the development of lung cancer. A quantitative job-exposure matrix (JEM) will be developed to assign exposures to five major lung carcinogens [asbestos, chromium, nickel, polycyclic aromatic hydrocarbons (PAH), and respirable crystalline silica (RCS)]. We assembled an exposure database, called ExpoSYN, to enable such a quantitative exposure assessment. Existing exposure databases were identified and European and Canadian research institutes were approached to identify pertinent exposure measurement data. Results of individual air measurements were entered anonymized according to a standardized protocol. The ExpoSYN database currently includes 356 551 measurements from 19 countries. In total, 140 666 personal and 215 885 stationary data points were available. Measurements were distributed over the five agents as follows: RCS (42%), asbestos (20%), chromium (16%), nickel (15%), and PAH (7%). The measurement data cover the time period from 1951 to present. However, only a small portion of measurements (1.4%) were performed prior to 1975. The major contributing countries for personal measurements were Germany (32%), UK (22%), France (14%), and Norway and Canada (both 11%). ExpoSYN is a unique occupational exposure database with measurements from 18 European countries and Canada covering a time period of >50 years. This database will be used to develop a country-, job-, and time period-specific quantitative JEM. This JEM will enable data-driven quantitative exposure assessment in a multinational pooled analysis of community-based lung cancer case-control studies.

Synthesising quantitative and qualitative research in evidence-based patient information.

PubMed

Goldsmith, Megan R; Bankhead, Clare R; Austoker, Joan

2007-03-01

Systematic reviews have, in the past, focused on quantitative studies and clinical effectiveness, while excluding qualitative evidence. Qualitative research can inform evidence-based practice independently of other research methodologies but methods for the synthesis of such data are currently evolving. Synthesising quantitative and qualitative research in a single review is an important methodological challenge. This paper describes the review methods developed and the difficulties encountered during the process of updating a systematic review of evidence to inform guidelines for the content of patient information related to cervical screening. Systematic searches of 12 electronic databases (January 1996 to July 2004) were conducted. Studies that evaluated the content of information provided to women about cervical screening or that addressed women's information needs were assessed for inclusion. A data extraction form and quality assessment criteria were developed from published resources. A non-quantitative synthesis was conducted and a tabular evidence profile for each important outcome (eg "explain what the test involves") was prepared. The overall quality of evidence for each outcome was then assessed using an approach published by the GRADE working group, which was adapted to suit the review questions and modified to include qualitative research evidence. Quantitative and qualitative studies were considered separately for every outcome. 32 papers were included in the systematic review following data extraction and assessment of methodological quality. The review questions were best answered by evidence from a range of data sources. The inclusion of qualitative research, which was often highly relevant and specific to many components of the screening information materials, enabled the production of a set of recommendations that will directly affect policy within the NHS Cervical Screening Programme. A practical example is provided of how quantitative and

Synthesising quantitative and qualitative research in evidence‐based patient information

PubMed Central

Goldsmith, Megan R; Bankhead, Clare R; Austoker, Joan

2007-01-01

Background Systematic reviews have, in the past, focused on quantitative studies and clinical effectiveness, while excluding qualitative evidence. Qualitative research can inform evidence‐based practice independently of other research methodologies but methods for the synthesis of such data are currently evolving. Synthesising quantitative and qualitative research in a single review is an important methodological challenge. Aims This paper describes the review methods developed and the difficulties encountered during the process of updating a systematic review of evidence to inform guidelines for the content of patient information related to cervical screening. Methods Systematic searches of 12 electronic databases (January 1996 to July 2004) were conducted. Studies that evaluated the content of information provided to women about cervical screening or that addressed women's information needs were assessed for inclusion. A data extraction form and quality assessment criteria were developed from published resources. A non‐quantitative synthesis was conducted and a tabular evidence profile for each important outcome (eg “explain what the test involves”) was prepared. The overall quality of evidence for each outcome was then assessed using an approach published by the GRADE working group, which was adapted to suit the review questions and modified to include qualitative research evidence. Quantitative and qualitative studies were considered separately for every outcome. Results 32 papers were included in the systematic review following data extraction and assessment of methodological quality. The review questions were best answered by evidence from a range of data sources. The inclusion of qualitative research, which was often highly relevant and specific to many components of the screening information materials, enabled the production of a set of recommendations that will directly affect policy within the NHS Cervical Screening Programme. Conclusions A

Stratified Charge Rotary Engine Critical Technology Enablement, Volume 1

NASA Technical Reports Server (NTRS)

Irion, C. E.; Mount, R. E.

1992-01-01

This report summarizes results of a critical technology enablement effort with the stratified charge rotary engine (SCRE) focusing on a power section of 0.67 liters (40 cu. in.) per rotor in single and two rotor versions. The work is a continuation of prior NASA Contracts NAS3-23056 and NAS3-24628. Technical objectives are multi-fuel capability, including civil and military jet fuel and DF-2, fuel efficiency of 0.355 Lbs/BHP-Hr. at best cruise condition above 50 percent power, altitude capability of up to 10Km (33,000 ft.) cruise, 2000 hour TBO and reduced coolant heat rejection. Critical technologies for SCRE's that have the potential for competitive performance and cost in a representative light-aircraft environment were examined. Objectives were: the development and utilization of advanced analytical tools, i.e. higher speed and enhanced three dimensional combustion modeling; identification of critical technologies; development of improved instrumentation, and to isolate and quantitatively identify the contribution to performance and efficiency of critical components or subsystems.

Quantitative model analysis with diverse biological data: applications in developmental pattern formation.

PubMed

Pargett, Michael; Umulis, David M

2013-07-15

Mathematical modeling of transcription factor and signaling networks is widely used to understand if and how a mechanism works, and to infer regulatory interactions that produce a model consistent with the observed data. Both of these approaches to modeling are informed by experimental data, however, much of the data available or even acquirable are not quantitative. Data that is not strictly quantitative cannot be used by classical, quantitative, model-based analyses that measure a difference between the measured observation and the model prediction for that observation. To bridge the model-to-data gap, a variety of techniques have been developed to measure model "fitness" and provide numerical values that can subsequently be used in model optimization or model inference studies. Here, we discuss a selection of traditional and novel techniques to transform data of varied quality and enable quantitative comparison with mathematical models. This review is intended to both inform the use of these model analysis methods, focused on parameter estimation, and to help guide the choice of method to use for a given study based on the type of data available. Applying techniques such as normalization or optimal scaling may significantly improve the utility of current biological data in model-based study and allow greater integration between disparate types of data. Copyright © 2013 Elsevier Inc. All rights reserved.

Semi-quantitative analysis of solid waste flows from nano-enabled consumer products in Europe, Denmark and the United Kingdom - Abundance, distribution and management.

PubMed

Heggelund, Laura; Hansen, Steffen Foss; Astrup, Thomas Fruergaard; Boldrin, Alessio

2016-10-01

Many nano-enabled consumer products are known to be in the global market. At the same, little is known about the quantity, type, location etc. of the engineered nanomaterials (ENMs) inside the products. This limits the scientific investigations of potential environmental effects of these materials, and especially the knowledge of ENM behaviour and potential effects at the end-of-life stage of the products is scarce. To gain a better understanding of the end-of-life waste treatment of nano-enabled consumer product, we provide an overview of the ENMs flowing into and throughout waste systems in Europe, Denmark and the United Kingdom. Using a nanoproduct inventory (nanodb.dk), we performed a four-step analysis to estimate the most abundant ENMs and in which waste fractions they are present. We found that in terms of number of products: (i) nano silver is the most used ENM in consumer products, and (ii) plastic from used product containers is the largest waste fraction also comprising a large variety of ENMs, though possibly in very small masses. Also, we showed that the local waste management system can influence the distribution of ENMs. It is recommended that future research focus on recycling and landfilling of nano-enabled products since these compartments represent hot spots for end-of-life nanoproducts. Copyright © 2016 Elsevier Ltd. All rights reserved.

PIQMIe: a web server for semi-quantitative proteomics data management and analysis

PubMed Central

Kuzniar, Arnold; Kanaar, Roland

2014-01-01

We present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates peptide and (non-redundant) protein identifications and quantitations from multiple experiments with additional biological information on the protein entries, and makes the linked data available in the form of a light-weight relational database, which enables dedicated data analyses (e.g. in R) and user-driven queries. Using the web interface, users are presented with a concise summary of their proteomics experiments in numerical and graphical forms, as well as with a searchable protein grid and interactive visualization tools to aid in the rapid assessment of the experiments and in the identification of proteins of interest. The web server not only provides data access through a web interface but also supports programmatic access through RESTful web service. The web server is available at http://piqmie.semiqprot-emc.cloudlet.sara.nl or http://www.bioinformatics.nl/piqmie. This website is free and open to all users and there is no login requirement. PMID:24861615

Toward Accurate and Quantitative Comparative Metagenomics.

PubMed

Nayfach, Stephen; Pollard, Katherine S

2016-08-25

Shotgun metagenomics and computational analysis are used to compare the taxonomic and functional profiles of microbial communities. Leveraging this approach to understand roles of microbes in human biology and other environments requires quantitative data summaries whose values are comparable across samples and studies. Comparability is currently hampered by the use of abundance statistics that do not estimate a meaningful parameter of the microbial community and biases introduced by experimental protocols and data-cleaning approaches. Addressing these challenges, along with improving study design, data access, metadata standardization, and analysis tools, will enable accurate comparative metagenomics. We envision a future in which microbiome studies are replicable and new metagenomes are easily and rapidly integrated with existing data. Only then can the potential of metagenomics for predictive ecological modeling, well-powered association studies, and effective microbiome medicine be fully realized. Copyright © 2016 Elsevier Inc. All rights reserved.

Hematocrit Measurement with R2* and Quantitative Susceptibility Mapping in Postmortem Brain.

PubMed

Walsh, A J; Sun, H; Emery, D J; Wilman, A H

2018-05-24

Noninvasive venous oxygenation quantification with MR imaging will improve the neurophysiologic investigation and the understanding of the pathophysiology in neurologic diseases. Available MR imaging methods are limited by sensitivity to flow and often require assumptions of the hematocrit level. In situ postmortem imaging enables evaluation of methods in a fully deoxygenated environment without flow artifacts, allowing direct calculation of hematocrit. This study compares 2 venous oxygenation quantification methods in in situ postmortem subjects. Transverse relaxation (R2*) mapping and quantitative susceptibility mapping were performed on a whole-body 4.7T MR imaging system. Intravenous measurements in major draining intracranial veins were compared between the 2 methods in 3 postmortem subjects. The quantitative susceptibility mapping technique was also applied in 10 healthy control subjects and compared with reference venous oxygenation values. In 2 early postmortem subjects, R2* mapping and quantitative susceptibility mapping measurements within intracranial veins had a significant and strong correlation ( R 2 = 0.805, P = .004 and R 2 = 0.836, P = .02). Higher R2* and susceptibility values were consistently demonstrated within gravitationally dependent venous segments during the early postmortem period. Hematocrit ranged from 0.102 to 0.580 in postmortem subjects, with R2* and susceptibility as large as 291 seconds -1 and 1.75 ppm, respectively. Measurements of R2* and quantitative susceptibility mapping within large intracranial draining veins have a high correlation in early postmortem subjects. This study supports the use of quantitative susceptibility mapping for evaluation of in vivo venous oxygenation and postmortem hematocrit concentrations. © 2018 by American Journal of Neuroradiology.

Algorithm-enabled partial-angular-scan configurations for dual-energy CT.

PubMed

Chen, Buxin; Zhang, Zheng; Xia, Dan; Sidky, Emil Y; Pan, Xiaochuan

2018-05-01

We seek to investigate an optimization-based one-step method for image reconstruction that explicitly compensates for nonlinear spectral response (i.e., the beam-hardening effect) in dual-energy CT, to investigate the feasibility of the one-step method for enabling two dual-energy partial-angular-scan configurations, referred to as the short- and half-scan configurations, on standard CT scanners without involving additional hardware, and to investigate the potential of the short- and half-scan configurations in reducing imaging dose and scan time in a single-kVp-switch full-scan configuration in which two full rotations are made for collection of dual-energy data. We use the one-step method to reconstruct images directly from dual-energy data through solving a nonconvex optimization program that specifies the images to be reconstructed in dual-energy CT. Dual-energy full-scan data are generated from numerical phantoms and collected from physical phantoms with the standard single-kVp-switch full-scan configuration, whereas dual-energy short- and half-scan data are extracted from the corresponding full-scan data. Besides visual inspection and profile-plot comparison, the reconstructed images are analyzed also in quantitative studies based upon tasks of linear-attenuation-coefficient and material-concentration estimation and of material differentiation. Following the performance of a computer-simulation study to verify that the one-step method can reconstruct numerically accurately basis and monochromatic images of numerical phantoms, we reconstruct basis and monochromatic images by using the one-step method from real data of physical phantoms collected with the full-, short-, and half-scan configurations. Subjective inspection based upon visualization and profile-plot comparison reveals that monochromatic images, which are used often in practical applications, reconstructed from the full-, short-, and half-scan data are largely visually comparable except for some

The Quantitative Methods Boot Camp: Teaching Quantitative Thinking and Computing Skills to Graduate Students in the Life Sciences

PubMed Central

Stefan, Melanie I.; Gutlerner, Johanna L.; Born, Richard T.; Springer, Michael

2015-01-01

The past decade has seen a rapid increase in the ability of biologists to collect large amounts of data. It is therefore vital that research biologists acquire the necessary skills during their training to visualize, analyze, and interpret such data. To begin to meet this need, we have developed a “boot camp” in quantitative methods for biology graduate students at Harvard Medical School. The goal of this short, intensive course is to enable students to use computational tools to visualize and analyze data, to strengthen their computational thinking skills, and to simulate and thus extend their intuition about the behavior of complex biological systems. The boot camp teaches basic programming using biological examples from statistics, image processing, and data analysis. This integrative approach to teaching programming and quantitative reasoning motivates students’ engagement by demonstrating the relevance of these skills to their work in life science laboratories. Students also have the opportunity to analyze their own data or explore a topic of interest in more detail. The class is taught with a mixture of short lectures, Socratic discussion, and in-class exercises. Students spend approximately 40% of their class time working through both short and long problems. A high instructor-to-student ratio allows students to get assistance or additional challenges when needed, thus enhancing the experience for students at all levels of mastery. Data collected from end-of-course surveys from the last five offerings of the course (between 2012 and 2014) show that students report high learning gains and feel that the course prepares them for solving quantitative and computational problems they will encounter in their research. We outline our course here which, together with the course materials freely available online under a Creative Commons License, should help to facilitate similar efforts by others. PMID:25880064

The quantitative methods boot camp: teaching quantitative thinking and computing skills to graduate students in the life sciences.

PubMed

Stefan, Melanie I; Gutlerner, Johanna L; Born, Richard T; Springer, Michael

2015-04-01

The past decade has seen a rapid increase in the ability of biologists to collect large amounts of data. It is therefore vital that research biologists acquire the necessary skills during their training to visualize, analyze, and interpret such data. To begin to meet this need, we have developed a "boot camp" in quantitative methods for biology graduate students at Harvard Medical School. The goal of this short, intensive course is to enable students to use computational tools to visualize and analyze data, to strengthen their computational thinking skills, and to simulate and thus extend their intuition about the behavior of complex biological systems. The boot camp teaches basic programming using biological examples from statistics, image processing, and data analysis. This integrative approach to teaching programming and quantitative reasoning motivates students' engagement by demonstrating the relevance of these skills to their work in life science laboratories. Students also have the opportunity to analyze their own data or explore a topic of interest in more detail. The class is taught with a mixture of short lectures, Socratic discussion, and in-class exercises. Students spend approximately 40% of their class time working through both short and long problems. A high instructor-to-student ratio allows students to get assistance or additional challenges when needed, thus enhancing the experience for students at all levels of mastery. Data collected from end-of-course surveys from the last five offerings of the course (between 2012 and 2014) show that students report high learning gains and feel that the course prepares them for solving quantitative and computational problems they will encounter in their research. We outline our course here which, together with the course materials freely available online under a Creative Commons License, should help to facilitate similar efforts by others.

In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing

PubMed Central

Aguilar, Carlos A.; Shcherbina, Anna; Ricke, Darrell O.; Pop, Ramona; Carrigan, Christopher T.; Gifford, Casey A.; Urso, Maria L.; Kottke, Melissa A.; Meissner, Alexander

2015-01-01

Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual’s muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing. PMID:26381351

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

Surface plasmon resonance microscopy: achieving a quantitative optical response

PubMed Central

Peterson, Alexander W.; Halter, Michael; Plant, Anne L.; Elliott, John T.

2016-01-01

Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction, and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based configuration. We carry out SPR imaging on a microscope by launching light into a sample, and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit, and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data, and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy. PMID:27782542

Quantitative MR imaging in fracture dating--Initial results.

PubMed

Baron, Katharina; Neumayer, Bernhard; Widek, Thomas; Schick, Fritz; Scheicher, Sylvia; Hassler, Eva; Scheurer, Eva

2016-04-01

significant changes could be detected for T2. MTR remained constant at 35.5 ± 8.0% over time. The study shows that the quantitative assessment of T1 and T2 behaviour over time in the fractured region enable the generation of a novel model allowing for an objective age determination of a fracture. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Dust control for Enabler

NASA Technical Reports Server (NTRS)

Hilton, Kevin; Karl, Chad; Litherland, Mark; Ritchie, David; Sun, Nancy

1992-01-01

The dust control group designed a system to restrict dust that is disturbed by the Enabler during its operation from interfering with astronaut or camera visibility. This design also considers the many different wheel positions made possible through the use of artinuation joints that provide the steering and wheel pitching for the Enabler. The system uses a combination of brushes and fenders to restrict the dust when the vehicle is moving in either direction and in a turn. This design also allows for each of maintenance as well as accessibility of the remainder of the vehicle.

Dust control for Enabler

NASA Technical Reports Server (NTRS)

Hilton, Kevin; Karl, Chad; Litherland, Mark; Ritchie, David; Sun, Nancy

1992-01-01

The dust control group designed a system to restrict dust that is disturbed by the Enabler during its operation from interfering with astronaut or camera visibility. This design also considers the many different wheel positions made possible through the use of artinuation joints that provide the steering and wheel pitching for the Enabler. The system uses a combination of brushes and fenders to restrict the dust when the vehicle is moving in either direction and in a turn. This design also allows for ease of maintenance as well as accessibility of the remainder of the vehicle.

A standardized kit for automated quantitative assessment of candidate protein biomarkers in human plasma.

PubMed

Percy, Andrew J; Mohammed, Yassene; Yang, Juncong; Borchers, Christoph H

2015-12-01

An increasingly popular mass spectrometry-based quantitative approach for health-related research in the biomedical field involves the use of stable isotope-labeled standards (SIS) and multiple/selected reaction monitoring (MRM/SRM). To improve inter-laboratory precision and enable more widespread use of this 'absolute' quantitative technique in disease-biomarker assessment studies, methods must be standardized. Results/methodology: Using this MRM-with-SIS-peptide approach, we developed an automated method (encompassing sample preparation, processing and analysis) for quantifying 76 candidate protein markers (spanning >4 orders of magnitude in concentration) in neat human plasma. The assembled biomarker assessment kit - the 'BAK-76' - contains the essential materials (SIS mixes), methods (for acquisition and analysis), and tools (Qualis-SIS software) for performing biomarker discovery or verification studies in a rapid and standardized manner.

A Strategy for Identifying Quantitative Trait Genes Using Gene Expression Analysis and Causal Analysis.

PubMed

Ishikawa, Akira

2017-11-27

Large numbers of quantitative trait loci (QTL) affecting complex diseases and other quantitative traits have been reported in humans and model animals. However, the genetic architecture of these traits remains elusive due to the difficulty in identifying causal quantitative trait genes (QTGs) for common QTL with relatively small phenotypic effects. A traditional strategy based on techniques such as positional cloning does not always enable identification of a single candidate gene for a QTL of interest because it is difficult to narrow down a target genomic interval of the QTL to a very small interval harboring only one gene. A combination of gene expression analysis and statistical causal analysis can greatly reduce the number of candidate genes. This integrated approach provides causal evidence that one of the candidate genes is a putative QTG for the QTL. Using this approach, I have recently succeeded in identifying a single putative QTG for resistance to obesity in mice. Here, I outline the integration approach and discuss its usefulness using my studies as an example.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

PubMed

Blakemore, Robert; Nabeta, Pamela; Davidow, Amy L; Vadwai, Viral; Tahirli, Rasim; Munsamy, Vanisha; Nicol, Mark; Jones, Martin; Persing, David H; Hillemann, Doris; Ruesch-Gerdes, Sabine; Leisegang, Felicity; Zamudio, Carlos; Rodrigues, Camilla; Boehme, Catharina C; Perkins, Mark D; Alland, David

2011-11-01

The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

PubMed

Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

2006-06-15

An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

Quantitative mass spectrometry of unconventional human biological matrices

NASA Astrophysics Data System (ADS)

Dutkiewicz, Ewelina P.; Urban, Pawel L.

2016-10-01

The development of sensitive and versatile mass spectrometric methodology has fuelled interest in the analysis of metabolites and drugs in unconventional biological specimens. Here, we discuss the analysis of eight human matrices-hair, nail, breath, saliva, tears, meibum, nasal mucus and skin excretions (including sweat)-by mass spectrometry (MS). The use of such specimens brings a number of advantages, the most important being non-invasive sampling, the limited risk of adulteration and the ability to obtain information that complements blood and urine tests. The most often studied matrices are hair, breath and saliva. This review primarily focuses on endogenous (e.g. potential biomarkers, hormones) and exogenous (e.g. drugs, environmental contaminants) small molecules. The majority of analytical methods used chromatographic separation prior to MS; however, such a hyphenated methodology greatly limits analytical throughput. On the other hand, the mass spectrometric methods that exclude chromatographic separation are fast but suffer from matrix interferences. To enable development of quantitative assays for unconventional matrices, it is desirable to standardize the protocols for the analysis of each specimen and create appropriate certified reference materials. Overcoming these challenges will make analysis of unconventional human biological matrices more common in a clinical setting. This article is part of the themed issue 'Quantitative mass spectrometry'.

Quantitative Imaging with a Mobile Phone Microscope

PubMed Central

Skandarajah, Arunan; Reber, Clay D.; Switz, Neil A.; Fletcher, Daniel A.

2014-01-01

Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone–based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications. PMID:24824072

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an

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

PubMed

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

2017-12-01

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

Quantitative characterization of nanoparticle agglomeration within biological media

NASA Astrophysics Data System (ADS)

Hondow, Nicole; Brydson, Rik; Wang, Peiyi; Holton, Mark D.; Brown, M. Rowan; Rees, Paul; Summers, Huw D.; Brown, Andy

2012-07-01

Quantitative analysis of nanoparticle dispersion state within biological media is essential to understanding cellular uptake and the roles of diffusion, sedimentation, and endocytosis in determining nanoparticle dose. The dispersion of polymer-coated CdTe/ZnS quantum dots in water and cell growth medium with and without fetal bovine serum was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Characterization by TEM of samples prepared by plunge freezing the blotted solutions into liquid ethane was sensitive to the dispersion state of the quantum dots and enabled measurement of agglomerate size distributions even in the presence of serum proteins where DLS failed. In addition, TEM showed a reduced packing fraction of quantum dots per agglomerate when dispersed in biological media and serum compared to just water, highlighting the effect of interactions between the media, serum proteins, and the quantum dots. The identification of a heterogeneous distribution of quantum dots and quantum dot agglomerates in cell growth medium and serum by TEM will enable correlation with the previously reported optical metrology of in vitro cellular uptake of this quantum dot dispersion. In this paper, we present a comparative study of TEM and DLS and show that plunge-freeze TEM provides a robust assessment of nanoparticle agglomeration state.

The Quantitative Criteria Based on the Fractal Dimensions, Entropy, and Lacunarity for the Spatial Distribution of Cancer Cell Nuclei Enable Identification of Low or High Aggressive Prostate Carcinomas

PubMed Central

Waliszewski, Przemyslaw

2016-01-01

relevant information. Two novel quantitative criteria based on the complexity and the diversity measures enabled the identification of low or high aggressive prostate carcinomas and should be verified in the future multicenter, randomized studies. PMID:26903883

Quantitation of Specific Barley, Rye, and Oat Marker Peptides by Targeted Liquid Chromatography-Mass Spectrometry To Determine Gluten Concentrations.

PubMed

Schalk, Kathrin; Koehler, Peter; Scherf, Katharina Anne

2018-04-04

Celiac disease is triggered by the ingestion of gluten from wheat, barley, rye, and possibly oats. Gluten is quantitated by DNA-based methods or enzyme-linked immunosorbent assays (ELISAs). ELISAs mostly detect the prolamin fraction and potentially over- or underestimate gluten contents. Therefore, a new independent method is required to comprehensively detect gluten. A targeted liquid chromatography-tandem mass spectrometry method was developed to quantitate seven barley, seven rye, and three oat marker peptides derived from each gluten protein fraction (prolamin and glutelin) and type (barley, B-, C-, D-, and γ-hordeins; rye, γ-75k-, γ-40k-, ω-, and HMW-secalins). The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference gluten protein type resulted in peptide-specific yields, which enabled the conversion of peptide into protein concentrations. This method was applied to quantitate gluten in samples from the brewing process, in raw materials for sourdough fermentation, and in dried sourdoughs.

Quantitative assessment of RNA-protein interactions with high-throughput sequencing-RNA affinity profiling.

PubMed

Ozer, Abdullah; Tome, Jacob M; Friedman, Robin C; Gheba, Dan; Schroth, Gary P; Lis, John T

2015-08-01

Because RNA-protein interactions have a central role in a wide array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay that couples sequencing on an Illumina GAIIx genome analyzer with the quantitative assessment of protein-RNA interactions. This assay is able to analyze interactions between one or possibly several proteins with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of the EGFP and negative elongation factor subunit E (NELF-E) proteins with their corresponding canonical and mutant RNA aptamers. Here we provide a detailed protocol for HiTS-RAP that can be completed in about a month (8 d hands-on time). This includes the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, HiTS and protein binding with a GAIIx instrument, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, quantitative analysis of RNA on a massively parallel array (RNA-MaP) and RNA Bind-n-Seq (RBNS), for quantitative analysis of RNA-protein interactions.

Apricot DNA as an indicator for persipan: detection and quantitation in marzipan using ligation-dependent probe amplification.

PubMed

Luber, Florian; Demmel, Anja; Hosken, Anne; Busch, Ulrich; Engel, Karl-Heinz

2012-06-13

The confectionery ingredient marzipan is exclusively prepared from almond kernels and sugar. The potential use of apricot kernels, so-called persipan, is an important issue for the quality assessment of marzipan. Therefore, a ligation-dependent probe amplification (LPA) assay was developed that enables a specific and sensitive detection of apricot DNA, as an indicator for the presence of persipan. The limit of detection was determined to be 0.1% persipan in marzipan. The suitability of the method was confirmed by the analysis of 20 commercially available food samples. The integration of a Prunus -specific probe in the LPA assay as a reference allowed for the relative quantitation of persipan in marzipan. The limit of quantitation was determined to be 0.5% persipan in marzipan. The analysis of two self-prepared mixtures of marzipan and persipan demonstrated the applicability of the quantitation method at concentration levels of practical relevance for quality control.

A Simple and Computationally Efficient Approach to Multifactor Dimensionality Reduction Analysis of Gene-Gene Interactions for Quantitative Traits

PubMed Central

Gui, Jiang; Moore, Jason H.; Williams, Scott M.; Andrews, Peter; Hillege, Hans L.; van der Harst, Pim; Navis, Gerjan; Van Gilst, Wiek H.; Asselbergs, Folkert W.; Gilbert-Diamond, Diane

2013-01-01

We present an extension of the two-class multifactor dimensionality reduction (MDR) algorithm that enables detection and characterization of epistatic SNP-SNP interactions in the context of a quantitative trait. The proposed Quantitative MDR (QMDR) method handles continuous data by modifying MDR’s constructive induction algorithm to use a T-test. QMDR replaces the balanced accuracy metric with a T-test statistic as the score to determine the best interaction model. We used a simulation to identify the empirical distribution of QMDR’s testing score. We then applied QMDR to genetic data from the ongoing prospective Prevention of Renal and Vascular End-Stage Disease (PREVEND) study. PMID:23805232

Getting quantitative about consequences of cross-ecosystem resource subsidies on recipient consumers

USGS Publications Warehouse

Richardson, John S.; Wipfli, Mark S.

2016-01-01

Most studies of cross-ecosystem resource subsidies have demonstrated positive effects on recipient consumer populations, often with very large effect sizes. However, it is important to move beyond these initial addition–exclusion experiments to consider the quantitative consequences for populations across gradients in the rates and quality of resource inputs. In our introduction to this special issue, we describe at least four potential models that describe functional relationships between subsidy input rates and consumer responses, most of them asymptotic. Here we aim to advance our quantitative understanding of how subsidy inputs influence recipient consumers and their communities. In the papers following, fish were either the recipient consumers or the subsidy as carcasses of anadromous species. Advancing general, predictive models will enable us to further consider what other factors are potentially co-limiting (e.g., nutrients, other population interactions, physical habitat, etc.) and better integrate resource subsidies into consumer–resource, biophysical dynamics models.

Behavioral and molecular studies of quantitative differences in hygienic behavior in honeybees.

PubMed

Gempe, Tanja; Stach, Silke; Bienefeld, Kaspar; Otte, Marianne; Beye, Martin

2016-10-21

Hygienic behavior (HB) enables honeybees to tolerate parasites, including infection with the parasitic mite Varroa destructor, and it is a well-known example of a quantitative genetic trait. The understanding of the molecular processes underpinning the quantitative differences in this behavior remains limited. We performed gene expression studies in worker bees that displayed quantitative genetic differences in HB. We established a high and low genetic source of HB performance and studied the engagements into HB of single worker bees under the same environmental conditions. We found that the percentage of worker bees that engaged in a hygienic behavioral task tripled in the high versus low HB sources, thus suggesting that genetic differences may mediate differences in stimulated states to perform HB. We found 501 differently expressed genes (DEGs) in the brains of hygienic and non-hygienic performing workers in the high HB source bees, and 342 DEGs in the brains of hygienic performing worker bees, relative to the gene expression in non-hygienic worker bees from the low HB source group. "Cell surface receptor ligand signal transduction" in the high and "negative regulation of cell communication" in the low HB source were overrepresented molecular processes, suggesting that these molecular processes in the brain may play a role in the regulation of quantitative differences in HB. Moreover, only 21 HB-associated DEGs were common between the high and low HB sources. The better HB colony performance is primarily achieved by a high number of bees engaging in the hygienic tasks that associate with distinct molecular processes in the brain. We propose that different gene products and pathways may mediate the quantitative genetic differences of HB.

Technical Note: Quantitative dynamic contrast-enhanced MRI of a 3-dimensional artificial capillary network.

PubMed

Gaass, Thomas; Schneider, Moritz Jörg; Dietrich, Olaf; Ingrisch, Michael; Dinkel, Julien

2017-04-01

Variability across devices, patients, and time still hinders widespread recognition of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) as quantitative biomarker. The purpose of this work was to introduce and characterize a dedicated microchannel phantom as a model for quantitative DCE-MRI measurements. A perfusable, MR-compatible microchannel network was constructed on the basis of sacrificial melt-spun sugar fibers embedded in a block of epoxy resin. Structural analysis was performed on the basis of light microscopy images before DCE-MRI experiments. During dynamic acquisition the capillary network was perfused with a standard contrast agent injection system. Flow-dependency, as well as inter- and intrascanner reproducibility of the computed DCE parameters were evaluated using a 3.0 T whole-body MRI. Semi-quantitative and quantitative flow-related parameters exhibited the expected proportionality to the set flow rate (mean Pearson correlation coefficient: 0.991, P < 2.5e-5). The volume fraction was approximately independent from changes of the applied flow rate through the phantom. Repeatability and reproducibility experiments yielded maximum intrascanner coefficients of variation (CV) of 4.6% for quantitative parameters. All evaluated parameters were well in the range of known in vivo results for the applied flow rates. The constructed phantom enables reproducible, flow-dependent, contrast-enhanced MR measurements with the potential to facilitate standardization and comparability of DCE-MRI examinations. © 2017 American Association of Physicists in Medicine.

Genome-Enabled Molecular Tools for Reductive Dehalogenation

DTIC Science & Technology

2011-11-01

Genome-Enabled Molecular Tools for Reductive Dehalogenation - A Shift in Paradigm for Bioremediation - Alfred M. Spormann Departments of Chemical...Genome-Enabled Molecular Tools for Reductive Dehalogenation 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...Applications Technical Session No. 3D C-77 GENOME-ENABLED MOLECULAR TOOLS FOR REDUCTIVE DEHALOGENATION PROFESSOR ALFRED SPORMANN Stanford

Microsystems Enabled Photovoltaics

ScienceCinema

Gupta, Vipin; Nielson, Greg; Okandan, Murat, Granata, Jennifer; Nelson, Jeff; Haney, Mike; Cruz-Campa, Jose Luiz

2018-06-07

Sandia's microsystems enabled photovoltaic advances combine mature technology and tools currently used in microsystem production with groundbreaking advances in photovoltaics cell design, decreasing production and system costs while improving energy conversion efficiency. The technology has potential applications in buildings, houses, clothing, portable electronics, vehicles, and other contoured structures.

Quantitative impact characterization of aeronautical CFRP materials with non-destructive testing methods

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

Kiefel, Denis, E-mail: Denis.Kiefel@airbus.com, E-mail: Rainer.Stoessel@airbus.com; Stoessel, Rainer, E-mail: Denis.Kiefel@airbus.com, E-mail: Rainer.Stoessel@airbus.com; Grosse, Christian, E-mail: Grosse@tum.de

2015-03-31

In recent years, an increasing number of safety-relevant structures are designed and manufactured from carbon fiber reinforced polymers (CFRP) in order to reduce weight of airplanes by taking the advantage of their specific strength into account. Non-destructive testing (NDT) methods for quantitative defect analysis of damages are liquid- or air-coupled ultrasonic testing (UT), phased array ultrasonic techniques, and active thermography (IR). The advantage of these testing methods is the applicability on large areas. However, their quantitative information is often limited on impact localization and size. In addition to these techniques, Airbus Group Innovations operates a micro x-ray computed tomography (μ-XCT)more » system, which was developed for CFRP characterization. It is an open system which allows different kinds of acquisition, reconstruction, and data evaluation. One main advantage of this μ-XCT system is its high resolution with 3-dimensional analysis and visualization opportunities, which enables to gain important quantitative information for composite part design and stress analysis. Within this study, different NDT methods will be compared at CFRP samples with specified artificial impact damages. The results can be used to select the most suitable NDT-method for specific application cases. Furthermore, novel evaluation and visualization methods for impact analyzes are developed and will be presented.« less

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy.

PubMed

Cheng, Cynthia; Lee, Chadd W; Daskalakis, Constantine

2015-10-27

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient's microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.(1) This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique.

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

PubMed Central

Daskalakis, Constantine

2015-01-01

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient’s microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.1 This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique. PMID:26554744

PIQMIe: a web server for semi-quantitative proteomics data management and analysis.

PubMed

Kuzniar, Arnold; Kanaar, Roland

2014-07-01

We present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates peptide and (non-redundant) protein identifications and quantitations from multiple experiments with additional biological information on the protein entries, and makes the linked data available in the form of a light-weight relational database, which enables dedicated data analyses (e.g. in R) and user-driven queries. Using the web interface, users are presented with a concise summary of their proteomics experiments in numerical and graphical forms, as well as with a searchable protein grid and interactive visualization tools to aid in the rapid assessment of the experiments and in the identification of proteins of interest. The web server not only provides data access through a web interface but also supports programmatic access through RESTful web service. The web server is available at http://piqmie.semiqprot-emc.cloudlet.sara.nl or http://www.bioinformatics.nl/piqmie. This website is free and open to all users and there is no login requirement. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Connecting qualitative observation and quantitative measurement for enhancing quantitative literacy in plant anatomy course

NASA Astrophysics Data System (ADS)

Nuraeni, E.; Rahmat, A.

2018-05-01

Forming of cognitive schemes of plant anatomy concepts is performed by processing of qualitative and quantitative data obtained from microscopic observations. To enhancing student’s quantitative literacy, strategy of plant anatomy course was modified by adding the task to analyze quantitative data produced by quantitative measurement of plant anatomy guided by material course. Participant in this study was 24 biology students and 35 biology education students. Quantitative Literacy test, complex thinking in plant anatomy test and questioner used to evaluate the course. Quantitative literacy capability data was collected by quantitative literacy test with the rubric from the Association of American Colleges and Universities, Complex thinking in plant anatomy by test according to Marzano and questioner. Quantitative literacy data are categorized according to modified Rhodes and Finley categories. The results showed that quantitative literacy of biology education students is better than biology students.

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography.

PubMed

Ruprecht, Benjamin; Koch, Heiner; Domasinska, Petra; Frejno, Martin; Kuster, Bernhard; Lemeer, Simone

2017-01-01

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO 2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

Commentary: Academic Enablers and School Learning.

ERIC Educational Resources Information Center

Keith, Timothy Z.

2002-01-01

This commentary presents academic enablers within the broader, overlapping context of school learning theory, including the theories of Carroll, Harnishfeger and Wiley, Walberg, and others. Multivariate models are needed to understand the influences of academic enabler and school learning variables on learning, as well as the influences of these…

Electronic Health Record Application Support Service Enablers.

PubMed

Neofytou, M S; Neokleous, K; Aristodemou, A; Constantinou, I; Antoniou, Z; Schiza, E C; Pattichis, C S; Schizas, C N

2015-08-01

There is a huge need for open source software solutions in the healthcare domain, given the flexibility, interoperability and resource savings characteristics they offer. In this context, this paper presents the development of three open source libraries - Specific Enablers (SEs) for eHealth applications that were developed under the European project titled "Future Internet Social and Technological Alignment Research" (FI-STAR) funded under the "Future Internet Public Private Partnership" (FI-PPP) program. The three SEs developed under the Electronic Health Record Application Support Service Enablers (EHR-EN) correspond to: a) an Electronic Health Record enabler (EHR SE), b) a patient summary enabler based on the EU project "European patient Summary Open Source services" (epSOS SE) supporting patient mobility and the offering of interoperable services, and c) a Picture Archiving and Communications System (PACS) enabler (PACS SE) based on the dcm4che open source system for the support of medical imaging functionality. The EHR SE follows the HL7 Clinical Document Architecture (CDA) V2.0 and supports the Integrating the Healthcare Enterprise (IHE) profiles (recently awarded in Connectathon 2015). These three FI-STAR platform enablers are designed to facilitate the deployment of innovative applications and value added services in the health care sector. They can be downloaded from the FI-STAR cataloque website. Work in progress focuses in the validation and evaluation scenarios for the proving and demonstration of the usability, applicability and adaptability of the proposed enablers.

Calibration methods influence quantitative material decomposition in photon-counting spectral CT

NASA Astrophysics Data System (ADS)

Curtis, Tyler E.; Roeder, Ryan K.

2017-03-01

Photon-counting detectors and nanoparticle contrast agents can potentially enable molecular imaging and material decomposition in computed tomography (CT). Material decomposition has been investigated using both simulated and acquired data sets. However, the effect of calibration methods on material decomposition has not been systematically investigated. Therefore, the objective of this study was to investigate the influence of the range and number of contrast agent concentrations within a modular calibration phantom on quantitative material decomposition. A commerciallyavailable photon-counting spectral micro-CT (MARS Bioimaging) was used to acquire images with five energy bins selected to normalize photon counts and leverage the contrast agent k-edge. Material basis matrix values were determined using multiple linear regression models and material decomposition was performed using a maximum a posteriori estimator. The accuracy of quantitative material decomposition was evaluated by the root mean squared error (RMSE), specificity, sensitivity, and area under the curve (AUC). An increased maximum concentration (range) in the calibration significantly improved RMSE, specificity and AUC. The effects of an increased number of concentrations in the calibration were not statistically significant for the conditions in this study. The overall results demonstrated that the accuracy of quantitative material decomposition in spectral CT is significantly influenced by calibration methods, which must therefore be carefully considered for the intended diagnostic imaging application.

Quantitative fluorescence nanoscopy for cancer biomedicine

NASA Astrophysics Data System (ADS)

Huang, Tao; Nickerson, Andrew; Peters, Alec; Nan, Xiaolin

2015-08-01

Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.

PubMed

Kline, Margaret C; Duewer, David L; Travis, John C; Smith, Melody V; Redman, Janette W; Vallone, Peter M; Decker, Amy E; Butler, John M

2009-06-01

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.

Quantitative stability of hematopoietic stem and progenitor cell clonal output in rhesus macaques receiving transplants

PubMed Central

Koelle, Samson J.

2017-01-01

Autologous transplantation of hematopoietic stem and progenitor cells lentivirally labeled with unique oligonucleotide barcodes flanked by sequencing primer targets enables quantitative assessment of the self-renewal and differentiation patterns of these cells in a myeloablative rhesus macaque model. Compared with other approaches to clonal tracking, this approach is highly quantitative and reproducible. We documented stable multipotent long-term hematopoietic clonal output of monocytes, granulocytes, B cells, and T cells from a polyclonal pool of hematopoietic stem and progenitor cells in 4 macaques observed for up to 49 months posttransplantation. A broad range of clonal behaviors characterized by contribution level and biases toward certain cell types were extremely stable over time. Correlations between granulocyte and monocyte clonalities were greatest, followed by correlations between these cell types and B cells. We also detected quantitative expansion of T cell–biased clones consistent with an adaptive immune response. In contrast to recent data from a nonquantitative murine model, there was little evidence for clonal succession after initial hematopoietic reconstitution. These findings have important implications for human hematopoiesis, given the similarities between macaque and human physiologies. PMID:28087539

GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.

PubMed

Halvey, Patrick J; Ferrone, Cristina R; Liebler, Daniel C

2012-07-06

Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 μg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/μg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.

Label-free imaging of intracellular motility by low-coherent quantitative phase microscope in reflection geometry

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2011-11-01

We demonstrate tomographic imaging of intracellular activity of living cells by a low-coherent quantitative phase microscope. The intracellular organelles, such as the nucleus, nucleolus, and mitochondria, are moving around inside living cells, driven by the cellular physiological activity. In order to visualize the intracellular motility in a label-free manner we have developed a reflection-type quantitative phase microscope which employs the phase shifting interferometric technique with a low-coherent light source. The phase shifting interferometry enables us to quantitatively measure the intensity and phase of the optical field, and the low-coherence interferometry makes it possible to selectively probe a specific sectioning plane in the cell volume. The results quantitatively revealed the depth-resolved fluctuations of intracellular surfaces so that the plasma membrane and the membranes of intracellular organelles were independently measured. The transversal and the vertical spatial resolutions were 0.56 μm and 0.93 μm, respectively, and the mechanical sensitivity of the phase measurement was 1.2 nanometers. The mean-squared displacement was applied as a statistical tool to analyze the temporal fluctuation of the intracellular organelles. To the best of our knowledge, our system visualized depth-resolved intracellular organelles motion for the first time in sub-micrometer resolution without contrast agents.

'Ethos' Enabling Organisational Knowledge Creation

NASA Astrophysics Data System (ADS)

Matsudaira, Yoshito

This paper examines knowledge creation in relation to improvements on the production line in the manufacturing department of Nissan Motor Company and aims to clarify embodied knowledge observed in the actions of organisational members who enable knowledge creation will be clarified. For that purpose, this study adopts an approach that adds a first, second, and third-person's viewpoint to the theory of knowledge creation. Embodied knowledge, observed in the actions of organisational members who enable knowledge creation, is the continued practice of 'ethos' (in Greek) founded in Nissan Production Way as an ethical basis. Ethos is knowledge (intangible) assets for knowledge creating companies. Substantiated analysis classifies ethos into three categories: the individual, team and organisation. This indicates the precise actions of the organisational members in each category during the knowledge creation process. This research will be successful in its role of showing the indispensability of ethos - the new concept of knowledge assets, which enables knowledge creation -for future knowledge-based management in the knowledge society.

Global Monitoring of the Mammalian Lipidome by Quantitative Shotgun Lipidomics.

PubMed

Nielsen, Inger Ødum; Maeda, Kenji; Bilgin, Mesut

2017-01-01

The emerging field of lipidomics presents the systems biology approach to identify and quantify the full lipid repertoire of cells, tissues, and organisms. The importance of the lipidome is demonstrated by a number of biological studies on dysregulation of lipid metabolism in human diseases such as cancer, diabetes, and neurodegenerative diseases. Exploring changes and regulations in the huge networks of lipids and their metabolic pathways requires a lipidomics methodology: Advanced mass spectrometry that resolves the complexity of the lipidome. Here, we report a comprehensive protocol of quantitative shotgun lipidomics that enables identification and quantification of hundreds of molecular lipid species, covering a wide range of lipid classes, extracted from cultured mammalian cells.

Some selected quantitative methods of thermal image analysis in Matlab.

PubMed

Koprowski, Robert

2016-05-01

The paper presents a new algorithm based on some selected automatic quantitative methods for analysing thermal images. It shows the practical implementation of these image analysis methods in Matlab. It enables to perform fully automated and reproducible measurements of selected parameters in thermal images. The paper also shows two examples of the use of the proposed image analysis methods for the area of ​​the skin of a human foot and face. The full source code of the developed application is also provided as an attachment. The main window of the program during dynamic analysis of the foot thermal image. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Agile SE Enablers and Quantification Project: Identification, Characterization, and Evaluation Criteria for Systems Engineering Agile Enablers

DTIC Science & Technology

2015-01-16

Enablers Draft Technical Report SERC -2015-049-1 January 16, 2015 Principal Investigator: Dr. Richard Turner, Stevens Institute of...Hudson, Hoboken, NJ 07030 1 Copyright © 2015 Stevens Institute of Technology The Systems Engineering Research Center ( SERC ) is a federally...inappropriate enablers are not pursued. The identification criteria developed for RT-124 are based on earlier SERC work. [4, 5, 6]: 1 Operated by DAU

Quantitative research.

PubMed

Watson, Roger

2015-04-01

This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

The Digital Thread as the Key Enabler

DTIC Science & Technology

2016-11-01

17 Defense AT&L: November-December 2016 The Digital Thread as the Key Enabler Col. Keith Bearden, USAF Bearden is the deputy director of...enabling you to do your job better, faster and cheaper. There is one initiative, the key enabler, to accomplish this goal—the digital thread . But let’s... process that would allow for rapid cross- domain analysis and technology transition prior to bending metal. • Re-establish a culture of “hands-on

Reorientation of health services: enablers and barriers faced by organisations when increasing health promotion capacity.

PubMed

McFarlane, K; Judd, J; Devine, S; Watt, K

2016-08-01

Issue addressed Primary healthcare settings are important providers of health promotion approaches. However, organisational challenges can affect their capacity to deliver these approaches. This review identified the common enablers and barriers health organisations faced and it aimed to explore the experiences health organisations, in particular Aboriginal organisations, had when increasing their health promotion capacity. Methods A systematic search of peer-reviewed literature was conducted. Articles published between 1990-2014 that focused on a health care-settings approach and discussed factors that facilitated or hindered an organisation's ability to increase health promotion capacity were included. Results Twenty-five articles met the inclusion criteria. Qualitative (n=18) and quantitative (n=7) study designs were included. Only one article described the experiences of an Aboriginal health organisation. Enablers included: management support, skilled staff, provision of external support to the organisation, committed staffing and financial resources, leadership and the availability of external partners to work with. Barriers included: lack of management support, lack of dedicated health promotion staff, staff lacking skills or confidence, competing priorities and a lack of time and resources allocated to health promotion activities. Conclusions While the literature highlighted the importance of health promotion work, barriers can limit the delivery of health promotion approaches within primary healthcare organisations. A gap in the literature exists about how Aboriginal health organisations face these challenges. So what? Primary healthcare organisations wanting to increase their health promotion capacity can pre-empt the common barriers and strengthen identified enablers through the shared learnings outlined in this review.

MsViz: A Graphical Software Tool for In-Depth Manual Validation and Quantitation of Post-translational Modifications.

PubMed

Martín-Campos, Trinidad; Mylonas, Roman; Masselot, Alexandre; Waridel, Patrice; Petricevic, Tanja; Xenarios, Ioannis; Quadroni, Manfredo

2017-08-04

Mass spectrometry (MS) has become the tool of choice for the large scale identification and quantitation of proteins and their post-translational modifications (PTMs). This development has been enabled by powerful software packages for the automated analysis of MS data. While data on PTMs of thousands of proteins can nowadays be readily obtained, fully deciphering the complexity and combinatorics of modification patterns even on a single protein often remains challenging. Moreover, functional investigation of PTMs on a protein of interest requires validation of the localization and the accurate quantitation of its changes across several conditions, tasks that often still require human evaluation. Software tools for large scale analyses are highly efficient but are rarely conceived for interactive, in-depth exploration of data on individual proteins. We here describe MsViz, a web-based and interactive software tool that supports manual validation of PTMs and their relative quantitation in small- and medium-size experiments. The tool displays sequence coverage information, peptide-spectrum matches, tandem MS spectra and extracted ion chromatograms through a single, highly intuitive interface. We found that MsViz greatly facilitates manual data inspection to validate PTM location and quantitate modified species across multiple samples.

Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

PubMed

Awasthi, Shivangi; Maity, Tapan; Oyler, Benjamin L; Qi, Yue; Zhang, Xu; Goodlett, David R; Guha, Udayan

2018-04-13

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR L858R mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR L858R/T790M mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and

A custom-built PET phantom design for quantitative imaging of printed distributions.

PubMed

Markiewicz, P J; Angelis, G I; Kotasidis, F; Green, M; Lionheart, W R; Reader, A J; Matthews, J C

2011-11-07

This note presents a practical approach to a custom-made design of PET phantoms enabling the use of digital radioactive distributions with high quantitative accuracy and spatial resolution. The phantom design allows planar sources of any radioactivity distribution to be imaged in transaxial and axial (sagittal or coronal) planes. Although the design presented here is specially adapted to the high-resolution research tomograph (HRRT), the presented methods can be adapted to almost any PET scanner. Although the presented phantom design has many advantages, a number of practical issues had to be overcome such as positioning of the printed source, calibration, uniformity and reproducibility of printing. A well counter (WC) was used in the calibration procedure to find the nonlinear relationship between digital voxel intensities and the actual measured radioactive concentrations. Repeated printing together with WC measurements and computed radiography (CR) using phosphor imaging plates (IP) were used to evaluate the reproducibility and uniformity of such printing. Results show satisfactory printing uniformity and reproducibility; however, calibration is dependent on the printing mode and the physical state of the cartridge. As a demonstration of the utility of using printed phantoms, the image resolution and quantitative accuracy of reconstructed HRRT images are assessed. There is very good quantitative agreement in the calibration procedure between HRRT, CR and WC measurements. However, the high resolution of CR and its quantitative accuracy supported by WC measurements made it possible to show the degraded resolution of HRRT brain images caused by the partial-volume effect and the limits of iterative image reconstruction.

Recent advances on multidimensional liquid chromatography-mass spectrometry for proteomics: from qualitative to quantitative analysis--a review.

PubMed

Wu, Qi; Yuan, Huiming; Zhang, Lihua; Zhang, Yukui

2012-06-20

With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography-mass spectrometry (MDLC-MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including "top-down" and "bottom-up" to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Multistrip western blotting to increase quantitative data output.

PubMed

Kiyatkin, Anatoly; Aksamitiene, Edita

2009-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.

A Targeted LC-MS/MS Method for the Simultaneous Detection and Quantitation of Egg, Milk, and Peanut Allergens in Sugar Cookies.

PubMed

Boo, Chelsea C; Parker, Christine H; Jackson, Lauren S

2018-01-01

Food allergy is a growing public health concern, with many individuals reporting allergies to multiple food sources. Compliance with food labeling regulations and prevention of inadvertent cross-contact in manufacturing requires the use of reliable methods for the detection and quantitation of allergens in processed foods. In this work, a novel liquid chromatography-tandem mass spectrometry multiple-reaction monitoring method for multiallergen detection and quantitation of egg, milk, and peanut was developed and evaluated in an allergen-incurred baked sugar cookie matrix. A systematic evaluation of method parameters, including sample extraction, concentration, and digestion, were optimized for candidate allergen peptide markers. The optimized method enabled the reliable detection and quantitation of egg, milk, and peanut allergens in sugar cookies, with allergen concentrations as low as 5 ppm allergen-incurred ingredient.

Quantitative chemical imaging with background-free multiplex coherent anti-Stokes Raman scattering by dual-soliton Stokes pulses

PubMed Central

Chen, Kun; Wu, Tao; Wei, Haoyun; Zhou, Tian; Li, Yan

2016-01-01

Coherent anti-Stokes Raman microscopy (CARS) is a quantitative, chemically specific, and label-free optical imaging technique for studying inhomogeneous systems. However, the complicating influence of the nonresonant response on the CARS signal severely limits its sensitivity and specificity and especially limits the extent to which CARS microscopy has been used as a fully quantitative imaging technique. On the basis of spectral focusing mechanism, we establish a dual-soliton Stokes based CARS microspectroscopy and microscopy scheme capable of quantifying the spatial information of densities and chemical composition within inhomogeneous samples, using a single fiber laser. Dual-soliton Stokes scheme not only removes the nonresonant background but also allows robust acquisition of multiple characteristic vibrational frequencies. This all-fiber based laser source can cover the entire fingerprint (800-2200 cm−1) region with a spectral resolution of 15 cm−1. We demonstrate that quantitative degree determination of lipid-chain unsaturation in the fatty acids mixture can be achieved by the characterization of C = C stretching and CH2 deformation vibrations. For microscopy purposes, we show that the spatially inhomogeneous distribution of lipid droplets can be further quantitatively visualized using this quantified degree of lipid unsaturation in the acyl chain for contrast in the hyperspectral CARS images. The combination of compact excitation source and background-free capability to facilitate extraction of quantitative composition information with multiplex spectral peaks will enable wider applications of quantitative chemical imaging in studying biological and material systems. PMID:27867704

Apparatus and method for quantitative assay of generic transuranic wastes from nuclear reactors

DOEpatents

Caldwell, J.T.; Kunz, W.E.; Atencio, J.D.

1982-03-31

A combination of passive and active neutron measurements which yields quantitative information about the isotopic composition of transuranic wastes from nuclear power or weapons material manufacture reactors is described. From the measurement of prompt and delayed neutron emission and the incidence of two coincidentally emitted neutrons from induced fission of fissile material in the sample, one can quantify /sup 233/U, /sup 235/U and /sup 239/Pu isotopes in waste samples. Passive coincidence counting, including neutron multiplicity measurement and determination of the overall passive neutron flux additionally enables the separate quantitative evaluation of spontaneous fission isotopes such as /sup 240/Pu, /sup 244/Cm and /sup 252/Cf, and the spontaneous alpha particle emitter /sup 241/Am. These seven isotopes are the most important constituents of wastes from nuclear power reactors and once the mass of each isotope present is determined by the apparatus and method of the instant invention, the overall alpha particle activity can be determined to better than 1 nCi/g from known radioactivity data. Therefore, in addition to the quantitative analysis of the waste sample useful for later reclamation purposes, the alpha particle activity can be determined to decide whether permanent low-level burial is appropriate for the waste sample.

Apparatus and method for quantitative assay of generic transuranic wastes from nuclear reactors

DOEpatents

Caldwell, John T.; Kunz, Walter E.; Atencio, James D.

1984-01-01

A combination of passive and active neutron measurements which yields quantitative information about the isotopic composition of transuranic wastes from nuclear power or weapons material manufacture reactors is described. From the measurement of prompt and delayed neutron emission and the incidence of two coincidentally emitted neutrons from induced fission of fissile material in the sample, one can quantify .sup.233 U, .sup.235 U and .sup.239 Pu isotopes in waste samples. Passive coincidence counting, including neutron multiplicity measurement and determination of the overall passive neutron flux additionally enables the separate quantitative evaluation of spontaneous fission isotopes such as .sup.240 Pu, .sup.244 Cm and .sup.252 Cf, and the spontaneous alpha particle emitter .sup.241 Am. These seven isotopes are the most important constituents of wastes from nuclear power reactors and once the mass of each isotope present is determined by the apparatus and method of the instant invention, the overall alpha particle activity can be determined to better than 1 nCi/g from known radioactivity data. Therefore, in addition to the quantitative analysis of the waste sample useful for later reclamation purposes, the alpha particle activity can be determined to decide whether "permanent" low-level burial is appropriate for the waste sample.

Nanoscale nuclear architecture for cancer diagnosis beyond pathology via spatial-domain low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Uttam, Shikhar; Staton, Kevin; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

2010-11-01

Definitive diagnosis of malignancy is often challenging due to limited availability of human cell or tissue samples and morphological similarity with certain benign conditions. Our recently developed novel technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM)-overcomes the technical difficulties and enables us to obtain quantitative information about cell nuclear architectural characteristics with nanoscale sensitivity. We explore its ability to improve the identification of malignancy, especially in cytopathologically non-cancerous-appearing cells. We perform proof-of-concept experiments with an animal model of colorectal carcinogenesis-APCMin mouse model and human cytology specimens of colorectal cancer. We show the ability of in situ nanoscale nuclear architectural characteristics in identifying cancerous cells, especially in those labeled as ``indeterminate or normal'' by expert cytopathologists. Our approach is based on the quantitative analysis of the cell nucleus on the original cytology slides without additional processing, which can be readily applied in a conventional clinical setting. Our simple and practical optical microscopy technique may lead to the development of novel methods for early detection of cancer.

Charting organellar importomes by quantitative mass spectrometry

PubMed Central

Peikert, Christian D.; Mani, Jan; Morgenstern, Marcel; Käser, Sandro; Knapp, Bettina; Wenger, Christoph; Harsman, Anke; Oeljeklaus, Silke; Schneider, André; Warscheid, Bettina

2017-01-01

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles. PMID:28485388

Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast

PubMed Central

Pang, Wei; Coghill, George M.

2015-01-01

In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. PMID:25864377

Rigour in quantitative research.

PubMed

Claydon, Leica Sarah

2015-07-22

This article which forms part of the research series addresses scientific rigour in quantitative research. It explores the basis and use of quantitative research and the nature of scientific rigour. It examines how the reader may determine whether quantitative research results are accurate, the questions that should be asked to determine accuracy and the checklists that may be used in this process. Quantitative research has advantages in nursing, since it can provide numerical data to help answer questions encountered in everyday practice.

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.

PubMed

von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana

2013-10-01

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

Quantitative microbiome profiling links gut community variation to microbial load.

PubMed

Vandeputte, Doris; Kathagen, Gunter; D'hoe, Kevin; Vieira-Silva, Sara; Valles-Colomer, Mireia; Sabino, João; Wang, Jun; Tito, Raul Y; De Commer, Lindsey; Darzi, Youssef; Vermeire, Séverine; Falony, Gwen; Raes, Jeroen

2017-11-23

Current sequencing-based analyses of faecal microbiota quantify microbial taxa and metabolic pathways as fractions of the sample sequence library generated by each analysis. Although these relative approaches permit detection of disease-associated microbiome variation, they are limited in their ability to reveal the interplay between microbiota and host health. Comparative analyses of relative microbiome data cannot provide information about the extent or directionality of changes in taxa abundance or metabolic potential. If microbial load varies substantially between samples, relative profiling will hamper attempts to link microbiome features to quantitative data such as physiological parameters or metabolite concentrations. Saliently, relative approaches ignore the possibility that altered overall microbiota abundance itself could be a key identifier of a disease-associated ecosystem configuration. To enable genuine characterization of host-microbiota interactions, microbiome research must exchange ratios for counts. Here we build a workflow for the quantitative microbiome profiling of faecal material, through parallelization of amplicon sequencing and flow cytometric enumeration of microbial cells. We observe up to tenfold differences in the microbial loads of healthy individuals and relate this variation to enterotype differentiation. We show how microbial abundances underpin both microbiota variation between individuals and covariation with host phenotype. Quantitative profiling bypasses compositionality effects in the reconstruction of gut microbiota interaction networks and reveals that the taxonomic trade-off between Bacteroides and Prevotella is an artefact of relative microbiome analyses. Finally, we identify microbial load as a key driver of observed microbiota alterations in a cohort of patients with Crohn's disease, here associated with a low-cell-count Bacteroides enterotype (as defined through relative profiling).

Frequency Modulation of Transcriptional Bursting Enables Sensitive and Rapid Gene Regulation.

PubMed

Li, Congxin; Cesbron, François; Oehler, Michael; Brunner, Michael; Höfer, Thomas

2018-04-25

Gene regulation is a complex non-equilibrium process. Here, we show that quantitating the temporal regulation of key gene states (transcriptionally inactive, active, and refractory) provides a parsimonious framework for analyzing gene regulation. Our theory makes two non-intuitive predictions. First, for transcription factors (TFs) that regulate transcription burst frequency, as opposed to amplitude or duration, weak TF binding is sufficient to elicit strong transcriptional responses. Second, refractoriness of a gene after a transcription burst enables rapid responses to stimuli. We validate both predictions experimentally by exploiting the natural, optogenetic-like responsiveness of the Neurospora GATA-type TF White Collar Complex (WCC) to blue light. Further, we demonstrate that differential regulation of WCC target genes is caused by different gene activation rates, not different TF occupancy, and that these rates are tuned by both the core promoter and the distance between TF-binding site and core promoter. In total, our work demonstrates the relevance of a kinetic, non-equilibrium framework for understanding transcriptional regulation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

Critical Quantitative Inquiry in Context

ERIC Educational Resources Information Center

Stage, Frances K.; Wells, Ryan S.

2014-01-01

This chapter briefly traces the development of the concept of critical quantitative inquiry, provides an expanded conceptualization of the tasks of critical quantitative research, offers theoretical explanation and justification for critical research using quantitative methods, and previews the work of quantitative criticalists presented in this…

Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast.

PubMed

Pang, Wei; Coghill, George M

2015-05-01

In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Quantitative dispersion microscopy

PubMed Central

Fu, Dan; Choi, Wonshik; Sung, Yongjin; Yaqoob, Zahid; Dasari, Ramachandra R.; Feld, Michael

2010-01-01

Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples. PMID:21113234

Using Computational Toxicology to Enable Risk-Based ...

EPA Pesticide Factsheets

presentation at Drug Safety Gordon Research Conference 2016 on research efforts in NCCT to enable Computational Toxicology to support risk assessment. Slide presentation at Drug Safety Gordon Research Conference 2016 on research efforts in NCCT to enable Computational Toxicology to support risk assessment.

P-MartCancer-Interactive Online Software to Enable Analysis of Shotgun Cancer Proteomic Datasets.

PubMed

Webb-Robertson, Bobbie-Jo M; Bramer, Lisa M; Jensen, Jeffrey L; Kobold, Markus A; Stratton, Kelly G; White, Amanda M; Rodland, Karin D

2017-11-01

P-MartCancer is an interactive web-based software environment that enables statistical analyses of peptide or protein data, quantitated from mass spectrometry-based global proteomics experiments, without requiring in-depth knowledge of statistical programming. P-MartCancer offers a series of statistical modules associated with quality assessment, peptide and protein statistics, protein quantification, and exploratory data analyses driven by the user via customized workflows and interactive visualization. Currently, P-MartCancer offers access and the capability to analyze multiple cancer proteomic datasets generated through the Clinical Proteomics Tumor Analysis Consortium at the peptide, gene, and protein levels. P-MartCancer is deployed as a web service (https://pmart.labworks.org/cptac.html), alternatively available via Docker Hub (https://hub.docker.com/r/pnnl/pmart-web/). Cancer Res; 77(21); e47-50. ©2017 AACR . ©2017 American Association for Cancer Research.

Biometrics Enabling Capability Increment 1 (BEC Inc 1)

DTIC Science & Technology

2016-03-01

2016 Major Automated Information System Annual Report Biometrics Enabling Capability Increment 1 (BEC Inc 1) Defense Acquisition Management...Phone: 227-3119 DSN Fax: Date Assigned: July 15, 2015 Program Information Program Name Biometrics Enabling Capability Increment 1 (BEC Inc 1) DoD...therefore, no Original Estimate has been established. BEC Inc 1 2016 MAR UNCLASSIFIED 4 Program Description The Biometrics Enabling Capability (BEC

Collaborating to improve the use of free-energy and other quantitative methods in drug discovery

NASA Astrophysics Data System (ADS)

Sherborne, Bradley; Shanmugasundaram, Veerabahu; Cheng, Alan C.; Christ, Clara D.; DesJarlais, Renee L.; Duca, Jose S.; Lewis, Richard A.; Loughney, Deborah A.; Manas, Eric S.; McGaughey, Georgia B.; Peishoff, Catherine E.; van Vlijmen, Herman

2016-12-01

In May and August, 2016, several pharmaceutical companies convened to discuss and compare experiences with Free Energy Perturbation (FEP). This unusual synchronization of interest was prompted by Schrödinger's FEP+ implementation and offered the opportunity to share fresh studies with FEP and enable broader discussions on the topic. This article summarizes key conclusions of the meetings, including a path forward of actions for this group to aid the accelerated evaluation, application and development of free energy and related quantitative, structure-based design methods.

Barriers to and enablers of implementing antimicrobial stewardship programs in veterinary practices.

PubMed

Hardefeldt, Laura Y; Gilkerson, J R; Billman-Jacobe, H; Stevenson, M A; Thursky, K; Bailey, K E; Browning, G F

2018-03-23

Antimicrobial stewardship (AMS) programs are yet to be widely implemented in veterinary practice and medical programs are unlikely to be directly applicable to veterinary settings. To gain an in-depth understanding of the factors that influence effective AMS in veterinary practices in Australia. A concurrent explanatory mixed methods design was used. The quantitative phase of the study consisted of an online questionnaire to assess veterinarians' attitudes to antimicrobial resistance (AMR) and antimicrobial use in animals, and the extent to which AMS currently is implemented (knowingly or unknowingly). The qualitative phase used semi-structured interviews to gain an understanding of the barriers to and enablers of AMS in veterinary practices. Data were collected and entered into NVivo v.11, openly coded and analyzed according to mixed methods data analysis principles. Companion animal, equine, and bovine veterinarians participated in the study. Veterinary practices rarely had antimicrobial prescribing policies. The key barriers were a lack of AMS governance structures, client expectations and competition between practices, cost of microbiological testing, and lack of access to education, training and AMS resources. The enablers were concern for the role of veterinary antimicrobial use in development of AMR in humans, a sense of pride in the service provided, and preparedness to change prescribing practices. Our study can guide development and establishment of AMS programs in veterinary practices by defining the major issues that influence the prescribing behavior of veterinarians. © 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

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

Transient deformation of a droplet near a microfluidic constriction: A quantitative analysis

NASA Astrophysics Data System (ADS)

Trégouët, Corentin; Salez, Thomas; Monteux, Cécile; Reyssat, Mathilde

2018-05-01

We report on experiments that consist of deforming a collection of monodisperse droplets produced by a microfluidic chip through a flow-focusing device. We show that a proper numerical modeling of the flow is necessary to access the stress applied by the latter on the droplet along its trajectory through the chip. This crucial step enables the full integration of the differential equation governing the dynamical deformation, and consequently the robust measurement of the interfacial tension by fitting the experiments with the calculated deformation. Our study thus demonstrates the feasibility of quantitative in situ rheology in microfluidic flows involving, e.g., droplets, capsules, or cells.

New Generation Sensor Web Enablement

PubMed Central

Bröring, Arne; Echterhoff, Johannes; Jirka, Simon; Simonis, Ingo; Everding, Thomas; Stasch, Christoph; Liang, Steve; Lemmens, Rob

2011-01-01

Many sensor networks have been deployed to monitor Earth’s environment, and more will follow in the future. Environmental sensors have improved continuously by becoming smaller, cheaper, and more intelligent. Due to the large number of sensor manufacturers and differing accompanying protocols, integrating diverse sensors into observation systems is not straightforward. A coherent infrastructure is needed to treat sensors in an interoperable, platform-independent and uniform way. The concept of the Sensor Web reflects such a kind of infrastructure for sharing, finding, and accessing sensors and their data across different applications. It hides the heterogeneous sensor hardware and communication protocols from the applications built on top of it. The Sensor Web Enablement initiative of the Open Geospatial Consortium standardizes web service interfaces and data encodings which can be used as building blocks for a Sensor Web. This article illustrates and analyzes the recent developments of the new generation of the Sensor Web Enablement specification framework. Further, we relate the Sensor Web to other emerging concepts such as the Web of Things and point out challenges and resulting future work topics for research on Sensor Web Enablement. PMID:22163760

Nanomaterial-Enabled Wearable Sensors for Healthcare.

PubMed

Yao, Shanshan; Swetha, Puchakayala; Zhu, Yong

2018-01-01

Highly sensitive wearable sensors that can be conformably attached to human skin or integrated with textiles to monitor the physiological parameters of human body or the surrounding environment have garnered tremendous interest. Owing to the large surface area and outstanding material properties, nanomaterials are promising building blocks for wearable sensors. Recent advances in the nanomaterial-enabled wearable sensors including temperature, electrophysiological, strain, tactile, electrochemical, and environmental sensors are presented in this review. Integration of multiple sensors for multimodal sensing and integration with other components into wearable systems are summarized. Representative applications of nanomaterial-enabled wearable sensors for healthcare, including continuous health monitoring, daily and sports activity tracking, and multifunctional electronic skin are highlighted. Finally, challenges, opportunities, and future perspectives in the field of nanomaterial-enabled wearable sensors are discussed. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enabling the Discovery of Gravitational Radiation

NASA Astrophysics Data System (ADS)

Isaacson, Richard

2017-01-01

The discovery of gravitational radiation was announced with the publication of the results of a physics experiment involving over a thousand participants. This was preceded by a century of theoretical work, involving a similarly large group of physicists, mathematicians, and computer scientists. This huge effort was enabled by a substantial commitment of resources, both public and private, to develop the different strands of this complex research enterprise, and to build a community of scientists to carry it out. In the excitement following the discovery, the role of key enablers of this success has not always been adequately recognized in popular accounts. In this talk, I will try to call attention to a few of the key ingredients that proved crucial to enabling the successful discovery of gravitational waves, and the opening of a new field of science.

Joint Enabling Capabilities Command

Science.gov Websites

Executive Director Chief of Staff Joint Planning Support Element Joint Communications Support Element mission Joint Enabling Capabilities Command provides decisive joint communications, planning and public and responsive support for joint communications, planning and public affairs. Priorities * Deliver

Analyzing the texture changes in the quantitative phase maps of adipocytes

NASA Astrophysics Data System (ADS)

Roitshtain, Darina; Sharabani-Yosef, Orna; Gefen, Amit; Shaked, Natan T.

2016-03-01

We present a new analysis tool for studying texture changes in the quantitative phase maps of live cells acquired by wide-field interferometry. The sensitivity of wide-field interferometry systems to small changes in refractive index enables visualizing cells and inner cell organelles without the using fluorescent dyes or other cell-invasive approaches, which may affect the measurement and require external labeling. Our label-free texture-analysis tool is based directly on the optical path delay profile of the sample and does not necessitate decoupling refractive index and thickness in the cell quantitative phase profile; thus, relevant parameters can be calculated using a single-frame acquisition. Our experimental system includes low-coherence wide-field interferometer, combined with simultaneous florescence microscopy system for validation. We used this system and analysis tool for studying lipid droplets formation in adipocytes. The latter demonstration is relevant for various cellular functions such as lipid metabolism, protein storage and degradation to viral replication. These processes are functionally linked to several physiological and pathological conditions, including obesity and metabolic diseases. Quantification of these biological phenomena based on the texture changes in the cell phase map has a potential as a new cellular diagnosis tool.

Spin-polarized scanning tunneling microscopy with quantitative insights into magnetic probes

NASA Astrophysics Data System (ADS)

Phark, Soo-hyon; Sander, Dirk

2017-04-01

Spin-polarized scanning tunneling microscopy and spectroscopy (spin-STM/S) have been successfully applied to magnetic characterizations of individual nanostructures. Spin-STM/S is often performed in magnetic fields of up to some Tesla, which may strongly influence the tip state. In spite of the pivotal role of the tip in spin-STM/S, the contribution of the tip to the differential conductance d I/d V signal in an external field has rarely been investigated in detail. In this review, an advanced analysis of spin-STM/S data measured on magnetic nanoislands, which relies on a quantitative magnetic characterization of tips, is discussed. Taking advantage of the uniaxial out-of-plane magnetic anisotropy of Co bilayer nanoisland on Cu(111), in-field spin-STM on this system has enabled a quantitative determination, and thereby, a categorization of the magnetic states of the tips. The resulting in-depth and conclusive analysis of magnetic characterization of the tip opens new venues for a clear-cut sub-nanometer scale spin ordering and spin-dependent electronic structure of the non-collinear magnetic state in bilayer high Fe nanoislands on Cu(111).

A quantitative model of optimal data selection in Wason's selection task.

PubMed

Hattori, Masasi

2002-10-01

The optimal data selection model proposed by Oaksford and Chater (1994) successfully formalized Wason's selection task (Wason, 1966). The model, however, involved some questionable assumptions and was also not sufficient as a model of the task because it could not provide quantitative predictions of the card selection frequencies. In this paper, the model was revised to provide quantitative fits to the data. The model can predict the selection frequencies of cards based on a selection tendency function (STF), or conversely, it enables the estimation of subjective probabilities from data. Past experimental data were first re-analysed based on the model. In Experiment 1, the superiority of the revised model was shown. However, when the relationship between antecedent and consequent was forced to deviate from the biconditional form, the model was not supported. In Experiment 2, it was shown that sufficient emphasis on probabilistic information can affect participants' performance. A detailed experimental method to sort participants by probabilistic strategies was introduced. Here, the model was supported by a subgroup of participants who used the probabilistic strategy. Finally, the results were discussed from the viewpoint of adaptive rationality.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging.

PubMed

Zhang, Liang; Thurber, Greg M

2016-02-01

Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type 1 diabetes. The glucagon-like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower-clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, down-regulation of the GLP-1 receptor and non-specific background uptake result in a higher target-to-background ratio for fast-clearing agents.

Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging

PubMed Central

Zhang, Liang; Thurber, Greg M.

2016-01-01

Purpose Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type-1 diabetes. The glucagon like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Procedures Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Results Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Conclusions Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, downregulation of the GLP-1 receptor and non-specific background uptake result in a higher TBR for fast-clearing agents. PMID:26194012

Unpacking the enabling factors for hand, cord and birth-surface hygiene in Zanzibar maternity units

PubMed Central

Gon, Giorgia; Ali, Said M; Towriss, Catriona; Kahabuka, Catherine; Ali, Ali O; Cavill, Sue; Dahoma, Mohammed; Faulkner, Sally; Haji, Haji S; Kabole, Ibrahim; Morrison, Emma; Said, Rukaiya M; Tajo, Amour; Velleman, Yael; Woodd, Susannah L; Graham, and Wendy J

2017-01-01

Abstract Recent national surveys in The United Republic of Tanzania have revealed poor standards of hygiene at birth in facilities. As more women opt for institutional delivery, improving basic hygiene becomes an essential part of preventative strategies for reducing puerperal and newborn sepsis. Our collaborative research in Zanzibar provides an in-depth picture of the state of hygiene on maternity wards to inform action. Hygiene was assessed in 2014 across all 37 facilities with a maternity unit in Zanzibar. We used a mixed methods approach, including structured and semi-structured interviews, and environmental microbiology. Data were analysed according to the WHO ‘cleans’ framework, focusing on the fundamental practices for prevention of newborn and maternal sepsis. For each ‘clean’ we explored the following enabling factors: knowledge, infrastructure (including equipment), staffing levels and policies. Composite indices were constructed for the enabling factors of the ‘cleans’ from the quantitative data: clean hands, cord cutting, and birth surface. Results from the qualitative tools were used to complement this information. Only 49% of facilities had the ‘infrastructural’ requirements to enable ‘clean hands’, with the availability of constant running water particularly lacking. Less than half (46%) of facilities met the ‘knowledge’ requirements for ensuring a ‘clean delivery surface’; six out of seven facilities had birthing surfaces that tested positive for multiple potential pathogens. Almost two thirds of facilities met the ‘infrastructure (equipment) requirement’ for ‘clean cord’; however, disposable cord clamps being frequently out of stock, often resulted in the use of non-sterile thread made of fabric. This mixed methods approach, and the analytical framework based on the WHO ‘cleans’ and the enabling factors, yielded practical information of direct relevance to action at local and ministerial levels. The same

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells.

PubMed

Park, Han Sang; Rinehart, Matthew T; Walzer, Katelyn A; Chi, Jen-Tsan Ashley; Wax, Adam

2016-01-01

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells

PubMed Central

Park, Han Sang; Rinehart, Matthew T.; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

2016-01-01

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection

Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

NASA Astrophysics Data System (ADS)

Esposito, Alessandro

2006-05-01

This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

The Xeno-glycomics database (XDB): a relational database of qualitative and quantitative pig glycome repertoire.

PubMed

Park, Hae-Min; Park, Ju-Hyeong; Kim, Yoon-Woo; Kim, Kyoung-Jin; Jeong, Hee-Jin; Jang, Kyoung-Soon; Kim, Byung-Gee; Kim, Yun-Gon

2013-11-15

In recent years, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans. Here we present a database named Xeno-glycomics database (XDB) that contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including a comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database will contribute in providing qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation and might eventually provide new targets in the α1,3-galactosyltransferase gene-knock out pigs era. The database can be accessed on the web at http://bioinformatics.snu.ac.kr/xdb.

Magnetite/Ceria-Codecorated Titanoniobate Nanosheet: A 2D Catalytic Nanoprobe for Efficient Enrichment and Programmed Dephosphorylation of Phosphopeptides.

PubMed

Min, Qianhao; Li, Siyuan; Chen, Xueqin; Abdel-Halim, E S; Jiang, Li-Ping; Zhu, Jun-Jie

2015-05-13

Global characterization and in-depth understanding of phosphoproteome based on mass spectrometry (MS) desperately needs a highly efficient affinity probe during sample preparation. In this work, a ternary nanocomposite of magnetite/ceria-codecorated titanoniobate nanosheet (MC-TiNbNS) was synthesized by the electrostatic assembly of Fe3O4 nanospheres and in situ growth of CeO 2 nanoparticles on pre-exfoliated titanoniobate and eventually utilized as the probe and catalyst for the enrichment and dephosphorylation of phosphopeptides. The two-dimensional (2D) structured titanoniobate nanosheet not only promoted the efficacy of capturing phosphopeptides with enlarged surface area, but also functioned as a substrate for embracing the magnetic anchor Fe3O4 to enable magnetic separation and mimic phosphatase CeO2 to produce identifying signatures of phosphopeptides. Compared to single-component TiNbNS or CeO2 nanoparticles, the ternary nanocomposite provided direct evidence of the number of phosphorylation sites while maintaining the enrichment efficiency. Moreover, by altering the on-sheet CeO2 coverage, the dephosphorylation activity could be fine-tuned, generating continuously adjustable signal intensities of both phosphopeptides and their dephosphorylated tags. Exhaustive detection of both mono- and multiphosphorylated peptides with precise counting of their phosphorylation sites was achieved in the primary mass spectra in the cases of digests of standard phosphoprotein and skim milk, as well as a more complex biological sample, human serum. With the resulting highly informative mass spectra, this multifunctional probe can be used as a promising tool for the fast and comprehensive characterization of phosphopeptides in MS-based phosphoproteomics.

Quantitative X-ray Differential Interference Contrast Microscopy

NASA Astrophysics Data System (ADS)

Nakamura, Takashi

Full-field soft x-ray microscopes are widely used in many fields of sciences. Advances in nanofabrication technology enabled short wavelength focusing elements with significantly improved spatial resolution. In the soft x-ray spectral region, samples as small as 12 nm can be resolved using micro zone-plates as the objective lens. In addition to conventional x-ray microscopy in which x-ray absorption difference provides the image contrast, phase contrast mechanisms such as differential phase contrast (DIC) and Zernike phase contrast have also been demonstrated These phase contrast imaging mechanisms are especially attractive at the x-ray wavelengths where phase contrast of most materials is typically 10 times stronger than the absorption contrast. With recent progresses in plasma-based x- ray sources and increasing accessibility to synchrotron user facilities, x-ray microscopes are quickly becoming standard measurement equipment in the laboratory. To further the usefulness of x-ray DIC microscopy this thesis explicitly addresses three known issues with this imaging modality by introducing new techniques and devices First, as opposed to its visible-light counterpart, no quantitative phase imaging technique exists for x-ray DIC microscopy. To address this issue, two nanoscale x-ray quantitative phase imaging techniques, using exclusive OR (XOR) patterns and zone-plate doublets, respectively, are proposed. Unlike existing x-ray quantitative phase imaging techniques such as Talbot interferometry and ptychography, no dedicated experimental setups or stringent illumination coherence are needed for quantitative phase retrieval. Second, to the best of our knowledge, no quantitative performance characterization of DIC microscopy exists to date. Therefore the imaging system's response to sample's spatial frequency is not known In order to gain in-depth understanding of this imaging modality, performance of x-ray DIC microscopy is quantified using modulation transfer function

Toward Quantitative Small Animal Pinhole SPECT: Assessment of Quantitation Accuracy Prior to Image Compensations

PubMed Central

Chen, Chia-Lin; Wang, Yuchuan; Lee, Jason J. S.; Tsui, Benjamin M. W.

2011-01-01

Purpose We assessed the quantitation accuracy of small animal pinhole single photon emission computed tomography (SPECT) under the current preclinical settings, where image compensations are not routinely applied. Procedures The effects of several common image-degrading factors and imaging parameters on quantitation accuracy were evaluated using Monte-Carlo simulation methods. Typical preclinical imaging configurations were modeled, and quantitative analyses were performed based on image reconstructions without compensating for attenuation, scatter, and limited system resolution. Results Using mouse-sized phantom studies as examples, attenuation effects alone degraded quantitation accuracy by up to −18% (Tc-99m or In-111) or −41% (I-125). The inclusion of scatter effects changed the above numbers to −12% (Tc-99m or In-111) and −21% (I-125), respectively, indicating the significance of scatter in quantitative I-125 imaging. Region-of-interest (ROI) definitions have greater impacts on regional quantitation accuracy for small sphere sources as compared to attenuation and scatter effects. For the same ROI, SPECT acquisitions using pinhole apertures of different sizes could significantly affect the outcome, whereas the use of different radii-of-rotation yielded negligible differences in quantitation accuracy for the imaging configurations simulated. Conclusions We have systematically quantified the influence of several factors affecting the quantitation accuracy of small animal pinhole SPECT. In order to consistently achieve accurate quantitation within 5% of the truth, comprehensive image compensation methods are needed. PMID:19048346

Quantitative laser diagnostic and modeling study of C2 and CH chemistry in combustion.

PubMed

Köhler, Markus; Brockhinke, Andreas; Braun-Unkhoff, Marina; Kohse-Höinghaus, Katharina

2010-04-15

Quantitative concentration measurements of CH and C(2) have been performed in laminar, premixed, flat flames of propene and cyclopentene with varying stoichiometry. A combination of cavity ring-down (CRD) spectroscopy and laser-induced fluorescence (LIF) was used to enable sensitive detection of these species with high spatial resolution. Previously, CH and C(2) chemistry had been studied, predominantly in methane flames, to understand potential correlations of their formation and consumption. For flames of larger hydrocarbon fuels, however, quantitative information on these small intermediates is scarce, especially under fuel-rich conditions. Also, the combustion chemistry of C(2) in particular has not been studied in detail, and although it has often been observed, its role in potential build-up reactions of higher hydrocarbon species is not well understood. The quantitative measurements performed here are the first to detect both species with good spatial resolution and high sensitivity in the same experiment in flames of C(3) and C(5) fuels. The experimental profiles were compared with results of combustion modeling to reveal details of the formation and consumption of these important combustion molecules, and the investigation was devoted to assist the further understanding of the role of C(2) and of its potential chemical interdependences with CH and other small radicals.

Correlative SEM SERS for quantitative analysis of dimer nanoparticles.

PubMed

Timmermans, F J; Lenferink, A T M; van Wolferen, H A G M; Otto, C

2016-11-14

A Raman microscope integrated with a scanning electron microscope was used to investigate plasmonic structures by correlative SEM-SERS analysis. The integrated Raman-SEM microscope combines high-resolution electron microscopy information with SERS signal enhancement from selected nanostructures with adsorbed Raman reporter molecules. Correlative analysis is performed for dimers of two gold nanospheres. Dimers were selected on the basis of SEM images from multi aggregate samples. The effect of the orientation of the dimer with respect to the polarization state of the laser light and the effect of the particle gap size on the Raman signal intensity is observed. Additionally, calculations are performed to simulate the electric near field enhancement. These simulations are based on the morphologies observed by electron microscopy. In this way the experiments are compared with the enhancement factor calculated with near field simulations and are subsequently used to quantify the SERS enhancement factor. Large differences between experimentally observed and calculated enhancement factors are regularly detected, a phenomenon caused by nanoscale differences between the real and 'simplified' simulated structures. Quantitative SERS experiments reveal the structure induced enhancement factor, ranging from ∼200 to ∼20 000, averaged over the full nanostructure surface. The results demonstrate correlative Raman-SEM microscopy for the quantitative analysis of plasmonic particles and structures, thus enabling a new analytical method in the field of SERS and plasmonics.

Exploiting induced variation to dissect quantitative traits in barley.

PubMed

Druka, Arnis; Franckowiak, Jerome; Lundqvist, Udda; Bonar, Nicola; Alexander, Jill; Guzy-Wrobelska, Justyna; Ramsay, Luke; Druka, Ilze; Grant, Iain; Macaulay, Malcolm; Vendramin, Vera; Shahinnia, Fahimeh; Radovic, Slobodanka; Houston, Kelly; Harrap, David; Cardle, Linda; Marshall, David; Morgante, Michele; Stein, Nils; Waugh, Robbie

2010-04-01

The identification of genes underlying complex quantitative traits such as grain yield by means of conventional genetic analysis (positional cloning) requires the development of several large mapping populations. However, it is possible that phenotypically related, but more extreme, allelic variants generated by mutational studies could provide a means for more efficient cloning of QTLs (quantitative trait loci). In barley (Hordeum vulgare), with the development of high-throughput genome analysis tools, efficient genome-wide identification of genetic loci harbouring mutant alleles has recently become possible. Genotypic data from NILs (near-isogenic lines) that carry induced or natural variants of genes that control aspects of plant development can be compared with the location of QTLs to potentially identify candidate genes for development--related traits such as grain yield. As yield itself can be divided into a number of allometric component traits such as tillers per plant, kernels per spike and kernel size, mutant alleles that both affect these traits and are located within the confidence intervals for major yield QTLs may represent extreme variants of the underlying genes. In addition, the development of detailed comparative genomic models based on the alignment of a high-density barley gene map with the rice and sorghum physical maps, has enabled an informed prioritization of 'known function' genes as candidates for both QTLs and induced mutant genes.

Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles

PubMed Central

Bousse, Tatiana; Shore, David A.; Goldsmith, Cynthia S.; Hossain, M. Jaber; Jang, Yunho; Davis, Charles T.; Donis, Ruben O.; Stevens, James

2017-01-01

Summary Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9 %. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus has greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed. PMID:23916678

Liquid metal enabled pump

PubMed Central

Tang, Shi-Yang; Khoshmanesh, Khashayar; Sivan, Vijay; Petersen, Phred; O’Mullane, Anthony P.; Abbott, Derek; Mitchell, Arnan; Kalantar-zadeh, Kourosh

2014-01-01

Small-scale pumps will be the heartbeat of many future micro/nanoscale platforms. However, the integration of small-scale pumps is presently hampered by limited flow rate with respect to the input power, and their rather complicated fabrication processes. These issues arise as many conventional pumping effects require intricate moving elements. Here, we demonstrate a system that we call the liquid metal enabled pump, for driving a range of liquids without mechanical moving parts, upon the application of modest electric field. This pump incorporates a droplet of liquid metal, which induces liquid flow at high flow rates, yet with exceptionally low power consumption by electrowetting/deelectrowetting at the metal surface. We present theory explaining this pumping mechanism and show that the operation is fundamentally different from other existing pumps. The presented liquid metal enabled pump is both efficient and simple, and thus has the potential to fundamentally advance the field of microfluidics. PMID:24550485

Smart Grid Enabled EVSE

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

None, None

2015-01-12

The combined team of GE Global Research, Federal Express, National Renewable Energy Laboratory, and Consolidated Edison has successfully achieved the established goals contained within the Department of Energy’s Smart Grid Capable Electric Vehicle Supply Equipment funding opportunity. The final program product, shown charging two vehicles in Figure 1, reduces by nearly 50% the total installed system cost of the electric vehicle supply equipment (EVSE) as well as enabling a host of new Smart Grid enabled features. These include bi-directional communications, load control, utility message exchange and transaction management information. Using the new charging system, Utilities or energy service providers willmore » now be able to monitor transportation related electrical loads on their distribution networks, send load control commands or preferences to individual systems, and then see measured responses. Installation owners will be able to authorize usage of the stations, monitor operations, and optimally control their electricity consumption. These features and cost reductions have been developed through a total system design solution.« less

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.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

PubMed

Ittig, Simon J; Schmutz, Christoph; Kasper, Christoph A; Amstutz, Marlise; Schmidt, Alexander; Sauteur, Loïc; Vigano, M Alessandra; Low, Shyan Huey; Affolter, Markus; Cornelis, Guy R; Nigg, Erich A; Arrieumerlou, Cécile

2015-11-23

Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network. © 2015 Ittig et al.

Measuring the Nonuniform Evaporation Dynamics of Sprayed Sessile Microdroplets with Quantitative Phase Imaging.

PubMed

Edwards, Chris; Arbabi, Amir; Bhaduri, Basanta; Wang, Xiaozhen; Ganti, Raman; Yunker, Peter J; Yodh, Arjun G; Popescu, Gabriel; Goddard, Lynford L

2015-10-13

We demonstrate real-time quantitative phase imaging as a new optical approach for measuring the evaporation dynamics of sessile microdroplets. Quantitative phase images of various droplets were captured during evaporation. The images enabled us to generate time-resolved three-dimensional topographic profiles of droplet shape with nanometer accuracy and, without any assumptions about droplet geometry, to directly measure important physical parameters that characterize surface wetting processes. Specifically, the time-dependent variation of the droplet height, volume, contact radius, contact angle distribution along the droplet's perimeter, and mass flux density for two different surface preparations are reported. The studies clearly demonstrate three phases of evaporation reported previously: pinned, depinned, and drying modes; the studies also reveal instances of partial pinning. Finally, the apparatus is employed to investigate the cooperative evaporation of the sprayed droplets. We observe and explain the neighbor-induced reduction in evaporation rate, that is, as compared to predictions for isolated droplets. In the future, the new experimental methods should stimulate the exploration of colloidal particle dynamics on the gas-liquid-solid interface.

Polymer on Top: Current Limits and Future Perspectives of Quantitatively Evaluating Surface Grafting.

PubMed

Michalek, Lukas; Barner, Leonie; Barner-Kowollik, Christopher

2018-03-07

Well-defined polymer strands covalently tethered onto solid substrates determine the properties of the resulting functional interface. Herein, the current approaches to determine quantitative grafting densities are assessed. Based on a brief introduction into the key theories describing polymer brush regimes, a user's guide is provided to estimating maximum chain coverage and-importantly-examine the most frequently employed approaches for determining grafting densities, i.e., dry thickness measurements, gravimetric assessment, and swelling experiments. An estimation of the reliability of these determination methods is provided via carefully evaluating their assumptions and assessing the stability of the underpinning equations. A practical access guide for comparatively and quantitatively evaluating the reliability of a given approach is thus provided, enabling the field to critically judge experimentally determined grafting densities and to avoid the reporting of grafting densities that fall outside the physically realistic parameter space. The assessment is concluded with a perspective on the development of advanced approaches for determination of grafting density, in particular, on single-chain methodologies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hyperspectral imaging with near-infrared-enabled mobile phones for tissue oximetry

NASA Astrophysics Data System (ADS)

Lin, Jonathan L.; Ghassemi, Pejhman; Chen, Yu; Pfefer, Joshua

2018-02-01

Hyperspectral reflectance imaging (HRI) is an emerging clinical tool for characterizing spatial and temporal variations in blood perfusion and oxygenation for applications such as burn assessment, wound healing, retinal exams and intraoperative tissue viability assessment. Since clinical HRI-based oximeters often use near-infrared (NIR) light, NIR-enabled mobile phones may provide a useful platform for future point-of-care devices. Furthermore, quantitative NIR imaging on mobile phones may dramatically increase the availability and accessibility of medical diagnostics for low-resource settings. We have evaluated the potential for phone-based NIR oximetry imaging and elucidated factors affecting performance using devices from two different manufacturers, as well as a scientific CCD. A broadband light source and liquid crystal tunable filter were used for imaging at 10 nm bands from 650 to 1000 nm. Spectral sensitivity measurements indicated that mobile phones with standard NIR blocking filters had minimal response beyond 700 nm, whereas one modified phone showed sensitivity to 800 nm and another to 1000 nm. Red pixel channels showed the greatest sensitivity up to 800 nm, whereas all channels provided essentially equivalent sensitivity at longer wavelengths. Referencing of blood oxygenation levels was performed with a CO-oximeter. HRI measurements were performed using cuvettes filled with hemoglobin solutions of different oxygen saturation levels. Good agreement between absorbance spectra measured with mobile phone and a CCD cameras were seen for wavelengths below 900 nm. Saturation estimates showed root-mean-squared-errors of 5.2% and 4.5% for the CCD and phone, respectively. Overall, this work provides strong evidence of the potential for mobile phones to provide quantitative spectral imaging in the NIR for applications such as oximetry, and generates practical insights into factors that impact performance as well as test methods for performance assessment.

Telepresence-enabled research and developing work practices

NASA Astrophysics Data System (ADS)

Mirmalek, Z.

2016-02-01

In the fall of 2014, a group of scientists and students conducted two weeks of telepresence-enabled research from the University of Rhode Island Inner Space Center and Woods Hole Oceanographic Institution with the Exploration Vessel Nautilus, which was at sea studying the Kick'em Jenny submarine volcano and Barbados Mud Volcanoes. The way that they conducted their work was not so different from other telepresence-enabled ocean science exploration. As a group, they spanned geographic distance, science expertise, exploration experience, and telepresence-enabled research experience. They were connected through technologies and work culture (e.g., shared habits, values, and practices particular to a community). Uniquely, their project included an NSF-sponsored cultural study on the workgroups' own use of technologies and social processes. The objective of the cultural study was, in part, to identify social and technical features of the work environment that present opportunities to better support science exploration via telepresence. Drawing from this case, and related research, I present some analysis on the developing work culture of telepresence-enabled research and highlight potential adjustments.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

PubMed

Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

2015-01-01

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

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

PubMed Central

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

2014-01-01

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

Physician communication via Internet-enabled technology: A systematic review.

PubMed

Barr, Neil G; Randall, Glen E; Archer, Norman P; Musson, David M

2017-10-01

The use of Internet-enabled technology (information and communication technology such as smartphone applications) may enrich information exchange among providers and, consequently, improve health care delivery. The purpose of this systematic review was to gain a greater understanding of the role that Internet-enabled technology plays in enhancing communication among physicians. Studies were identified through a search in three electronic platforms: the Association for Computing Machinery Digital Library, ProQuest, and Web of Science. The search identified 5140 articles; of these, 21 met all inclusion criteria. In general, physicians were satisfied with Internet-enabled technology, but consensus was lacking regarding whether Internet-enabled technology improved efficiency or made a difference to clinical decision-making. Internet-enabled technology can play an important role in enhancing communication among physicians, but the extent of that benefit is influenced by (1) the impact of Internet-enabled technology on existing work practices, (2) the availability of adequate resources, and (3) the nature of institutional elements, such as privacy legislation.

The ENABLER - Based on proven NERVA technology

NASA Astrophysics Data System (ADS)

Livingston, Julie M.; Pierce, Bill L.

The ENABLER reactor for use in a nuclear thermal propulsion engine uses the technology developed in the NERVA/Rover program, updated to incorporate advances in the technology. Using composite fuel, higher power densities per fuel element, improved radiation resistant control components and the advancements in use of carbon-carbon materials; the ENABLER can provide a specific impulse of 925 seconds, an engine thrust to weight (excluding reactor shield) approaching five, an improved initial mass in low Earth orbit and a consequent reduction in launch costs and logistics problems. This paper describes the 75,000 lbs thrust ENABLER design which is a low cost, low risk approach to meeting tommorrow's space propulsion needs.

Internet-enabled collaborative agent-based supply chains

NASA Astrophysics Data System (ADS)

Shen, Weiming; Kremer, Rob; Norrie, Douglas H.

2000-12-01

This paper presents some results of our recent research work related to the development of a new Collaborative Agent System Architecture (CASA) and an Infrastructure for Collaborative Agent Systems (ICAS). Initially being proposed as a general architecture for Internet based collaborative agent systems (particularly complex industrial collaborative agent systems), the proposed architecture is very suitable for managing the Internet enabled complex supply chain for a large manufacturing enterprise. The general collaborative agent system architecture with the basic communication and cooperation services, domain independent components, prototypes and mechanisms are described. Benefits of implementing Internet enabled supply chains with the proposed infrastructure are discussed. A case study on Internet enabled supply chain management is presented.

Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring.

PubMed

Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi

2008-08-01

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Comparison of salivary collection and processing methods for quantitative HHV-8 detection.

PubMed

Speicher, D J; Johnson, N W

2014-10-01

Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown. Stabilising fluids impede nucleic acid degradation, compared with collection onto ice and then freezing, and we have shown that the DNA Genotek P-021 prototype kit (P-021) can produce high-quality DNA after 14 months of storage at room temperature. Here we evaluate the quantitative capability of 10 collection/processing methods. Unstimulated whole mouth fluid was spiked with a mixture of HHV-8 cloned constructs, 10-fold serial dilutions were produced, and samples were extracted and then examined with quantitative PCR (qPCR). Calibration curves were compared by linear regression and qPCR dynamics. All methods extracted with commercial spin columns produced linear calibration curves with large dynamic range and gave accurate viral loads. Ethanol precipitation of the P-021 does not produce a linear standard curve, and virus is lost in the cell pellet. DNA extractions from the P-021 using commercial spin columns produced linear standard curves with wide dynamic range and excellent limit of detection. When extracted with spin columns, the P-021 enables accurate viral loads down to 23 copies μl(-1) DNA. The quantitative and long-term storage capability of this system makes it ideal for study of salivary DNA viruses in resource-poor settings. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantitative evaluation of brain development using anatomical MRI and diffusion tensor imaging☆

PubMed Central

Oishi, Kenichi; Faria, Andreia V.; Yoshida, Shoko; Chang, Linda; Mori, Susumu

2013-01-01

The development of the brain is structure-specific, and the growth rate of each structure differs depending on the age of the subject. Magnetic resonance imaging (MRI) is often used to evaluate brain development because of the high spatial resolution and contrast that enable the observation of structure-specific developmental status. Currently, most clinical MRIs are evaluated qualitatively to assist in the clinical decision-making and diagnosis. The clinical MRI report usually does not provide quantitative values that can be used to monitor developmental status. Recently, the importance of image quantification to detect and evaluate mild-to-moderate anatomical abnormalities has been emphasized because these alterations are possibly related to several psychiatric disorders and learning disabilities. In the research arena, structural MRI and diffusion tensor imaging (DTI) have been widely applied to quantify brain development of the pediatric population. To interpret the values from these MR modalities, a “growth percentile chart,” which describes the mean and standard deviation of the normal developmental curve for each anatomical structure, is required. Although efforts have been made to create such a growth percentile chart based on MRI and DTI, one of the greatest challenges is to standardize the anatomical boundaries of the measured anatomical structures. To avoid inter- and intra-reader variability about the anatomical boundary definition, and hence, to increase the precision of quantitative measurements, an automated structure parcellation method, customized for the neonatal and pediatric population, has been developed. This method enables quantification of multiple MR modalities using a common analytic framework. In this paper, the attempt to create an MRI- and a DTI-based growth percentile chart, followed by an application to investigate developmental abnormalities related to cerebral palsy, Williams syndrome, and Rett syndrome, have been introduced

Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.

PubMed

Yu, Clinton; Huszagh, Alexander; Viner, Rosa; Novitsky, Eric J; Rychnovsky, Scott D; Huang, Lan

2016-10-18

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

PubMed Central

Gunning, Yvonne; Watson, Andrew D.; Rigby, Neil M.; Philo, Mark; Peazer, Joshua K.; Kemsley, E. Kate

2016-01-01

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture. PMID:27685654

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication.

PubMed

Gunning, Yvonne; Watson, Andrew D; Rigby, Neil M; Philo, Mark; Peazer, Joshua K; Kemsley, E Kate

2016-09-20

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

Accelerating simultaneous algebraic reconstruction technique with motion compensation using CUDA-enabled GPU.

PubMed

Pang, Wai-Man; Qin, Jing; Lu, Yuqiang; Xie, Yongming; Chui, Chee-Kong; Heng, Pheng-Ann

2011-03-01

To accelerate the simultaneous algebraic reconstruction technique (SART) with motion compensation for speedy and quality computed tomography reconstruction by exploiting CUDA-enabled GPU. Two core techniques are proposed to fit SART into the CUDA architecture: (1) a ray-driven projection along with hardware trilinear interpolation, and (2) a voxel-driven back-projection that can avoid redundant computation by combining CUDA shared memory. We utilize the independence of each ray and voxel on both techniques to design CUDA kernel to represent a ray in the projection and a voxel in the back-projection respectively. Thus, significant parallelization and performance boost can be achieved. For motion compensation, we rectify each ray's direction during the projection and back-projection stages based on a known motion vector field. Extensive experiments demonstrate the proposed techniques can provide faster reconstruction without compromising image quality. The process rate is nearly 100 projections s (-1), and it is about 150 times faster than a CPU-based SART. The reconstructed image is compared against ground truth visually and quantitatively by peak signal-to-noise ratio (PSNR) and line profiles. We further evaluate the reconstruction quality using quantitative metrics such as signal-to-noise ratio (SNR) and mean-square-error (MSE). All these reveal that satisfactory results are achieved. The effects of major parameters such as ray sampling interval and relaxation parameter are also investigated by a series of experiments. A simulated dataset is used for testing the effectiveness of our motion compensation technique. The results demonstrate our reconstructed volume can eliminate undesirable artifacts like blurring. Our proposed method has potential to realize instantaneous presentation of 3D CT volume to physicians once the projection data are acquired.

Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins.

PubMed

Schalk, Kathrin; Koehler, Peter; Scherf, Katharina Anne

2018-01-01

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods.

Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins

PubMed Central

Schalk, Kathrin; Koehler, Peter

2018-01-01

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods. PMID:29425234

Quantitative photoacoustic elastography of Young's modulus in humans

NASA Astrophysics Data System (ADS)

Hai, Pengfei; Zhou, Yong; Gong, Lei; Wang, Lihong V.

2017-03-01

Elastography can noninvasively map the elasticity distribution of biological tissue, which is often altered in pathological states. In this work, we report quantitative photoacoustic elastography (QPAE), capable of measuring Young's modulus of human tissue in vivo. By combining photoacoustic elastography with a stress sensor having known stress-strain behavior, QPAE can simultaneously measure strain and stress, from which Young's modulus is calculated. We first applied QPAE to quantify the Young's modulus of tissue-mimicking agar phantoms with different concentrations. The measured values fitted well with both the empirical expectations based on the agar concentrations and those measured in independent standard compression tests. We then demonstrated the feasibility of QPAE by measuring the Young's modulus of human skeletal muscle in vivo. The data showed a linear relationship between muscle stiffness and loading. The results proved that QPAE can noninvasively quantify the absolute elasticity of biological tissue, thus enabling longitudinal imaging of tissue elasticity. QPAE can be exploited for both preclinical biomechanics studies and clinical applications.

Airborne radar and radiometer experiment for quantitative remote measurements of rain

NASA Technical Reports Server (NTRS)

Kozu, Toshiaki; Meneghini, Robert; Boncyk, Wayne; Wilheit, Thomas T.; Nakamura, Kenji

1989-01-01

An aircraft experiment has been conducted with a dual-frequency (10 GHz and 35 GHz) radar/radiometer system and an 18-GHz radiometer to test various rain-rate retrieval algorithms from space. In the experiment, which took place in the fall of 1988 at the NASA Wallops Flight Facility, VA, both stratiform and convective storms were observed. A ground-based radar and rain gauges were also used to obtain truth data. An external radar calibration is made with rain gauge data, thereby enabling quantitative reflectivity measurements. Comparisons between path attenuations derived from the surface return and from the radar reflectivity profile are made to test the feasibility of a technique to estimate the raindrop size distribution from simultaneous radar and path-attenuation measurements.

Customized Molecular Phenotyping by Quantitative Gene Expression and Pattern Recognition Analysis

PubMed Central

Akilesh, Shreeram; Shaffer, Daniel J.; Roopenian, Derry

2003-01-01

Description of the molecular phenotypes of pathobiological processes in vivo is a pressing need in genomic biology. We have implemented a high-throughput real-time PCR strategy to establish quantitative expression profiles of a customized set of target genes. It enables rapid, reproducible data acquisition from limited quantities of RNA, permitting serial sampling of mouse blood during disease progression. We developed an easy to use statistical algorithm—Global Pattern Recognition—to readily identify genes whose expression has changed significantly from healthy baseline profiles. This approach provides unique molecular signatures for rheumatoid arthritis, systemic lupus erythematosus, and graft versus host disease, and can also be applied to defining the molecular phenotype of a variety of other normal and pathological processes. PMID:12840047

Distributed Cognition and Process Management Enabling Individualized Translational Research: The NIH Undiagnosed Diseases Program Experience

PubMed Central

Links, Amanda E.; Draper, David; Lee, Elizabeth; Guzman, Jessica; Valivullah, Zaheer; Maduro, Valerie; Lebedev, Vlad; Didenko, Maxim; Tomlin, Garrick; Brudno, Michael; Girdea, Marta; Dumitriu, Sergiu; Haendel, Melissa A.; Mungall, Christopher J.; Smedley, Damian; Hochheiser, Harry; Arnold, Andrew M.; Coessens, Bert; Verhoeven, Steven; Bone, William; Adams, David; Boerkoel, Cornelius F.; Gahl, William A.; Sincan, Murat

2016-01-01

The National Institutes of Health Undiagnosed Diseases Program (NIH UDP) applies translational research systematically to diagnose patients with undiagnosed diseases. The challenge is to implement an information system enabling scalable translational research. The authors hypothesized that similar complex problems are resolvable through process management and the distributed cognition of communities. The team, therefore, built the NIH UDP integrated collaboration system (UDPICS) to form virtual collaborative multidisciplinary research networks or communities. UDPICS supports these communities through integrated process management, ontology-based phenotyping, biospecimen management, cloud-based genomic analysis, and an electronic laboratory notebook. UDPICS provided a mechanism for efficient, transparent, and scalable translational research and thereby addressed many of the complex and diverse research and logistical problems of the NIH UDP. Full definition of the strengths and deficiencies of UDPICS will require formal qualitative and quantitative usability and process improvement measurement. PMID:27785453

Quantitative measurements of electromechanical response with a combined optical beam and interferometric atomic force microscope

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

Labuda, Aleksander; Proksch, Roger

An ongoing challenge in atomic force microscope (AFM) experiments is the quantitative measurement of cantilever motion. The vast majority of AFMs use the optical beam deflection (OBD) method to infer the deflection of the cantilever. The OBD method is easy to implement, has impressive noise performance, and tends to be mechanically robust. However, it represents an indirect measurement of the cantilever displacement, since it is fundamentally an angular rather than a displacement measurement. Here, we demonstrate a metrological AFM that combines an OBD sensor with a laser Doppler vibrometer (LDV) to enable accurate measurements of the cantilever velocity and displacement.more » The OBD/LDV AFM allows a host of quantitative measurements to be performed, including in-situ measurements of cantilever oscillation modes in piezoresponse force microscopy. As an example application, we demonstrate how this instrument can be used for accurate quantification of piezoelectric sensitivity—a longstanding goal in the electromechanical community.« less

Quantitative Measurement of Local Infrared Absorption and Dielectric Function with Tip-Enhanced Near-Field Microscopy.

PubMed

Govyadinov, Alexander A; Amenabar, Iban; Huth, Florian; Carney, P Scott; Hillenbrand, Rainer

2013-05-02

Scattering-type scanning near-field optical microscopy (s-SNOM) and Fourier transform infrared nanospectroscopy (nano-FTIR) are emerging tools for nanoscale chemical material identification. Here, we push s-SNOM and nano-FTIR one important step further by enabling them to quantitatively measure local dielectric constants and infrared absorption. Our technique is based on an analytical model, which allows for a simple inversion of the near-field scattering problem. It yields the dielectric permittivity and absorption of samples with 2 orders of magnitude improved spatial resolution compared to far-field measurements and is applicable to a large class of samples including polymers and biological matter. We verify the capabilities by determining the local dielectric permittivity of a PMMA film from nano-FTIR measurements, which is in excellent agreement with far-field ellipsometric data. We further obtain local infrared absorption spectra with unprecedented accuracy in peak position and shape, which is the key to quantitative chemometrics on the nanometer scale.

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

PubMed

LeBlanc, André; Michaud, Sarah A; Percy, Andrew J; Hardie, Darryl B; Yang, Juncong; Sinclair, Nicholas J; Proudfoot, Jillaine I; Pistawka, Adam; Smith, Derek S; Borchers, Christoph H

2017-07-07

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

AFC-Enabled Vertical Tail System Integration Study

NASA Technical Reports Server (NTRS)

Mooney, Helen P.; Brandt, John B.; Lacy, Douglas S.; Whalen, Edward A.

2014-01-01

This document serves as the final report for the SMAAART AFC-Enabled Vertical Tail System Integration Study. Included are the ground rule assumptions which have gone into the study, layouts of the baseline and AFC-enabled configurations, critical sizing information, system requirements and architectures, and assumed system properties that result in an NPV assessment of the two candidate AFC technologies.

Quantitative 3D analysis of bone in hip osteoarthritis using clinical computed tomography.

PubMed

Turmezei, Tom D; Treece, Graham M; Gee, Andrew H; Fotiadou, Anastasia F; Poole, Kenneth E S

2016-07-01

To assess the relationship between proximal femoral cortical bone thickness and radiological hip osteoarthritis using quantitative 3D analysis of clinical computed tomography (CT) data. Image analysis was performed on clinical CT imaging data from 203 female volunteers with a technique called cortical bone mapping (CBM). Colour thickness maps were created for each proximal femur. Statistical parametric mapping was performed to identify statistically significant differences in cortical bone thickness that corresponded with the severity of radiological hip osteoarthritis. Kellgren and Lawrence (K&L) grade, minimum joint space width (JSW) and a novel CT-based osteophyte score were also blindly assessed from the CT data. For each increase in K&L grade, cortical thickness increased by up to 25 % in distinct areas of the superolateral femoral head-neck junction and superior subchondral bone plate. For increasing severity of CT osteophytes, the increase in cortical thickness was more circumferential, involving a wider portion of the head-neck junction, with up to a 7 % increase in cortical thickness per increment in score. Results were not significant for minimum JSW. These findings indicate that quantitative 3D analysis of the proximal femur can identify changes in cortical bone thickness relevant to structural hip osteoarthritis. • CT is being increasingly used to assess bony involvement in osteoarthritis • CBM provides accurate and reliable quantitative analysis of cortical bone thickness • Cortical bone is thicker at the superior femoral head-neck with worse osteoarthritis • Regions of increased thickness co-locate with impingement and osteophyte formation • Quantitative 3D bone analysis could enable clinical disease prediction and therapy development.

High-Throughput Quantitative Lipidomics Analysis of Nonesterified Fatty Acids in Human Plasma.

PubMed

Christinat, Nicolas; Morin-Rivron, Delphine; Masoodi, Mojgan

2016-07-01

We present a high-throughput, nontargeted lipidomics approach using liquid chromatography coupled to high-resolution mass spectrometry for quantitative analysis of nonesterified fatty acids. We applied this method to screen a wide range of fatty acids from medium-chain to very long-chain (8 to 24 carbon atoms) in human plasma samples. The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important ω-3 and ω-6 polyunsaturated fatty acids. We used 51 fatty acid species to demonstrate the quantitative capability of this method with quantification limits in the nanomolar range; however, this method is not limited only to these fatty acid species. High-throughput sample preparation was developed and carried out on a robotic platform that allows extraction of 96 samples simultaneously within 3 h. This high-throughput platform was used to assess the influence of different types of human plasma collection and preparation on the nonesterified fatty acid profile of healthy donors. Use of the anticoagulants EDTA and heparin has been compared with simple clotting, and only limited changes have been detected in most nonesterified fatty acid concentrations.

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

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

Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein,more » we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.« less

Nanomaterial-Enabled Neural Stimulation

PubMed Central

Wang, Yongchen; Guo, Liang

2016-01-01

Neural stimulation is a critical technique in treating neurological diseases and investigating brain functions. Traditional electrical stimulation uses electrodes to directly create intervening electric fields in the immediate vicinity of neural tissues. Second-generation stimulation techniques directly use light, magnetic fields or ultrasound in a non-contact manner. An emerging generation of non- or minimally invasive neural stimulation techniques is enabled by nanotechnology to achieve a high spatial resolution and cell-type specificity. In these techniques, a nanomaterial converts a remotely transmitted primary stimulus such as a light, magnetic or ultrasonic signal to a localized secondary stimulus such as an electric field or heat to stimulate neurons. The ease of surface modification and bio-conjugation of nanomaterials facilitates cell-type-specific targeting, designated placement and highly localized membrane activation. This review focuses on nanomaterial-enabled neural stimulation techniques primarily involving opto-electric, opto-thermal, magneto-electric, magneto-thermal and acousto-electric transduction mechanisms. Stimulation techniques based on other possible transduction schemes and general consideration for these emerging neurotechnologies are also discussed. PMID:27013938

Affinity for Quantitative Tools: Undergraduate Marketing Students Moving beyond Quantitative Anxiety

ERIC Educational Resources Information Center

Tarasi, Crina O.; Wilson, J. Holton; Puri, Cheenu; Divine, Richard L.

2013-01-01

Marketing students are known as less likely to have an affinity for the quantitative aspects of the marketing discipline. In this article, we study the reasons why this might be true and develop a parsimonious 20-item scale for measuring quantitative affinity in undergraduate marketing students. The scale was administered to a sample of business…