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Sample records for rapid nmr-based protein

  1. PINE-SPARKY.2 for automated NMR-based protein structure research.

    Science.gov (United States)

    Lee, Woonghee; Markley, John L

    2018-05-01

    Nuclear magnetic resonance (NMR) spectroscopy, along with X-ray crystallography and cryoelectron microscopy, is one of the three major tools that enable the determination of atomic-level structural models of biological macromolecules. Of these, NMR has the unique ability to follow important processes in solution, including conformational changes, internal dynamics and protein-ligand interactions. As a means for facilitating the handling and analysis of spectra involved in these types of NMR studies, we have developed PINE-SPARKY.2, a software package that integrates and automates discrete tasks that previously required interaction with separate software packages. The graphical user interface of PINE-SPARKY.2 simplifies chemical shift assignment and verification, automated detection of secondary structural elements, predictions of flexibility and hydrophobic cores, and calculation of three-dimensional structural models. PINE-SPARKY.2 is available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). For a detailed description of the program, see http://www.nmrfam.wisc.edu/pine-sparky2.htm. whlee@nmrfam.wisc.edu or markley@nmrfam.wisc.edu. Supplementary data are available at Bioinformatics online.

  2. Rapid discrimination of strain-dependent fermentation characteristics among Lactobacillus strains by NMR-based metabolomics of fermented vegetable juice.

    Directory of Open Access Journals (Sweden)

    Satoru Tomita

    Full Text Available In this study, we investigated the applicability of NMR-based metabolomics to discriminate strain-dependent fermentation characteristics of lactic acid bacteria (LAB, which are important microorganisms for fermented food production. To evaluate the discrimination capability, six type strains of Lactobacillus species and six additional L. brevis strains were used focusing on i the difference between homo- and hetero-lactic fermentative species and ii strain-dependent characteristics within L. brevis. Based on the differences in the metabolite profiles of fermented vegetable juices, non-targeted principal component analysis (PCA clearly separated the samples into those inoculated with homo- and hetero-lactic fermentative species. The separation was primarily explained by the different levels of dominant metabolites (lactic acid, acetic acid, ethanol, and mannitol. Orthogonal partial least squares discrimination analysis, based on a regions-of-interest (ROIs approach, revealed the contribution of low-abundance metabolites: acetoin, phenyllactic acid, p-hydroxyphenyllactic acid, glycerophosphocholine, and succinic acid for homolactic fermentation; and ornithine, tyramine, and γ-aminobutyric acid (GABA for heterolactic fermentation. Furthermore, ROIs-based PCA of seven L. brevis strains separated their strain-dependent fermentation characteristics primarily based on their ability to utilize sucrose and citric acid, and convert glutamic acid and tyrosine into GABA and tyramine, respectively. In conclusion, NMR metabolomics successfully discriminated the fermentation characteristics of the tested strains and provided further information on metabolites responsible for these characteristics, which may impact the taste, aroma, and functional properties of fermented foods.

  3. A rapid NMR-based method for discrimination of strain-specific cell wall teichoic acid structures reveals a third backbone type in Lactobacillus plantarum.

    Science.gov (United States)

    Tomita, Satoru; Tanaka, Naoto; Okada, Sanae

    2017-03-01

    The lactic acid bacterium Lactobacillus plantarum is capable of producing strain-specific structures of cell wall teichoic acid (WTA), an anionic polysaccharide found in the Gram-positive bacterial cell wall. In this study, we established a rapid, NMR-based procedure to discriminate WTA structures in this species, and applied it to 94 strains of L. plantarum. Six previously reported glycerol- and ribitol-containing WTA subtypes were successfully identified from 78 strains, suggesting that these were the dominant structures. However, the level of structural variety differed markedly among bacterial sources, possibly reflecting differences in strain-level microbial diversity. WTAs from eight strains were not identified based on NMR spectra and were classified into three groups. Structural analysis of a partial degradation product of an unidentified WTA produced by strain TUA 1496L revealed that the WTA was 1-O-β-d-glucosylglycerol. Two-dimensional NMR analysis of the polymer structure showed phosphodiester bonds between C-3 and C-6 of the glycerol and glucose residues, suggesting a polymer structure of 3,6΄-linked poly(1-O-β-d-glucosyl-sn-glycerol phosphate). This is the third WTA backbone structure in L. plantarum, following 3,6΄-linked poly(1-O-α-d-glucosyl-sn-glycerol phosphate) and 1,5-linked poly(ribitol phosphate). © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

    Science.gov (United States)

    Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

    2010-05-05

    Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  5. Characterization of the Raf kinase inhibitory protein (RKIP binding pocket: NMR-based screening identifies small-molecule ligands.

    Directory of Open Access Journals (Sweden)

    Anne N Shemon

    2010-05-01

    Full Text Available Raf kinase inhibitory protein (RKIP, also known as phoshaptidylethanolamine binding protein (PEBP, has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE. In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

  6. Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

    Science.gov (United States)

    Baxa, Michael C.; Haddadian, Esmael J.; Jumper, John M.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol−1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix−sheet = 0.5 kcal⋅mol−1), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR. PMID:25313044

  7. Hepatitis B virus X protein (HBx)-induced abnormalities of nucleic acid metabolism revealed by (1)H-NMR-based metabonomics.

    Science.gov (United States)

    Dan Yue; Zhang, Yuwei; Cheng, Liuliu; Ma, Jinhu; Xi, Yufeng; Yang, Liping; Su, Chao; Shao, Bin; Huang, Anliang; Xiang, Rong; Cheng, Ping

    2016-04-14

    Hepatitis B virus X protein (HBx) plays an important role in HBV-related hepatocarcinogenesis; however, mechanisms underlying HBx-mediated carcinogenesis remain unclear. In this study, an NMR-based metabolomics approach was applied to systematically investigate the effects of HBx on cell metabolism. EdU incorporation assay was conducted to examine the effects of HBx on DNA synthesis, an important feature of nucleic acid metabolism. The results revealed that HBx disrupted metabolism of glucose, lipids, and amino acids, especially nucleic acids. To understand the potential mechanism of HBx-induced abnormalities of nucleic acid metabolism, gene expression profiles of HepG2 cells expressing HBx were investigated. The results showed that 29 genes involved in DNA damage and DNA repair were differentially expressed in HBx-expressing HepG2 cells. HBx-induced DNA damage was further demonstrated by karyotyping, comet assay, Western blotting, immunofluorescence and immunohistochemistry analyses. Many studies have previously reported that DNA damage can induce abnormalities of nucleic acid metabolism. Thus, our results implied that HBx initially induces DNA damage, and then disrupts nucleic acid metabolism, which in turn blocks DNA repair and induces the occurrence of hepatocellular carcinoma (HCC). These findings further contribute to our understanding of the occurrence of HCC.

  8. An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by cold shock protein A.

    Science.gov (United States)

    Alexandrescu, A T

    2001-01-01

    Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (approximately 1 x 10(-3) min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10(-5) min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are approximately 10(3), and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10(5) on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

  9. NMR-based milk metabolomics

    DEFF Research Database (Denmark)

    Sundekilde, Ulrik; Larsen, Lotte Bach; Bertram, Hanne Christine S.

    2013-01-01

    and processing capabilities of bovine milk is closely associated to milk composition. Metabolomics is ideal in the study of the low-molecular-weight compounds in milk, and this review focuses on the recent nuclear magnetic resonance (NMR)-based metabolomics trends in milk research, including applications linking...... compounds. Furthermore, metabolomics applications elucidating how the differential regulated genes affects milk composition are also reported. This review will highlight the recent advances in NMR-based metabolomics on milk, as well as give a brief summary of when NMR spectroscopy can be useful for gaining...

  10. Rapid comparison of properties on protein surface.

    Science.gov (United States)

    Sael, Lee; La, David; Li, Bin; Rustamov, Raif; Kihara, Daisuke

    2008-10-01

    The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM beta/alpha barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure.

  11. International NMR-based Environmental Metabolomics Intercomparison Exercise

    Science.gov (United States)

    Several fundamental requirements must be met so that NMR-based metabolomics and the related technique of metabonomics can be formally adopted into environmental monitoring and chemical risk assessment. Here we report an intercomparison exercise which has evaluated the effectivene...

  12. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E.; Korgsdam, A.-M.; Jørgensen, H.F.

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  13. Hydrophobic Collapse of Ubiquitin Generates Rapid Protein-Water Motions.

    Science.gov (United States)

    Wirtz, Hanna; Schäfer, Sarah; Hoberg, Claudius; Reid, Korey M; Leitner, David M; Havenith, Martina

    2018-06-04

    We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (<500 ns) initial phase of the hydrophobic collapse from the elongated protein to a molten globule structure precedes secondary structure formation. We find that these very first steps, including large-amplitude changes within the unfolded manifold, are accompanied by a rapid (<500 ns) pronounced change of the coupled protein-solvent response. The KITA response upon secondary structure formation exhibits an opposite sign, which indicates a distinct effect on the solvent-exposed surface.

  14. Rapid and reliable protein structure determination via chemical shift threading.

    Science.gov (United States)

    Hafsa, Noor E; Berjanskii, Mark V; Arndt, David; Wishart, David S

    2018-01-01

    Protein structure determination using nuclear magnetic resonance (NMR) spectroscopy can be both time-consuming and labor intensive. Here we demonstrate how chemical shift threading can permit rapid, robust, and accurate protein structure determination using only chemical shift data. Threading is a relatively old bioinformatics technique that uses a combination of sequence information and predicted (or experimentally acquired) low-resolution structural data to generate high-resolution 3D protein structures. The key motivations behind using NMR chemical shifts for protein threading lie in the fact that they are easy to measure, they are available prior to 3D structure determination, and they contain vital structural information. The method we have developed uses not only sequence and chemical shift similarity but also chemical shift-derived secondary structure, shift-derived super-secondary structure, and shift-derived accessible surface area to generate a high quality protein structure regardless of the sequence similarity (or lack thereof) to a known structure already in the PDB. The method (called E-Thrifty) was found to be very fast (often chemical shift refinement, these results suggest that protein structure determination, using only NMR chemical shifts, is becoming increasingly practical and reliable. E-Thrifty is available as a web server at http://ethrifty.ca .

  15. Association of protein C23 with rapidly labeled nucleolar RNA

    International Nuclear Information System (INIS)

    Herrera, A.H.; Olson, M.O.

    1986-01-01

    The association of nucleolar phosphoprotein C23 with preribosomal ribonucleoprotein (RNP) particles was examined in Novikoff hepatoma nucleoli. RNA was labeled with [ 3 H]uridine for various times in cell suspensions, and RNP particles were extracted from isolated nucleoli and fractionated by sucrose gradient ultracentrifugation. The majority of protein C23 cosedimented with fractions containing rapidly labeled RNA (RL fraction). To determine whether there was a direct association of RNA with protein C23, the RL fraction was exposed to ultraviolet (UV) light (254 nm) for short periods of time. After 2 min of exposure there was a 50% decrease in C23 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, with no significant further decrease at longer times. When UV-treated fractions were subjected to phenol/chloroform extractions, as much as 30% of the labeled RNA was found in the phenol (protein) layer, indicating that RNA became cross-linked to protein. Similarly, there was an increase in protein C23 extracted into the water layer after irradiation. By SDS-PAGE analyses the cross-linked species migrated more slowly than protein C23, appearing as a smear detected either by [ 3 H]uridine radioactivity or by anti-C23 antibody. With anti-C23 antibodies, up to 25% of the labeled RNA was precipitated from the RL fraction. Dot-blot hybridizations, using cloned rDNA fragments as probes, indicated that the RNA in the RL fraction and the immunoprecipitated RNA contained sequences from 18S and 28S ribosomal RNA

  16. Small acid soluble proteins for rapid spore identification.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  17. Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

    Science.gov (United States)

    Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

    2006-01-01

    Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

  18. Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, J.E.A. [CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Erny, G.L. [CESAM - Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Barros, A.S. [QOPNAA-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Esteves, V.I. [CESAM - Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal); Brandao, T.; Ferreira, A.A. [UNICER, Bebidas de Portugal, Leca do Balio, 4466-955 S. Mamede de Infesta (Portugal); Cabrita, E. [Department of Chemistry, New University of Lisbon, 2825-114 Caparica (Portugal); Gil, A.M., E-mail: agil@ua.pt [CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro (Portugal)

    2010-08-03

    The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

  19. Quantification of organic acids in beer by nuclear magnetic resonance (NMR)-based methods

    International Nuclear Information System (INIS)

    Rodrigues, J.E.A.; Erny, G.L.; Barros, A.S.; Esteves, V.I.; Brandao, T.; Ferreira, A.A.; Cabrita, E.; Gil, A.M.

    2010-01-01

    The organic acids present in beer provide important information on the product's quality and history, determining organoleptic properties and being useful indicators of fermentation performance. NMR spectroscopy may be used for rapid quantification of organic acids in beer and different NMR-based methodologies are hereby compared for the six main acids found in beer (acetic, citric, lactic, malic, pyruvic and succinic). The use of partial least squares (PLS) regression enables faster quantification, compared to traditional integration methods, and the performance of PLS models built using different reference methods (capillary electrophoresis (CE), both with direct and indirect UV detection, and enzymatic essays) was investigated. The best multivariate models were obtained using CE/indirect detection and enzymatic essays as reference and their response was compared with NMR integration, either using an internal reference or an electrical reference signal (Electronic REference To access In vivo Concentrations, ERETIC). NMR integration results generally agree with those obtained by PLS, with some overestimation for malic and pyruvic acids, probably due to peak overlap and subsequent integral errors, and an apparent relative underestimation for citric acid. Overall, these results make the PLS-NMR method an interesting choice for organic acid quantification in beer.

  20. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

    Science.gov (United States)

    Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

    2016-02-01

    Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. NMR-Based Identification of Metabolites in Polar and Non-Polar Extracts of Avian Liver.

    Science.gov (United States)

    Fathi, Fariba; Brun, Antonio; Rott, Katherine H; Falco Cobra, Paulo; Tonelli, Marco; Eghbalnia, Hamid R; Caviedes-Vidal, Enrique; Karasov, William H; Markley, John L

    2017-11-16

    Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue. We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer. Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.7 mm cryogenic probe. In order to evaluate the feasibility of this protocol for metabolomics studies, we analyzed the metabolic profile of livers from house sparrow ( Passer domesticus ) nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate diet (HC) for 1 d. The protocol enabled the detection of 52 polar and nine non-polar metabolites in ¹H NMR spectra of the extracts. We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups. The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of liver tissue (approx. 100 mg wet mass) obtainable from small animals.

  2. Rapid Sampling of Hydrogen Bond Networks for Computational Protein Design.

    Science.gov (United States)

    Maguire, Jack B; Boyken, Scott E; Baker, David; Kuhlman, Brian

    2018-05-08

    Hydrogen bond networks play a critical role in determining the stability and specificity of biomolecular complexes, and the ability to design such networks is important for engineering novel structures, interactions, and enzymes. One key feature of hydrogen bond networks that makes them difficult to rationally engineer is that they are highly cooperative and are not energetically favorable until the hydrogen bonding potential has been satisfied for all buried polar groups in the network. Existing computational methods for protein design are ill-equipped for creating these highly cooperative networks because they rely on energy functions and sampling strategies that are focused on pairwise interactions. To enable the design of complex hydrogen bond networks, we have developed a new sampling protocol in the molecular modeling program Rosetta that explicitly searches for sets of amino acid mutations that can form self-contained hydrogen bond networks. For a given set of designable residues, the protocol often identifies many alternative sets of mutations/networks, and we show that it can readily be applied to large sets of residues at protein-protein interfaces or in the interior of proteins. The protocol builds on a recently developed method in Rosetta for designing hydrogen bond networks that has been experimentally validated for small symmetric systems but was not extensible to many larger protein structures and complexes. The sampling protocol we describe here not only recapitulates previously validated designs with performance improvements but also yields viable hydrogen bond networks for cases where the previous method fails, such as the design of large, asymmetric interfaces relevant to engineering protein-based therapeutics.

  3. Thermodynamic properties of an extremely rapid protein folding reaction.

    Science.gov (United States)

    Schindler, T; Schmid, F X

    1996-12-24

    The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to

  4. Rapid and efficient protein enzymatic digestion: An experimental comparison

    Czech Academy of Sciences Publication Activity Database

    Dyčka, Filip; Bobáľ, P.; Mazanec, Karel; Bobálová, Janette

    2012-01-01

    Roč. 33, č. 2 (2012), s. 288-295 ISSN 0173-0835 R&D Projects: GA MŠk 1M0570; GA MŠk 1M06030; GA AV ČR IAA600040701 Institutional research plan: CEZ:AV0Z40310501 Keywords : in-gel digestion * MALDI - TOF MS * protein Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.261, year: 2012

  5. Rapid mapping of protein functional epitopes by combinatorial alanine scanning

    OpenAIRE

    Weiss, GA; Watanabe, CK; Zhong, A; Goddard, A; Sidhu, SS

    2000-01-01

    A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine ...

  6. Candidate protein biomarkers as rapid indicators of radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Horn, Simon, E-mail: sjh.horn@gmail.com [Health Protection Agency, Centre for Radiation, Chemical and Environmental Hazards, Chilton, Oxfordshire OX11 0RQ (United Kingdom); Queen' s University Belfast, Centre for Cancer Research and Cell Biology, Belfast BT9 7BL (United Kingdom); Rothkamm, Kai [Health Protection Agency, Centre for Radiation, Chemical and Environmental Hazards, Chilton, Oxfordshire OX11 0RQ (United Kingdom)

    2011-09-15

    For large scale exposures of the human population to ionising radiation, there is a need for cost-effective high throughput assessment of radiation exposure levels from biological samples to allow triage decisions to be made. Here we assess the usefulness of H2AX phosphorylation, 53BP1 foci formation, p53 induction and caspase activation as tools for biological dosimetry. Peripheral blood lymphocytes from healthy donors were isolated and exposed to X-rays. Cells were fixed, permeabilised and then stained with primary antibodies for {gamma}-H2AX and/or 53BP1, p53 or FLICA caspase detection kit followed by fluorescently tagged secondary antibodies. Cell nuclei were DAPI or propidium iodide counterstained for microscopy or cytometry respectively. Average {gamma}-H2AX/53BP1 foci numbers and {gamma}-H2AX fluorescence intensities increased with dose. Foci loss occurred over a period of 24 h post exposure with foci levels remaining above baseline levels for at least 24 h following exposure to 0.5 Gy or more of X-rays. p53 levels increased with dose and over time, peaking at 48 h post exposure. Apoptotic cells were highlighted with greatly increased levels of activated caspases. A single dose of 4 Gy increased the percentage of apoptotic lymphocytes to over 60% at 96 h post exposure. The finding that the biomarkers analysed here have different temporal dynamics following radiation exposure suggests that they could be combined to enable detection of exposures over a period of hours to several days after a radiation incident to help facilitate rapid triage.

  7. Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

    Directory of Open Access Journals (Sweden)

    Kevin R Ramkissoon

    Full Text Available The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

  8. Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

    Science.gov (United States)

    Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

    2013-01-01

    The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

  9. Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells

    International Nuclear Information System (INIS)

    Canut, H.; Alibert, G.; Carrasco, A.; Boudet, A.M.

    1986-01-01

    In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([ 14 C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([ 14 C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole

  10. New Computational Approaches for NMR-based Drug Design: A Protocol for Ligand Docking to Flexible Target Sites

    International Nuclear Information System (INIS)

    Gracia, Luis; Speidel, Joshua A.; Weinstein, Harel

    2006-01-01

    NMR-based drug design has met with some success in the last decade, as illustrated in numerous instances by Fesik's ''ligand screening by NMR'' approach. Ongoing efforts to generalize this success have led us to the development of a new paradigm in which quantitative computational approaches are being integrated with NMR derived data and biological assays. The key component of this work is the inclusion of the intrinsic dynamic quality of NMR structures in theoretical models and its use in docking. A new computational protocol is introduced here, designed to dock small molecule ligands to flexible proteins derived from NMR structures. The algorithm makes use of a combination of simulated annealing monte carlo simulations (SA/MC) and a mean field potential informed by the NMR data. The new protocol is illustrated in the context of an ongoing project aimed at developing new selective inhibitors for the PCAF bromodomains that interact with HIV Tat

  11. Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly.

    Science.gov (United States)

    Aird, Steven D; Aggarwal, Shikha; Villar-Briones, Alejandro; Tin, Mandy Man-Ying; Terada, Kouki; Mikheyev, Alexander S

    2015-08-28

    While many studies have shown that extracellular proteins evolve rapidly, how selection acts on them remains poorly understood. We used snake venoms to understand the interaction between ecology, expression level, and evolutionary rate in secreted protein systems. Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. Venoms are generally optimized to subdue preferred prey more effectively than non-prey, and many venom protein families manifest positive selection and rapid gene family diversification. Although previous studies have illuminated how individual venom protein families evolve, how selection acts on venoms as integrated systems, is unknown. Using next-generation transcriptome sequencing and mass spectrometry, we examined microevolution in two pitvipers, allopatrically separated for at least 1.6 million years, and their hybrids. Transcriptomes of parental species had generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. For instance, a phospholipase A2 transcript comprising 73.4 % of the Protobothrops elegans transcriptome, was barely present in the P. flavoviridis transcriptome (king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms. Looking more broadly at Protobothrops, we show that rapid evolution of the most abundant components is due to positive selection, suggesting an interplay between abundance and adaptation. Given log-scale differences in toxin abundance, which are likely correlated with biosynthetic costs, we hypothesize that as a result of natural selection, snakes optimize return on energetic investment by producing more of venom proteins that increase their fitness. Natural selection then acts on the additive genetic variance of these components, in proportion to their contributions to overall fitness. Adaptive

  12. Biospecific protein immobilization for rapid analysis of weak protein interactions using self-interaction nanoparticle spectroscopy.

    Science.gov (United States)

    Bengali, Aditya N; Tessier, Peter M

    2009-10-01

    "Reversible" protein interactions govern diverse biological behavior ranging from intracellular transport and toxic protein aggregation to protein crystallization and inactivation of protein therapeutics. Much less is known about weak protein interactions than their stronger counterparts since they are difficult to characterize, especially in a parallel format (in contrast to a sequential format) necessary for high-throughput screening. We have recently introduced a highly efficient approach of characterizing protein self-association, namely self-interaction nanoparticle spectroscopy (SINS; Tessier et al., 2008; J Am Chem Soc 130:3106-3112). This approach exploits the separation-dependent optical properties of gold nanoparticles to detect weak self-interactions between proteins immobilized on nanoparticles. A limitation of our previous work is that differences in the sequence and structure of proteins can lead to significant differences in their affinity to adsorb to nanoparticle surfaces, which complicates analysis of the corresponding protein self-association behavior. In this work we demonstrate a highly specific approach for coating nanoparticles with proteins using biotin-avidin interactions to generate protein-nanoparticle conjugates that report protein self-interactions through changes in their optical properties. Using lysozyme as a model protein that is refractory to characterization by conventional SINS, we demonstrate that surface Plasmon wavelengths for gold-avidin-lysozyme conjugates over a range of solution conditions (i.e., pH and ionic strength) are well correlated with lysozyme osmotic second virial coefficient measurements. Since SINS requires orders of magnitude less protein and time than conventional methods (e.g., static light scattering), we envision this approach will find application in large screens of protein self-association aimed at either preventing (e.g., protein aggregation) or promoting (e.g., protein crystallization) these

  13. NMR-based metabolomics of mammalian cell and tissue cultures

    International Nuclear Information System (INIS)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan; Niemitz, Matthias; Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D.

    2011-01-01

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  14. NMR-based metabolomics of mammalian cell and tissue cultures

    Energy Technology Data Exchange (ETDEWEB)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan [Bristol-Myers Squibb Company (United States); Niemitz, Matthias [PERCH Solutions Ltd. (Finland); Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D., E-mail: michael.reily@bms.com [Bristol-Myers Squibb Company (United States)

    2011-04-15

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  15. Sensitive rapid analysis of iodine-labelled protein mixture on flat substrates with high spatial resolution

    International Nuclear Information System (INIS)

    Zanevskij, Yu.V.; Ivanov, A.B.; Movchan, S.A.; Peshekhonov, V.D.; Chan Dyk Tkhan'; Chernenko, S.P.; Kaminir, L.B.; Krejndlin, Eh.Ya.; Chernyj, A.A.

    1983-01-01

    Usability of rapid analysis by electrophoresis of the admixture of I 125 -labelled proteins on flat samples by means of URAN type installation developed using a multiwire proportional chamber is studied. The sensitivity of the method is better than 200 cpm/cm 2 and the spatial resolution is approximately 1 mm. The procedure of the rapid analysis is no longer than several tens of minutes

  16. Ischemic stroke progress evaluation by {sup 31}P NMR-based metabonomic of human serum

    Energy Technology Data Exchange (ETDEWEB)

    Grandizoli, Caroline W.P.S.; Barison, Andersson, E-mail: andernmr@ufpr.br [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Departamento de Quimica. Centro de RMN; Lange, Marcos C.; Novak, Felipe T. M. [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Hospital de Clínicas. Divisao de Neurologia; Campos, Francinete R. [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Departmento de Farmacia

    2014-07-01

    In this work, chemometric analyses over {sup 31}P{"1"H} NMR (nuclear magnetic resonance) spectra of human blood serum permitted to discriminated ischemic stroke patients from health individuals due to changes in the chemical composition of phosphorus-containing compounds. These results indicate that {sup 31}P NMR-based metabonomic allowed insights over the mechanism triggered by ischemic stroke. (author)

  17. NMR-based metabolomics for identification of α-amylase inhibitors in rowan berries (Sorbus spp.)

    DEFF Research Database (Denmark)

    Broholm, Sofie L.; Gramsbergen, Simone; Nyberg, Nils

    Type 2 diabetes is a metabolic disorder estimated to affect millions of people all over the world.1 One way of reducing diabetes-related complications is to control postprandial glucose.2 Inhibition of the carbohydrate digestive enzyme α-amylase is a therapeutic target for maintaining low blood g...... a 1H-NMR method suitable for NMR-based metabolomics...

  18. Ischemic stroke progress evaluation by 31P NMR-based metabonomic of human serum

    International Nuclear Information System (INIS)

    Grandizoli, Caroline W.P.S.; Barison, Andersson; Lange, Marcos C.; Novak, Felipe T. M.; Campos, Francinete R.

    2014-01-01

    In this work, chemometric analyses over 31 P{ 1H } NMR (nuclear magnetic resonance) spectra of human blood serum permitted to discriminated ischemic stroke patients from health individuals due to changes in the chemical composition of phosphorus-containing compounds. These results indicate that 31 P NMR-based metabonomic allowed insights over the mechanism triggered by ischemic stroke. (author)

  19. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    Directory of Open Access Journals (Sweden)

    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  20. A multi-directional rapidly exploring random graph (mRRG) for protein folding

    KAUST Repository

    Nath, Shuvra Kanti; Thomas, Shawna; Ekenna, Chinwe; Amato, Nancy M.

    2012-01-01

    Modeling large-scale protein motions, such as those involved in folding and binding interactions, is crucial to better understanding not only how proteins move and interact with other molecules but also how proteins misfold, thus causing many devastating diseases. Robotic motion planning algorithms, such as Rapidly Exploring Random Trees (RRTs), have been successful in simulating protein folding pathways. Here, we propose a new multi-directional Rapidly Exploring Random Graph (mRRG) specifically tailored for proteins. Unlike traditional RRGs which only expand a parent conformation in a single direction, our strategy expands the parent conformation in multiple directions to generate new samples. Resulting samples are connected to the parent conformation and its nearest neighbors. By leveraging multiple directions, mRRG can model the protein motion landscape with reduced computational time compared to several other robotics-based methods for small to moderate-sized proteins. Our results on several proteins agree with experimental hydrogen out-exchange, pulse-labeling, and F-value analysis. We also show that mRRG covers the conformation space better as compared to the other computation methods. Copyright © 2012 ACM.

  1. Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

    Science.gov (United States)

    Yong, K J; Scott, D J

    2015-03-01

    Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

  2. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    International Nuclear Information System (INIS)

    Sun, L W; Zhao, Y; Jiang, R; Song, Y; Feng, H; Feng, K; Niu, L P; Qi, C

    2011-01-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  3. Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

    Science.gov (United States)

    Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

    2017-06-20

    Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

  4. Positive selection and propeptide repeats promote rapid interspecific divergence of a gastropod sperm protein.

    Science.gov (United States)

    Hellberg, M E; Moy, G W; Vacquier, V D

    2000-03-01

    Male-specific proteins have increasingly been reported as targets of positive selection and are of special interest because of the role they may play in the evolution of reproductive isolation. We report the rapid interspecific divergence of cDNA encoding a major acrosomal protein of unknown function (TMAP) of sperm from five species of teguline gastropods. A mitochondrial DNA clock (calibrated by congeneric species divided by the Isthmus of Panama) estimates that these five species diverged 2-10 MYA. Inferred amino acid sequences reveal a propeptide that has diverged rapidly between species. The mature protein has diverged faster still due to high nonsynonymous substitution rates (> 25 nonsynonymous substitutions per site per 10(9) years). cDNA encoding the mature protein (89-100 residues) shows evidence of positive selection (Dn/Ds > 1) for 4 of 10 pairwise species comparisons. cDNA and predicted secondary-structure comparisons suggest that TMAP is neither orthologous nor paralogous to abalone lysin, and thus marks a second, phylogenetically independent, protein subject to strong positive selection in free-spawning marine gastropods. In addition, an internal repeat in one species (Tegula aureotincta) produces a duplicated cleavage site which results in two alternatively processed mature proteins differing by nine amino acid residues. Such alternative processing may provide a mechanism for introducing novel amino acid sequence variation at the amino-termini of proteins. Highly divergent TMAP N-termini from two other tegulines (Tegula regina and Norrisia norrisii) may have originated by such a mechanism.

  5. A rapid, ensemble and free energy based method for engineering protein stabilities.

    Science.gov (United States)

    Naganathan, Athi N

    2013-05-02

    Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile-thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.

  6. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  7. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  8. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    Energy Technology Data Exchange (ETDEWEB)

    Scaife, R.M. (Fred Hutchinson Cancer Research Center, Seattle, WA (United States)); Wilson, L. (Univ. of California, Santa Barbara (United States)); Purich, D.L. (Univ. of Florida, Gainesville (United States))

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  9. Rapid changes in protein phosphorylation associated with gravity perception in corn roots

    International Nuclear Information System (INIS)

    McFadden, J.J.; Poovaiah, B.W.

    1987-01-01

    A previous paper from this laboratory showed calcium- and calmodulin-dependent in vivo protein phosphorylation in corn root tips. The authors show that rapid changes in calcium-dependent protein phosphorylation are involved in light-dependent graviperception in corn root tips. Corn seedlings (Zea mays L, cv Merit) were grown in the dark for 3 d, then apical root segments were harvested in dim green light to measure in vivo protein phosphorylation. Segments were incubated with 0.5 mCi 32 P for 1 h, then immediately frozen in liquid N 2 or first treated with either 7 min light, or 7 min light plus 1 mM EGTA and 10 μM A23187. Labeled proteins were separated by 2D gel electrophoresis and detected by autoradiography. Light caused rapid and specific promotion of phosphorylation of 5 polypeptides. The increases in protein phosphorylation were reversed by treating with EGTA and A23187. The authors postulate that these changes in protein phosphorylation are an essential part of the light-dependent gravity response in Merit roots

  10. Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics

    OpenAIRE

    Rist, Manuela; Muhle-Goll, Claudia; Görling, Benjamin; Bub, Achim; Heissler, Stefan; Watzl, Bernhard; Luy, Burkhard

    2013-01-01

    It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine sample...

  11. Automics: an integrated platform for NMR-based metabonomics spectral processing and data analysis

    Directory of Open Access Journals (Sweden)

    Qu Lijia

    2009-03-01

    Full Text Available Abstract Background Spectral processing and post-experimental data analysis are the major tasks in NMR-based metabonomics studies. While there are commercial and free licensed software tools available to assist these tasks, researchers usually have to use multiple software packages for their studies because software packages generally focus on specific tasks. It would be beneficial to have a highly integrated platform, in which these tasks can be completed within one package. Moreover, with open source architecture, newly proposed algorithms or methods for spectral processing and data analysis can be implemented much more easily and accessed freely by the public. Results In this paper, we report an open source software tool, Automics, which is specifically designed for NMR-based metabonomics studies. Automics is a highly integrated platform that provides functions covering almost all the stages of NMR-based metabonomics studies. Automics provides high throughput automatic modules with most recently proposed algorithms and powerful manual modules for 1D NMR spectral processing. In addition to spectral processing functions, powerful features for data organization, data pre-processing, and data analysis have been implemented. Nine statistical methods can be applied to analyses including: feature selection (Fisher's criterion, data reduction (PCA, LDA, ULDA, unsupervised clustering (K-Mean and supervised regression and classification (PLS/PLS-DA, KNN, SIMCA, SVM. Moreover, Automics has a user-friendly graphical interface for visualizing NMR spectra and data analysis results. The functional ability of Automics is demonstrated with an analysis of a type 2 diabetes metabolic profile. Conclusion Automics facilitates high throughput 1D NMR spectral processing and high dimensional data analysis for NMR-based metabonomics applications. Using Automics, users can complete spectral processing and data analysis within one software package in most cases

  12. Automics: an integrated platform for NMR-based metabonomics spectral processing and data analysis.

    Science.gov (United States)

    Wang, Tao; Shao, Kang; Chu, Qinying; Ren, Yanfei; Mu, Yiming; Qu, Lijia; He, Jie; Jin, Changwen; Xia, Bin

    2009-03-16

    Spectral processing and post-experimental data analysis are the major tasks in NMR-based metabonomics studies. While there are commercial and free licensed software tools available to assist these tasks, researchers usually have to use multiple software packages for their studies because software packages generally focus on specific tasks. It would be beneficial to have a highly integrated platform, in which these tasks can be completed within one package. Moreover, with open source architecture, newly proposed algorithms or methods for spectral processing and data analysis can be implemented much more easily and accessed freely by the public. In this paper, we report an open source software tool, Automics, which is specifically designed for NMR-based metabonomics studies. Automics is a highly integrated platform that provides functions covering almost all the stages of NMR-based metabonomics studies. Automics provides high throughput automatic modules with most recently proposed algorithms and powerful manual modules for 1D NMR spectral processing. In addition to spectral processing functions, powerful features for data organization, data pre-processing, and data analysis have been implemented. Nine statistical methods can be applied to analyses including: feature selection (Fisher's criterion), data reduction (PCA, LDA, ULDA), unsupervised clustering (K-Mean) and supervised regression and classification (PLS/PLS-DA, KNN, SIMCA, SVM). Moreover, Automics has a user-friendly graphical interface for visualizing NMR spectra and data analysis results. The functional ability of Automics is demonstrated with an analysis of a type 2 diabetes metabolic profile. Automics facilitates high throughput 1D NMR spectral processing and high dimensional data analysis for NMR-based metabonomics applications. Using Automics, users can complete spectral processing and data analysis within one software package in most cases. Moreover, with its open source architecture, interested

  13. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

    Science.gov (United States)

    Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

    2014-12-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

  14. Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis

    International Nuclear Information System (INIS)

    Heidenreich, K.A.; Toledo, S.P.

    1989-01-01

    In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of [3H]leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of [3H]aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that (1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; (2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and (3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons

  15. Toxic responses of Perna viridis hepatopancreas exposed to DDT, benzo(a)pyrene and their mixture uncovered by iTRAQ-based proteomics and NMR-based metabolomics.

    Science.gov (United States)

    Song, Qinqin; Zhou, Hailong; Han, Qian; Diao, Xiaoping

    2017-11-01

    Dichlorodiphenyltrichloroethane (DDT) and benzo(a)pyrene (BaP) are environmental estrogens (EEs) that are ubiquitous in the marine environment. In the present study, we integrated isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic and nuclear magnetic resonance (NMR)-based metabolomic approaches to explore the toxic responses of green mussel hepatopancreas exposed to DDT (10μg/L), BaP (10μg/L) and their mixture. The metabolic responses indicated that BaP primarily disturbed energy metabolism and osmotic regulation in the hepatopancreas of the male green mussel P. viridis. Both DDT and the mixture of DDT and BaP perturbed the energy metabolism and osmotic regulation in P. viridis. The proteomic responses revealed that BaP affected the proteins involved in energy metabolism, material transformation, cytoskeleton, stress responses, reproduction and development in green mussels. DDT exposure could change the proteins involved in primary metabolism, stress responses, cytoskeleton and signal transduction. However, the mixture of DDT and BaP altered proteins associated with material and energy metabolism, stress responses, signal transduction, reproduction and development, cytoskeleton and apoptosis. This study showed that iTRAQ-based proteomic and NMR-based metabolomic approaches could effectively elucidate the essential molecular mechanism of disturbances in hepatopancreas function of green mussels exposed to environmental estrogens. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    Science.gov (United States)

    Li, Shikuo; Shen, Yuhua; Xie, Anjian; Yu, Xuerong; Zhang, Xiuzhen; Yang, Liangbao; Li, Chuanhao

    2007-10-01

    We describe the formation of amorphous selenium (α-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO32-) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO32- ions to Se0, but also controls the nucleation and growth of Se0, and even participates in the formation of α-Se/protein composites. The size and shell thickness of the α-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO32- ions by Capsicum annuum L extract.

  17. Rapid evolution of the sequences and gene repertoires of secreted proteins in bacteria.

    Directory of Open Access Journals (Sweden)

    Teresa Nogueira

    Full Text Available Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation.

  18. A rapid and cost-effective fluorescence detection in tube (FDIT) method to analyze protein phosphorylation.

    Science.gov (United States)

    Jin, Xiao; Gou, Jin-Ying

    2016-01-01

    Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life. Here we adapted Pro-Q ® Diamond (Pro-Q ® Diamond Phosphoprotein Gel Stain), a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT) method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q ® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1) on a thylakoid ascorbate peroxidase (tAPX), an established phosphorylation target in our earlier study. The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays.

  19. A rapid and cost-effective fluorescence detection in tube (FDIT method to analyze protein phosphorylation

    Directory of Open Access Journals (Sweden)

    Xiao Jin

    2016-11-01

    Full Text Available Abstract Background Protein phosphorylation is one of the most important post-translational modifications catalyzed by protein kinases in living organisms. The advance of genome sequencing provided the information of protein kinase families in many organisms, including both model and non-model plants. The development of proteomics technologies also enabled scientists to efficiently reveal a large number of protein phosphorylations of an organism. However, kinases and phosphorylation targets are still to be connected to illustrate the complicated network in life. Results Here we adapted Pro-Q® Diamond (Pro-Q® Diamond Phosphoprotein Gel Stain, a widely used phosphoprotein gel-staining fluorescence dye, to establish a rapid, economical and non-radioactive fluorescence detection in tube (FDIT method to analyze phosphorylated proteins. Taking advantages of high sensitivity and specificity of Pro-Q® diamond, the FDIT method is also demonstrated to be rapid and reliable, with a suitable linear range for in vitro protein phosphorylation. A significant and satisfactory protein kinase reaction was detected as fast as 15 min from Wheat Kinase START 1.1 (WKS1.1 on a thylakoid ascorbate peroxidase (tAPX, an established phosphorylation target in our earlier study. Conclusion The FDIT method saves up to 95% of the dye consumed in a gel staining method. The FDIT method is remarkably quick, highly reproducible, unambiguous and capable to be scaled up to dozens of samples. The FDIT method could serve as a simple and sensitive alternative procedure to determine protein kinase reactions with zero radiation exposure, as a supplementation to other widely used radioactive and in-gel assays.

  20. Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

    Science.gov (United States)

    Urmann, Katharina; Reich, Peggy; Walter, Johanna-Gabriela; Beckmann, Dieter; Segal, Ester; Scheper, Thomas

    2017-09-10

    Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23μM. The apparent dissociation constant was determined as 13.98μM and the LoD is 3.17μM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Rapid microscale in-gel processing and digestion of proteins using surface acoustic waves.

    Science.gov (United States)

    Kulkarni, Ketav P; Ramarathinam, Sri H; Friend, James; Yeo, Leslie; Purcell, Anthony W; Perlmutter, Patrick

    2010-06-21

    A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.

  2. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    International Nuclear Information System (INIS)

    Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

    2007-01-01

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

  3. Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

    International Nuclear Information System (INIS)

    Ducat, Thierry; Declerck, Nathalie; Gostan, Thierry; Kochoyan, Michel; Demene, Helene

    2006-01-01

    Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters

  4. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  5. Super: a web server to rapidly screen superposable oligopeptide fragments from the protein data bank

    Science.gov (United States)

    Collier, James H.; Lesk, Arthur M.; Garcia de la Banda, Maria; Konagurthu, Arun S.

    2012-01-01

    Searching for well-fitting 3D oligopeptide fragments within a large collection of protein structures is an important task central to many analyses involving protein structures. This article reports a new web server, Super, dedicated to the task of rapidly screening the protein data bank (PDB) to identify all fragments that superpose with a query under a prespecified threshold of root-mean-square deviation (RMSD). Super relies on efficiently computing a mathematical bound on the commonly used structural similarity measure, RMSD of superposition. This allows the server to filter out a large proportion of fragments that are unrelated to the query; >99% of the total number of fragments in some cases. For a typical query, Super scans the current PDB containing over 80 500 structures (with ∼40 million potential oligopeptide fragments to match) in under a minute. Super web server is freely accessible from: http://lcb.infotech.monash.edu.au/super. PMID:22638586

  6. Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples

    DEFF Research Database (Denmark)

    Poulsen, Nicklas N; Andersen, Nina Z; Østergaard, Jesper

    2015-01-01

    Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject...... to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system...... in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator...

  7. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  8. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  9. Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rongyue; Li, Qiang; Li, Juan; Zhou, Weiqing; Ye, Peili; Gao, Yang; Ma, Guanghui, E-mail: ghma@home.ipe.ac.cn; Su, Zhiguo

    2012-12-01

    Protein denaturation and nonspecific adsorption on polymer media as a chromatographic support have been a problem which needs to be overcome. Macroporous poly(glycidyl methacrylate-divinylbezene) (PGMA-DVB) microspheres prepared in this study were firstly covalently coated with dextran through a three-step method. The dextran was firstly adsorbed onto the microspheres and then covalently bound to the PGMA-DVB microsphere through ether bonds which were formed by hydroxyl group reacting with epoxy group at the presence of 4-(Dimethylamino) pyridine. Finally, the coating dextran layer was crosslinked by ethylene glycol diglycidyl ether to form the continuous network coating. The coated microspheres were characterized by Fourier transform infrared spectra, scanning electron microscope, mercury porosimetry measurements, laser scanning confocal microscope, and protein adsorption experiments. Results showed that PGMA-DVB microspheres coated with dextran successfully maintained the macroporous structure and high permeability. The backpressure was only 1.69 MPa at a high flow rate of 2891 cm/h. Consequently, the hydrophilicity and biocompatibility of modified microspheres were greatly improved, and the contact angle decreased from 184 Degree-Sign to 13 Degree-Sign , and nonspecific adsorption of proteins was decreased to little or none. The clad dextran coating with large amounts of hydroxyl group was easily derived to be various functional groups. The derived media have great potential applications in rapid protein chromatography. - Highlights: Black-Right-Pointing-Pointer Macroporous PGMA-DVB microspheres were covalently coated with dextran. Black-Right-Pointing-Pointer The hydrophilicity of the coated microspheres was significantly improved. Black-Right-Pointing-Pointer The irreversible adsorption of proteins was reduced to zero. Black-Right-Pointing-Pointer The coated microspheres can maintain the macropore structure. Black-Right-Pointing-Pointer The coated microspheres

  10. Covalently coating dextran on macroporous polyglycidyl methacrylate microsphere enabled rapid protein chromatographic separation

    International Nuclear Information System (INIS)

    Zhang, Rongyue; Li, Qiang; Li, Juan; Zhou, Weiqing; Ye, Peili; Gao, Yang; Ma, Guanghui; Su, Zhiguo

    2012-01-01

    Protein denaturation and nonspecific adsorption on polymer media as a chromatographic support have been a problem which needs to be overcome. Macroporous poly(glycidyl methacrylate–divinylbezene) (PGMA–DVB) microspheres prepared in this study were firstly covalently coated with dextran through a three-step method. The dextran was firstly adsorbed onto the microspheres and then covalently bound to the PGMA–DVB microsphere through ether bonds which were formed by hydroxyl group reacting with epoxy group at the presence of 4-(Dimethylamino) pyridine. Finally, the coating dextran layer was crosslinked by ethylene glycol diglycidyl ether to form the continuous network coating. The coated microspheres were characterized by Fourier transform infrared spectra, scanning electron microscope, mercury porosimetry measurements, laser scanning confocal microscope, and protein adsorption experiments. Results showed that PGMA–DVB microspheres coated with dextran successfully maintained the macroporous structure and high permeability. The backpressure was only 1.69 MPa at a high flow rate of 2891 cm/h. Consequently, the hydrophilicity and biocompatibility of modified microspheres were greatly improved, and the contact angle decreased from 184° to 13°, and nonspecific adsorption of proteins was decreased to little or none. The clad dextran coating with large amounts of hydroxyl group was easily derived to be various functional groups. The derived media have great potential applications in rapid protein chromatography. - Highlights: ► Macroporous PGMA–DVB microspheres were covalently coated with dextran. ► The hydrophilicity of the coated microspheres was significantly improved. ► The irreversible adsorption of proteins was reduced to zero. ► The coated microspheres can maintain the macropore structure. ► The coated microspheres were applied to rapid protein separation.

  11. Rapid labeling of intracellular His-tagged proteins in living cells.

    Science.gov (United States)

    Lai, Yau-Tsz; Chang, Yuen-Yan; Hu, Ligang; Yang, Ya; Chao, Ailun; Du, Zhi-Yan; Tanner, Julian A; Chye, Mee-Len; Qian, Chengmin; Ng, Kwan-Ming; Li, Hongyan; Sun, Hongzhe

    2015-03-10

    Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.

  12. NMR-based urine analysis in rats: prediction of proximal tubule kidney toxicity and phospholipidosis.

    Science.gov (United States)

    Lienemann, Kai; Plötz, Thomas; Pestel, Sabine

    2008-01-01

    The aim of safety pharmacology is early detection of compound-induced side-effects. NMR-based urine analysis followed by multivariate data analysis (metabonomics) identifies efficiently differences between toxic and non-toxic compounds; but in most cases multiple administrations of the test compound are necessary. We tested the feasibility of detecting proximal tubule kidney toxicity and phospholipidosis with metabonomics techniques after single compound administration as an early safety pharmacology approach. Rats were treated orally, intravenously, inhalatively or intraperitoneally with different test compounds. Urine was collected at 0-8 h and 8-24 h after compound administration, and (1)H NMR-patterns were recorded from the samples. Variation of post-processing and feature extraction methods led to different views on the data. Support Vector Machines were trained on these different data sets and then aggregated as experts in an Ensemble. Finally, validity was monitored with a cross-validation study using a training, validation, and test data set. Proximal tubule kidney toxicity could be predicted with reasonable total classification accuracy (85%), specificity (88%) and sensitivity (78%). In comparison to alternative histological studies, results were obtained quicker, compound need was reduced, and very importantly fewer animals were needed. In contrast, the induction of phospholipidosis by the test compounds could not be predicted using NMR-based urine analysis or the previously published biomarker PAG. NMR-based urine analysis was shown to effectively predict proximal tubule kidney toxicity after single compound administration in rats. Thus, this experimental design allows early detection of toxicity risks with relatively low amounts of compound in a reasonably short period of time.

  13. Metabolic Effect of Dietary Taurine Supplementation on Nile Tilapia (Oreochromis nilotictus) Evaluated by NMR-Based Metabolomics.

    Science.gov (United States)

    Shen, Guiping; Huang, Ying; Dong, Jiyang; Wang, Xuexi; Cheng, Kian-Kai; Feng, Jianghua; Xu, Jingjing; Ye, Jidan

    2018-01-10

    Taurine is indispensable in aquatic diets that are based solely on plant protein, and it promotes growth of many fish species. However, the physiological and metabolome effects of taurine on fish have not been well described. In this study, 1 H NMR-based metabolomics approaches were applied to investigate the metabolite variations in Nile tilapia (Oreochromis nilotictus) muscle in order to visualize the metabolic trajectory and reveal the possible mechanisms of metabolic effects of dietary taurine supplementation on tilapia growth. After extraction using aqueous and organic solvents, 19 taurine-induced metabolic changes were evaluated in our study. The metabolic changes were characterized by differences in carbohydrate, amino acid, lipid, and nucleotide contents. The results indicate that taurine supplementation could significantly regulate the physiological state of fish and promote growth and development. These results provide a basis for understanding the mechanism of dietary taurine supplementation in fish feeding. 1 H NMR spectroscopy, coupled with multivariate pattern recognition technologies, is an efficient and useful tool to map the fish metabolome and identify metabolic responses to different dietary nutrients in aquaculture.

  14. Chemical Composition and Seasonality of Aromatic Mediterranean Plant Species by NMR-Based Metabolomics

    Directory of Open Access Journals (Sweden)

    Monica Scognamiglio

    2015-01-01

    Full Text Available An NMR-based metabolomic approach has been applied to analyse seven aromatic Mediterranean plant species used in traditional cuisine. Based on the ethnobotanical use of these plants, the approach has been employed in order to study the metabolic changes during different seasons. Primary and secondary metabolites have been detected and quantified. Flavonoids (apigenin, quercetin, and kaempferol derivatives and phenylpropanoid derivatives (e.g., chlorogenic and rosmarinic acid are the main identified polyphenols. The richness in these metabolites could explain the biological properties ascribed to these plant species.

  15. Chemical Composition and Seasonality of Aromatic Mediterranean Plant Species by NMR-Based Metabolomics.

    Science.gov (United States)

    Scognamiglio, Monica; D'Abrosca, Brigida; Esposito, Assunta; Fiorentino, Antonio

    2015-01-01

    An NMR-based metabolomic approach has been applied to analyse seven aromatic Mediterranean plant species used in traditional cuisine. Based on the ethnobotanical use of these plants, the approach has been employed in order to study the metabolic changes during different seasons. Primary and secondary metabolites have been detected and quantified. Flavonoids (apigenin, quercetin, and kaempferol derivatives) and phenylpropanoid derivatives (e.g., chlorogenic and rosmarinic acid) are the main identified polyphenols. The richness in these metabolites could explain the biological properties ascribed to these plant species.

  16. Specific changes in rapidly transported proteins during regeneration of the goldfish optic nerve

    International Nuclear Information System (INIS)

    Benowitz, L.I.; Shashoua, V.E.; Yoon, M.G.

    1981-01-01

    Double labeling methods were used to identify changes in the complement of proteins synthesized in the retinal ganglion cells and transported down the optic nerve during the process of axonal regeneration. Eight to 62 days after goldfish underwent a unilateral optic nerve crush, one eye was labeled with [3H]-, the other with [14C]proline. Control and regenerating optic nerves were dissected out and homogenized together after 5 hr, a time which allowed us to examine selectively membrane-bound components which migrate in the rapid phase of axoplasmic transport. Proteins from the two sides were so-purified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the 3H and 14C incorporation patterns along the gels revealed a radical shift away from the normal labeling spectrum during regeneration, with selective changes in labeling at particular molecular weights varying over a 3-fold range. Eight days after crushing the optic nerve, the greatest increases in labeling were seen for material with apparent molecular weights of 24,000 to 27,000, 44,000, and 210,000 daltons. These peaks declined thereafter, and on days 29 to 39, the most prominent increases were at 110,000 to 140,000 daltons. These studies indicate a continuously changing pattern in the synthesis and/or degradation of proteins that are rapidly transported down the optic nerve during regeneration and point to molecular species potential significance in the establishment of the visual map upon the brain

  17. Rapid, Labile, and Protein Synthesis– Independent Short-Term Memory in Conditioned Taste Aversion

    OpenAIRE

    Houpt, Thomas A.; Berlin, RoseAnn

    1999-01-01

    Short-term memory is a rapid, labile, and protein-synthesis-independent phase of memory. The existence of short-term memory in conditioned taste aversion (CTA) learning has not been demonstrated formally. To determine the earliest time at which a CTA is expressed, we measured intraoral intake of sucrose at 15 min, 1 hr, 6 hr, or 48 h after contingent pairing of an intraoral infusion of 5% sucrose (6.6 ml over 6 min) and toxic lithium chloride injection (76 mg/kg). Rats were implanted with int...

  18. Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

    Directory of Open Access Journals (Sweden)

    Lihong Jiang

    2018-06-01

    Full Text Available Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors to speed up design-build-test (DBT cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

  19. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray.

    Directory of Open Access Journals (Sweden)

    Bettina Stieber

    Full Text Available S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.

  20. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

    Directory of Open Access Journals (Sweden)

    Thiel Cora S

    2012-01-01

    Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

  1. Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Rasia, Rodolfo M. [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France); Lescop, Ewen [CNRS, Institut de Chimie des Substances Naturelles (France); Palatnik, Javier F. [Universidad Nacional de Rosario, Instituto de Biologia Molecular y Celular de Rosario, Facultad de Ciencias Bioquimicas y Farmaceuticas (Argentina); Boisbouvier, Jerome, E-mail: jerome.boisbouvier@ibs.fr; Brutscher, Bernhard, E-mail: Bernhard.brutscher@ibs.fr [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France)

    2011-11-15

    It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.

  2. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    Energy Technology Data Exchange (ETDEWEB)

    Li Shikuo; Shen Yuhua; Xie Anjian; Yu Xuerong; Zhang Xiuzhen; Yang Liangbao; Li Chuanhao [School of Chemistry and Chemical Engineering, Anhui University, Hefei 230039 (China)

    2007-10-10

    We describe the formation of amorphous selenium ({alpha}-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO{sub 3}{sup 2-}) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO{sub 3}{sup 2-} ions to Se{sup 0}, but also controls the nucleation and growth of Se{sup 0}, and even participates in the formation of {alpha}-Se/protein composites. The size and shell thickness of the {alpha}-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO{sub 3}{sup 2-} ions by Capsicum annuum L extract.

  3. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    International Nuclear Information System (INIS)

    Li Shikuo; Shen Yuhua; Xie Anjian; Yu Xuerong; Zhang Xiuzhen; Yang Liangbao; Li Chuanhao

    2007-01-01

    We describe the formation of amorphous selenium (α-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO 3 2- ) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO 3 2- ions to Se 0 , but also controls the nucleation and growth of Se 0 , and even participates in the formation of α-Se/protein composites. The size and shell thickness of the α-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO 3 2- ions by Capsicum annuum L extract

  4. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping

    International Nuclear Information System (INIS)

    Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P.A.; Vogt, S.

    2010-01-01

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

  5. Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

    Science.gov (United States)

    Manikwar, Prakash; Zimmerman, Tahl; Blanco, Francisco J; Williams, Todd D; Siahaan, Teruna J

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

  6. TRDistiller: a rapid filter for enrichment of sequence datasets with proteins containing tandem repeats.

    Science.gov (United States)

    Richard, François D; Kajava, Andrey V

    2014-06-01

    The dramatic growth of sequencing data evokes an urgent need to improve bioinformatics tools for large-scale proteome analysis. Over the last two decades, the foremost efforts of computer scientists were devoted to proteins with aperiodic sequences having globular 3D structures. However, a large portion of proteins contain periodic sequences representing arrays of repeats that are directly adjacent to each other (so called tandem repeats or TRs). These proteins frequently fold into elongated fibrous structures carrying different fundamental functions. Algorithms specific to the analysis of these regions are urgently required since the conventional approaches developed for globular domains have had limited success when applied to the TR regions. The protein TRs are frequently not perfect, containing a number of mutations, and some of them cannot be easily identified. To detect such "hidden" repeats several algorithms have been developed. However, the most sensitive among them are time-consuming and, therefore, inappropriate for large scale proteome analysis. To speed up the TR detection we developed a rapid filter that is based on the comparison of composition and order of short strings in the adjacent sequence motifs. Tests show that our filter discards up to 22.5% of proteins which are known to be without TRs while keeping almost all (99.2%) TR-containing sequences. Thus, we are able to decrease the size of the initial sequence dataset enriching it with TR-containing proteins which allows a faster subsequent TR detection by other methods. The program is available upon request. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Rapid and Efficient Protein Digestion using Trypsin Coated Magnetic Nanoparticles under Pressure Cycles

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Byoungsoo; Lopez-Ferrer, Daniel; Kim, Byoung Chan; Na, Hyon Bin; Park, Yong Il; Weitz, Karl K.; Warner, Marvin G.; Hyeon, Taeghwan; Lee, Sang-Won; Smith, Richard D.; Kim, Jungbae

    2011-01-01

    Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple crosslinking of the enzyme to magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, while a control sample of covalently-attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. Digestions were carried out on a single model protein, a five protein mixture, and a whole mouse brain proteome, and also compared for digestion at atmospheric pressure and 37 ºC for 12 h, and in combination with pressure cycling technology (PCT) at room temperature for 1 min. In all cases, the EC-TR/NPs performed equally as well or better than free trypsin in terms of the number of peptide/protein identifications and reproducibility across technical replicates. However, the concomitant use of EC-TR/NPs and PCT resulted in very fast (~1 min) and more reproducible digestions.

  8. Rapid generation of protein aerosols and nanoparticles via surface acoustic wave atomization

    International Nuclear Information System (INIS)

    Alvarez, Mar; Friend, James; Yeo, Leslie Y

    2008-01-01

    We describe the fabrication of a surface acoustic wave (SAW) atomizer and show its ability to generate monodisperse aerosols and particles for drug delivery applications. In particular, we demonstrate the generation of insulin liquid aerosols for pulmonary delivery and solid protein nanoparticles for transdermal and gastrointestinal delivery routes using 20 MHz SAW devices. Insulin droplets around 3 μm were obtained, matching the optimum range for maximizing absorption in the alveolar region. A new approach is provided to explain these atomized droplet diameters by returning to fundamental physical analysis and considering viscous-capillary and inertial-capillary force balance rather than employing modifications to the Kelvin equation under the assumption of parametric forcing that has been extended to these frequencies in past investigations. In addition, we consider possible mechanisms by which the droplet ejections take place with the aid of high-speed flow visualization. Finally, we show that nanoscale protein particles (50-100 nm in diameter) were obtained through an evaporative process of the initial aerosol, the final size of which could be controlled merely by modifying the initial protein concentration. These results illustrate the feasibility of using SAW as a novel method for rapidly producing particles and droplets with a controlled and narrow size distribution.

  9. Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

    Science.gov (United States)

    Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

    1997-12-01

    In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

  10. Vibratory Reaction Unit for the Rapid Analysis of Proteins and Glycochains

    Directory of Open Access Journals (Sweden)

    Yukie Sasakura

    2007-01-01

    Full Text Available A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF, an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG. Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF would be effective for the rapid structural analysis of glycoproteins.Abbreviations: BSA: bovine serum albumin; MS: mass spectrometry; NGF: glycopeptidase F; IgG: immunoglobulin G; PTM: post-translational modification; HPLC: high-performance liquid chromatography; PBS: phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; DTT: dithiothreitol; RT: retention time; ABOE: p-aminobenzoic acid octyl ester; PDMS: polydimethylsiloxane; ArgC: endoprotease Arginine C.

  11. Rapid, labile, and protein synthesis-independent short-term memory in conditioned taste aversion.

    Science.gov (United States)

    Houpt, T A; Berlin, R

    1999-01-01

    Short-term memory is a rapid, labile, and protein-synthesis-independent phase of memory. The existence of short-term memory in conditioned taste aversion (CTA) learning has not been demonstrated formally. To determine the earliest time at which a CTA is expressed, we measured intraoral intake of sucrose at 15 min, 1 hr, 6 hr, or 48 h after contingent pairing of an intraoral infusion of 5% sucrose (6.6 ml over 6 min) and toxic lithium chloride injection (76 mg/kg). Rats were implanted with intraoral catheters to allow presentation of taste solutions at arbitrary times. Intraoral intake was measured under conditions of long-delay, single-trial learning typical of CTA. Rats decreased intraoral intake of sucrose at 15 min after contingent pairing of sucrose and LiCl, but not after noncontingent LiCl or sucrose. Thus CTA learning can be expressed rapidly. To determine if short-term CTA memory is labile and decays in the absence of long-term memory, we measured intraoral intake of sucrose after pairing sucrose with low doses of LiCl. Rats received an intraoral infusion of 5% sucrose (6 ml/6 min); 30 min later LiCl was injected at three different doses (19, 38, or 76 mg/kg). A second intraoral infusion of sucrose was administered 15 min, 1 hr, 3 hr, 4.5 hr, 6 hr, or 48 hr later. The formation of long-term CTA memory was dependent on the dose of LiCl paired with sucrose during acquisition. Low doses of LiCl induced a CTA that decayed within 6 hr after pairing. Central administration of the protein synthesis inhibitor cycloheximide prior to LiCl injection blocked long-term CTA expression at 6 and 48 hr, but not short-term CTA expression at 1 hr. Thus, short-term memory for CTA learning exists that is acquired rapidly and independent of protein synthesis, but labile in the absence of long-term memory formation.

  12. NMR-based metabolomics in human disease diagnosis: Applications, limitations, and recommendations

    KAUST Repository

    Emwas, Abdul-Hamid M.

    2013-04-03

    Metabolomics is a dynamic and emerging research field, similar to proteomics, transcriptomics and genomics in affording global understanding of biological systems. It is particularly useful in functional genomic studies in which metabolism is thought to be perturbed. Metabolomics provides a snapshot of the metabolic dynamics that reflect the response of living systems to both pathophysiological stimuli and/or genetic modification. Because this approach makes possible the examination of interactions between an organism and its diet or environment, it is particularly useful for identifying biomarkers of disease processes that involve the environment. For example, the interaction of a high fat diet with cardiovascular disease can be studied via such a metabolomics approach by modeling the interaction between genes and diet. The high reproducibility of NMR-based techniques gives this method a number of advantages over other analytical techniques in large-scale and long-term metabolomic studies, such as epidemiological studies. This approach has been used to study a wide range of diseases, through the examination of biofluids, including blood plasma/serum, urine, blister fluid, saliva and semen, as well as tissue extracts and intact tissue biopsies. However, complicating the use of NMR spectroscopy in biomarker discovery is the fact that numerous variables can effect metabolic composition including, fasting, stress, drug administration, diet, gender, age, physical activity, life style and the subject\\'s health condition. To minimize the influence of these variations in the datasets, all experimental conditions including sample collection, storage, preparation as well as NMR spectroscopic parameters and data analysis should be optimized carefully and conducted in an identical manner as described by the local standard operating protocol. This review highlights the potential applications of NMR-based metabolomics studies and gives some recommendations to improve sample

  13. Comparison of Fruits of Forsythia suspensa at Two Different Maturation Stages by NMR-Based Metabolomics

    Directory of Open Access Journals (Sweden)

    Jinping Jia

    2015-05-01

    Full Text Available Forsythiae Fructus (FF, the dried fruit of Forsythia suspensa, has been widely used as a heat-clearing and detoxifying herbal medicine in China. Green FF (GF and ripe FF (RF are fruits of Forsythia suspensa at different maturity stages collected about a month apart. FF undergoes a complex series of physical and biochemical changes during fruit ripening. However, the clinical uses of GF and RF have not been distinguished to date. In order to comprehensively compare the chemical compositions of GF and RF, NMR-based metabolomics coupled with HPLC and UV spectrophotometry methods were adopted in this study. Furthermore, the in vitro antioxidant and antibacterial activities of 50% methanol extracts of GF and RF were also evaluated. A total of 27 metabolites were identified based on NMR data, and eight of them were found to be different between the GF and RF groups. The GF group contained higher levels of forsythoside A, forsythoside C, cornoside, rutin, phillyrin and gallic acid and lower levels of rengyol and β-glucose compared with the RF group. The antioxidant activity of GF was higher than that of RF, but no significant difference was observed between the antibacterial activities of GF and RF. Given our results showing their distinct chemical compositions, we propose that NMR-based metabolic profiling can be used to discriminate between GF and RF. Differences in the chemical and biological activities of GF and RF, as well as their clinical efficacies in traditional Chinese medicine should be systematically investigated in future studies.

  14. Toxicological effects of cinnabar in rats by NMR-based metabolic profiling of urine and serum

    International Nuclear Information System (INIS)

    Wei Lai; Liao Peiqiu; Wu Huifeng; Li Xiaojing; Pei Fengkui; Li Weisheng; Wu Yijie

    2008-01-01

    Cinnabar, an important traditional Chinese mineral medicine, has been widely used as a Chinese patent medicine ingredient for sedative therapy. However, the pharmaceutical and toxicological effects of cinnabar, especially in the whole organism, were subjected to few investigations. In this study, an NMR-based metabolomics approach has been applied to investigate the toxicological effects of cinnabar after intragastrical administration (dosed at 0.5, 2 and 5 g/kg body weight) on male Wistar rats. Liver and kidney histopathology examinations and serum clinical chemistry analyses were also performed. The 1 H NMR spectra were analyzed using multivariate pattern recognition techniques to show the time- and dose-dependent biochemical variations induced by cinnabar. The metabolic signature of urinalysis from cinnabar-treated animals exhibited an increase in the levels of creatinine, acetate, acetoacetate, taurine, hippurate and phenylacetylglycine, together with a decrease in the levels of trimethyl-N-oxide, dimethylglycine and Kreb's cycle intermediates (citrate, 2-oxoglutarate and succinate). The metabolomics analyses of serum showed elevated concentrations of ketone bodies (3-D-hydroxybutyrate and acetoacetate), branched-chain amino acids (valine, leucine and isoleucine), choline and creatine as well as decreased glucose, lipids and lipoproteins from cinnabar-treated animals. These findings indicated cinnabar induced disturbance in energy metabolism, amino acid metabolism and gut microflora environment as well as slight injury in liver and kidney, which might indirectly result from cinnabar induced oxidative stress. This work illustrated the high reliability of NMR-based metabolomic approach on the study of the biochemical effects induced by traditional Chinese medicine

  15. NMR-based lipidomic analysis of blood lipoproteins differentiates the progression of coronary heart disease.

    Science.gov (United States)

    Kostara, Christina E; Papathanasiou, Athanasios; Psychogios, Nikolaos; Cung, Manh Thong; Elisaf, Moses S; Goudevenos, John; Bairaktari, Eleni T

    2014-05-02

    Abnormal lipid composition and metabolism of plasma lipoproteins play a crucial role in the pathogenesis of coronary heart disease (CHD). A (1)H NMR-based lipidomic approach was used to investigate the correlation of coronary artery stenosis with the atherogenic (non-HDL) and atheroprotective (HDL) lipid profiles in 99 patients with CHD of various stages of disease and compared with 60 patients with normal coronary arteries (NCA), all documented in coronary angiography. The pattern recognition models created from lipid profiles predicted the presence of CHD with a sensitivity of 87% and a specificity of 88% in the HDL model and with 90% and 89% in the non-HDL model, respectively. Patients with mild, moderate, and severe coronary artery stenosis were progressively differentiated from those with NCA in the non-HDL model with a statistically significant separation of severe stage from both mild and moderate. In the HDL model, the progressive differentiation of the disease stages was statistically significant only between patients with mild and severe coronary artery stenosis. The lipid constituents of lipoproteins that mainly characterized the initial stages and then the progression of the disease were the high levels of saturated fatty acids in lipids in both HDL and non-HDL particles, the low levels of HDL-phosphatidylcholine, HDL-sphingomyelin, and omega-3 fatty acids and linoleic acid in lipids in non-HDL particles. The conventional lipid marker, total cholesterol, found in low levels in HDL and in high levels in non-HDL, also contributed to the onset of the disease but with a much lower coefficient of significance. (1)H NMR-based lipidomic analysis of atherogenic and atheroprotective lipoproteins could contribute to the early evaluation of the onset of coronary artery disease and possibly to the establishment of an appropriate therapeutic option.

  16. ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

    Directory of Open Access Journals (Sweden)

    Jarom Heijmans

    2013-04-01

    Full Text Available Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER stress and activity of the unfolded protein response (UPR are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

  17. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    Science.gov (United States)

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

  18. Fluorescent QDs-polystyrene composite nanospheres for highly efficient and rapid protein antigen detection

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Changhua; Mao, Mao [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Yuan, Hang [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Shen, Huaibin [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China); Wu, Feng; Ma, Lan, E-mail: malan@sz.tsinghua.edu.cn [Tsinghua University, Life Science Division, Graduate School at Shenzhen (China); Li, Lin Song, E-mail: lsli@henu.edu.cn [Henan University, Key Laboratory for Special Functional Materials of the Ministry of Education (China)

    2013-09-15

    In this paper, high-quality carboxyl-functionalized fluorescent (red, green, and blue emitting) nanospheres (46-103 nm) consisting of hydrophobic quantum dots (QDs) and polystyrene were prepared by a miniemulsion polymerization approach. This miniemulsion polymerization approach induced a homogeneous distribution and high aqueous-phase transport efficiency of fluorescent QDs in composite nanospheres, which proved the success of our encoding QDs strategy. The obtained fluorescent nanospheres exhibited high stability in aqueous solution under a wide range of pH, different salt concentrations, PBS buffer, and thermal treatment at 80 Degree-Sign C. Based on the red emitting composite nanosphere, we performed fluorescent lateral flow immunoassay (LFIA) strips for high-sensitivity and rapid alpha-fetal protein detection. The detection limit reached 0.1 ng/mL, which was 200 times higher than commercial colloidal gold-labeled LFIA strips, and it reached similar detection level in enzyme-linked immunosorbent assay kit.

  19. Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange.

    Science.gov (United States)

    Chien, Chih-Ta Henry; Ho, Meng-Ru; Lin, Chung-Hung; Hsu, Shang-Te Danny

    2017-08-16

    Galectins are β-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8 NTD and hGal8 CTD , respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8 NTD . We further demonstrated using the NMR-based hydrogen-deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8 NTD , indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8 CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8 CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.

  20. Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

    Science.gov (United States)

    Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

    2016-03-01

    In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

  1. A rapid chemical method for lysing Arabidopsis cells for protein analysis

    Directory of Open Access Journals (Sweden)

    Takano Tetsuo

    2011-07-01

    Full Text Available Abstract Background Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once. Results We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them. Conclusions Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.

  2. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    Directory of Open Access Journals (Sweden)

    Zhang PF

    2015-09-01

    Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

  3. Rapid label-free profiling of oral cancer biomarker proteins using nano-UPLC-Q-TOF ion mobility mass spectrometry.

    Science.gov (United States)

    Nassar, Ala F; Williams, Brad J; Yaworksy, Dustin C; Patel, Vyomesh; Rusling, James F

    2016-03-01

    It has become quite clear that single cancer biomarkers cannot in general provide high sensitivity and specificity for reliable clinical cancer diagnostics. This paper explores the feasibility of rapid detection of multiple biomarker proteins in model oral cancer samples using label-free protein relative quantitation. MS-based label-free quantitative proteomics offer a rapid alternative that bypasses the need for stable isotope containing compounds to chemically bind and label proteins. Total protein content in oral cancer cell culture conditioned media was precipitated, subjected to proteolytic digestion, and then analyzed using a nano-UPLC (where UPLC is ultra-performance liquid chromatography) coupled to a hybrid Q-Tof ion-mobility mass spectrometry (MS). Rapid, simultaneous identification and quantification of multiple possible cancer biomarker proteins was achieved. In a comparative study between cancer and noncancer samples, approximately 952 proteins were identified using a high-throughput 1D ion mobility assisted data independent acquisition (IM-DIA) approach. As we previously demonstrated that interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A) were readily detected in oral cancer cell conditioned media(1), we targeted these biomarker proteins to validate our approach. Target biomarker protein IL-8 was found between 3.5 and 8.8 fmol, while VEGF-A was found at 1.45 fmol in the cancer cell media. Overall, our data suggest that the nano-UPLC-IM-DIA bioassay is a feasible approach to identify and quantify proteins in complex samples without the need for stable isotope labeling. These results have significant implications for rapid tumor diagnostics and prognostics by monitoring proteins such as IL-8 and VEGF-A implicated in cancer development and progression. The analysis in tissue or plasma is not possible at this time, but the subsequent work would be needed for validation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  5. Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips

    Directory of Open Access Journals (Sweden)

    Cheng X

    2014-12-01

    Full Text Available Xianglin Cheng,1,* Xu Pu,2,* Pen Jun,3 XiaoBo Zhu,3 Di Zhu,4 Ming Chen1 1Department of Laboratory Medicine, First Affiliated Hospital of Yangtze University, Jingzhou, 2Department of Laboratory Medicine, RenMin Hospital of Wuhan University, Wuhan, 3Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei, People’s Republic of China; 4Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA *These authors contributed equally to this study and share first authorship Background: Rapid immunochromatographic tests can detect disease markers in 10–15 minutes, which facilitates clinical diagnosis and treatment programs. However, most immunochromatographic tests employ gold nanoparticles as reporters, and these have only moderate sensitivity and act as qualitative methods for analyzing high biomarker concentrations. Methods: In this study, we introduce quantum dots (QDs as fluorescent probes and immunochromatographic strips to develop quantitative fluorescence point-of-care tests (QF-POCT to analyze C-reactive protein (CRP levels. Goat anti-rabbit IgG and rabbit IgG were used as control antibodies, and mouse monoclonal CRP antibody pairs were used for disease marker detection. One monoclonal CRP antibody was conjugated with QDs and served as a signal antibody, and the other monoclonal CRP antibody was dispensed onto the nitrocellulose membrane and served as a capturing antibody. In the presence of CRP, the fluorescence intensity of the monoclonal antibody-CRP-monoclonal antibody sandwich complex captured on the nitrocellulose membrane was determined using the fluorescence strip reader. Results: QF-POCT assays could quantitatively analyze the concentration of CRP in 15 minutes had a detection limit of 0.25 mg/L, and had a wide detection linearity range (0.5–300 mg/L. The intra-assay and interassay

  6. EzMol: A Web Server Wizard for the Rapid Visualization and Image Production of Protein and Nucleic Acid Structures.

    Science.gov (United States)

    Reynolds, Christopher R; Islam, Suhail A; Sternberg, Michael J E

    2018-01-31

    EzMol is a molecular visualization Web server in the form of a software wizard, located at http://www.sbg.bio.ic.ac.uk/ezmol/. It is designed for easy and rapid image manipulation and display of protein molecules, and is intended for users who need to quickly produce high-resolution images of protein molecules but do not have the time or inclination to use a software molecular visualization system. EzMol allows the upload of molecular structure files in PDB format to generate a Web page including a representation of the structure that the user can manipulate. EzMol provides intuitive options for chain display, adjusting the color/transparency of residues, side chains and protein surfaces, and for adding labels to residues. The final adjusted protein image can then be downloaded as a high-resolution image. There are a range of applications for rapid protein display, including the illustration of specific areas of a protein structure and the rapid prototyping of images. Copyright © 2018. Published by Elsevier Ltd.

  7. Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

    International Nuclear Information System (INIS)

    Roder, H.; Wuethrich, K.

    1986-01-01

    A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin, which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and 1 H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using 1 H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes

  8. Rapidly evolving zona pellucida domain proteins are a major component of the vitelline envelope of abalone eggs

    Science.gov (United States)

    Aagaard, Jan E.; Yi, Xianhua; MacCoss, Michael J.; Swanson, Willie J.

    2006-01-01

    Proteins harboring a zona pellucida (ZP) domain are prominent components of vertebrate egg coats. Although less well characterized, the egg coat of the non-vertebrate marine gastropod abalone (Haliotis spp.) is also known to contain a ZP domain protein, raising the possibility of a common molecular basis of metazoan egg coat structures. Egg coat proteins from vertebrate as well as non-vertebrate taxa have been shown to evolve under positive selection. Studied most extensively in the abalone system, coevolution between adaptively diverging egg coat and sperm proteins may contribute to the rapid development of reproductive isolation. Thus, identifying the pattern of evolution among egg coat proteins is important in understanding the role these genes may play in the speciation process. The purpose of the present study is to characterize the constituent proteins of the egg coat [vitelline envelope (VE)] of abalone eggs and to provide preliminary evidence regarding how selection has acted on VE proteins during abalone evolution. A proteomic approach is used to match tandem mass spectra of peptides from purified VE proteins with abalone ovary EST sequences, identifying 9 of 10 ZP domain proteins as components of the VE. Maximum likelihood models of codon evolution suggest positive selection has acted among a subset of amino acids for 6 of these genes. This work provides further evidence of the prominence of ZP proteins as constituents of the egg coat, as well as the prominent role of positive selection in diversification of these reproductive proteins. PMID:17085584

  9. Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

    Science.gov (United States)

    Zoltowski, Brian D.; Crane, Brian R.

    2009-01-01

    The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

  10. Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

    Science.gov (United States)

    Zoltowski, Brian D; Crane, Brian R

    2008-07-08

    The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

  11. Impact of metal pollution on shrimp Crangon affinis by NMR-based metabolomics

    International Nuclear Information System (INIS)

    Ji, Chenglong; Yu, Deliang; Wang, Qing; Li, Fei; Zhao, Jianmin; Wu, Huifeng

    2016-01-01

    Both cadmium and arsenic are the important metal/metalloid pollutants in the Bohai Sea. In this work, we sampled the dominant species, shrimp Crangon affinis, from three sites, the Middle of the Bohai Sea (MBS), the Yellow River Estuary (YRE) and the Laizhou Bay (LZB) along the Bohai Sea. The concentrations of metals/metalloids in shrimps C. affinis indicated that the YRE site was polluted by Cd and Pb, while the LZB site was contaminated by As. The metabolic differences between shrimps C. affinis from the reference site (MBS) and metal-pollution sites (YRE and LZB) were characterized using NMR-based metabolomics. Results indicated that the metal pollutions in YRE and LZB induced disturbances in osmotic regulation and energy metabolism via different metabolic pathways. In addition, a combination of alanine and arginine might be the biomarker of Cd contamination, while BCAAs and tyrosine could be the biomarkers of arsenic contamination in C. affinis. - Highlights: •YRE and LZB are mainly polluted by Cd and As, respectively. •Metal pollutions caused differential effects in C. affinis from different sites. •Metabolomics is useful to elucidate metal pollution-induced biological effects.

  12. Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics

    Directory of Open Access Journals (Sweden)

    Burkhard Luy

    2013-04-01

    Full Text Available It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at −20 °C, on dry ice, at −80 °C or in liquid nitrogen and then stored at −20 °C, −80 °C or in liquid nitrogen vapor phase for 1–5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at −20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.

  13. Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics.

    Science.gov (United States)

    Rist, Manuela J; Muhle-Goll, Claudia; Görling, Benjamin; Bub, Achim; Heissler, Stefan; Watzl, Bernhard; Luy, Burkhard

    2013-04-09

    It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at -20 °C, on dry ice, at -80 °C or in liquid nitrogen and then stored at -20 °C, -80 °C or in liquid nitrogen vapor phase for 1-5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at -20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.

  14. NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with chronic atrophic gastritis.

    Science.gov (United States)

    Cui, Jiajia; Liu, Yuetao; Hu, Yinghuan; Tong, Jiayu; Li, Aiping; Qu, Tingli; Qin, Xuemei; Du, Guanhua

    2017-01-05

    Chronic atrophic gastritis (CAG) is one of the most important pre-cancerous states with a high prevalence. Exploring of the underlying mechanism and potential biomarkers is of significant importance for CAG. In the present work, 1 H NMR-based metabonomics with correlative analysis was performed to analyze the metabolic features of CAG. 19 plasma metabolites and 18 urine metabolites were enrolled to construct the circulatory and excretory metabolome of CAG, which was in response to alterations of energy metabolism, inflammation, immune dysfunction, as well as oxidative stress. 7 plasma biomarkers and 7 urine biomarkers were screened to elucidate the pathogenesis of CAG based on the further correlation analysis with biochemical indexes. Finally, 3 plasma biomarkers (arginine, succinate and 3-hydroxybutyrate) and 2 urine biomarkers (α-ketoglutarate and valine) highlighted the potential to indicate risks of CAG in virtue of correlation with pepsin activity and ROC analysis. Here, our results paved a way for elucidating the underlying mechanisms in the development of CAG, and provided new avenues for the diagnosis of CAG and presented potential drug targets for treatment of CAG. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. [Monitoring of chemical components with different color traits of Tussilago farfara using NMR-based metabolomics].

    Science.gov (United States)

    Mi, Xi; Li, Zhen-yu; Qin, Xue-mei; Zhang, Li-zeng

    2013-11-01

    The quality and grade of traditional Chinese medicinal herbs were assessed by their characteristics traditionally. According to traditional experience, the quality of the purple Flos Farfarae is better than that of yellow buds. NMR-based metabolomic approach combined with significant analysis of microarray (SAM) and Spearman rank correlation analysis were used to investigate the different metabolites of the Flos Farfarae with different color feature. Principal component analysis (PCA) showed clear distinction between the purple and yellow flower buds of Tussilago farfara. The S-plot of orthogonal PLS-DA (OPLS-DA) and t test revealed that the levels of threonine, proline, phosphatidylcholine, creatinine, 4, 5-dicaffeoylquinic acid, rutin, caffeic acid, kaempferol analogues, and tussilagone were higher in the purple flower buds than that in the yellow buds, in agreement with the results of SAM and Spearman rank correlation analysis. The results confirmed the traditional medication experience that "purple flower bud is better than the yellow ones", and provide a scientific basis for assessing the quality of Flos Farfarae by the color features.

  16. An efficient and rapid method for protein detection with an example ...

    African Journals Online (AJOL)

    AJL

    2012-05-15

    May 15, 2012 ... protein expressed in Esherichia coli by staining and destaining in under 30 min. The CMW method .... the saturated solutions reached a state of dynamic ... M, Protein molecular marker; 1, the control vector; 2, the SQR protein.

  17. Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

    Science.gov (United States)

    Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

    2015-01-01

    Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

  18. Ehrlich and sarcoma 180 tumour characterisation and early detection by 1H NMR-based metabonomics of mice serum

    International Nuclear Information System (INIS)

    Grandizoli, Caroline W.P. da S.; Simonelli, Fabio; Nagata, Noemi; Barison, Andersson; Carrenho, Luise Z.B.; Francisco, Thais M.G. de; Campos, Francinete R.; Santana Filho, Arquimedes P. de; Sassaki, Guilherme L.; Kreuger, Maria R.O.

    2014-01-01

    The success of cancer treatment is directly related to early detection before symptoms emerge, although nowadays few cancers can be detected early. In this sense, 1 H nuclear magnetic resonance ( 1 H NMR)-based metabonomics was used to identify metabolic changes in biofluid as a consequence of tumours growing in mice. Through partial least squares discriminant analysis (PLS-DA) analysis of 1 H NMR spectra from serum samples it was possible to diagnose Ehrlich ascites and Sarcoma 180 tumours five and ten days after cell inoculation, respectively. Lipids, lipoproteins and lactate were the main biomarkers at onset as well as in the progress of carcinogenic process. Thus, NMR-based metabonomics can be a valuable tool to study the effects of tumour establishment on the chemical composition of biofluids. (author)

  19. Ehrlich and sarcoma 180 tumour characterisation and early detection by {sup 1}H NMR-based metabonomics of mice serum

    Energy Technology Data Exchange (ETDEWEB)

    Grandizoli, Caroline W.P. da S.; Simonelli, Fabio; Nagata, Noemi; Barison, Andersson [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Dept. de Quimica; Carrenho, Luise Z.B.; Francisco, Thais M.G. de; Campos, Francinete R. [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Dept. de Farmacia; Santana Filho, Arquimedes P. de; Sassaki, Guilherme L. [Universidade Federal do Parana (UFPR), Curitiba, PR (Brazil). Dept. de Bioquimica; Kreuger, Maria R.O. [Universidade do Vale do Itajai (UNIVALI), (Brazil). Centro de Ciencias da Saude

    2014-05-15

    The success of cancer treatment is directly related to early detection before symptoms emerge, although nowadays few cancers can be detected early. In this sense, {sup 1}H nuclear magnetic resonance ({sup 1}H NMR)-based metabonomics was used to identify metabolic changes in biofluid as a consequence of tumours growing in mice. Through partial least squares discriminant analysis (PLS-DA) analysis of {sup 1}H NMR spectra from serum samples it was possible to diagnose Ehrlich ascites and Sarcoma 180 tumours five and ten days after cell inoculation, respectively. Lipids, lipoproteins and lactate were the main biomarkers at onset as well as in the progress of carcinogenic process. Thus, NMR-based metabonomics can be a valuable tool to study the effects of tumour establishment on the chemical composition of biofluids. (author)

  20. Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

    Science.gov (United States)

    Ashok, Yashwanth; Jaakola, Veli-Pekka

    2016-01-01

    Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

  1. P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing.

    Science.gov (United States)

    Jekat, Stephan B; Ernst, Antonia M; von Bohl, Andreas; Zielonka, Sascia; Twyman, Richard M; Noll, Gundula A; Prüfer, Dirk

    2013-01-01

    Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing.

  2. P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing

    Directory of Open Access Journals (Sweden)

    Stephan B Jekat

    2013-07-01

    Full Text Available Structural phloem proteins (P-proteins are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently evidenced to be encoded by the widespread SEO gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing. 

  3. 1H NMR- based metabolomics approaches as non- invasive tools for diagnosis of endometriosis

    Directory of Open Access Journals (Sweden)

    Negar Ghazi

    2016-01-01

    Full Text Available Background: So far, non-invasive diagnostic approaches such as ultrasound, magnetic resonance imaging, or blood tests do not have sufficient diagnostic power for endometriosis disease. Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. Objective: The present study focuses on the identification of predictive biomarkers in serum by pattern recognition techniques and uses partial least square discriminant analysis, multi-layer feed forward artificial neural networks (ANNs and quadratic discriminant analysis (QDA modeling tools for the early diagnosis of endometriosis in a minimally invasive manner by 1H- NMR based metabolomics. Materials and Methods: This prospective cohort study was done in Pasteur Institute, Iran in June 2013. Serum samples of 31 infertile women with endometriosis (stage II and III who confirmed by diagnostic laparoscopy and 15 normal women were collected and analyzed by nuclear magnetic resonance spectroscopy. The model was built by using partial least square discriminant analysis, QDA, and ANNs to determine classifier metabolites for early prediction risk of disease. Results: The levels of 2- methoxyestron, 2-methoxy estradiol, dehydroepiandrostion androstendione, aldosterone, and deoxy corticosterone were enhanced significantly in infertile group. While cholesterol and primary bile acids levels were decreased. QDA model showed significant difference between two study groups. Positive and negative predict value levels obtained about 71% and 78%, respectively. ANNs provided also criteria for detection of endometriosis. Conclusion: The QDA and ANNs modeling can be used as computational tools in noninvasive diagnose of endometriosis. However, the model designed by QDA methods is more efficient compared to ANNs in diagnosis of endometriosis patients.

  4. Automatic NMR-based identification of chemical reaction types in mixtures of co-occurring reactions.

    Directory of Open Access Journals (Sweden)

    Diogo A R S Latino

    Full Text Available The combination of chemoinformatics approaches with NMR techniques and the increasing availability of data allow the resolution of problems far beyond the original application of NMR in structure elucidation/verification. The diversity of applications can range from process monitoring, metabolic profiling, authentication of products, to quality control. An application related to the automatic analysis of complex mixtures concerns mixtures of chemical reactions. We encoded mixtures of chemical reactions with the difference between the (1H NMR spectra of the products and the reactants. All the signals arising from all the reactants of the co-occurring reactions were taken together (a simulated spectrum of the mixture of reactants and the same was done for products. The difference spectrum is taken as the representation of the mixture of chemical reactions. A data set of 181 chemical reactions was used, each reaction manually assigned to one of 6 types. From this dataset, we simulated mixtures where two reactions of different types would occur simultaneously. Automatic learning methods were trained to classify the reactions occurring in a mixture from the (1H NMR-based descriptor of the mixture. Unsupervised learning methods (self-organizing maps produced a reasonable clustering of the mixtures by reaction type, and allowed the correct classification of 80% and 63% of the mixtures in two independent test sets of different similarity to the training set. With random forests (RF, the percentage of correct classifications was increased to 99% and 80% for the same test sets. The RF probability associated to the predictions yielded a robust indication of their reliability. This study demonstrates the possibility of applying machine learning methods to automatically identify types of co-occurring chemical reactions from NMR data. Using no explicit structural information about the reactions participants, reaction elucidation is performed without structure

  5. ¹H NMR-based metabolic profiling of human rectal cancer tissue

    Science.gov (United States)

    2013-01-01

    Background Rectal cancer is one of the most prevalent tumor types. Understanding the metabolic profile of rectal cancer is important for developing therapeutic approaches and molecular diagnosis. Methods Here, we report a metabonomics profiling of tissue samples on a large cohort of human rectal cancer subjects (n = 127) and normal controls (n = 43) using 1H nuclear magnetic resonance (1H NMR) based metabonomics assay, which is a highly sensitive and non-destructive method for the biomarker identification in biological systems. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA) were applied to analyze the 1H-NMR profiling data to identify the distinguishing metabolites of rectal cancer. Results Excellent separation was obtained and distinguishing metabolites were observed among the different stages of rectal cancer tissues (stage I = 35; stage II = 37; stage III = 37 and stage IV = 18) and normal controls. A total of 38 differential metabolites were identified, 16 of which were closely correlated with the stage of rectal cancer. The up-regulation of 10 metabolites, including lactate, threonine, acetate, glutathione, uracil, succinate, serine, formate, lysine and tyrosine, were detected in the cancer tissues. On the other hand, 6 metabolites, including myo-inositol, taurine, phosphocreatine, creatine, betaine and dimethylglycine were decreased in cancer tissues. These modified metabolites revealed disturbance of energy, amino acids, ketone body and choline metabolism, which may be correlated with the progression of human rectal cancer. Conclusion Our findings firstly identify the distinguishing metabolites in different stages of rectal cancer tissues, indicating possibility of the attribution of metabolites disturbance to the progression of rectal cancer. The altered metabolites may be as potential biomarkers, which would

  6. Automatic NMR-based identification of chemical reaction types in mixtures of co-occurring reactions.

    Science.gov (United States)

    Latino, Diogo A R S; Aires-de-Sousa, João

    2014-01-01

    The combination of chemoinformatics approaches with NMR techniques and the increasing availability of data allow the resolution of problems far beyond the original application of NMR in structure elucidation/verification. The diversity of applications can range from process monitoring, metabolic profiling, authentication of products, to quality control. An application related to the automatic analysis of complex mixtures concerns mixtures of chemical reactions. We encoded mixtures of chemical reactions with the difference between the (1)H NMR spectra of the products and the reactants. All the signals arising from all the reactants of the co-occurring reactions were taken together (a simulated spectrum of the mixture of reactants) and the same was done for products. The difference spectrum is taken as the representation of the mixture of chemical reactions. A data set of 181 chemical reactions was used, each reaction manually assigned to one of 6 types. From this dataset, we simulated mixtures where two reactions of different types would occur simultaneously. Automatic learning methods were trained to classify the reactions occurring in a mixture from the (1)H NMR-based descriptor of the mixture. Unsupervised learning methods (self-organizing maps) produced a reasonable clustering of the mixtures by reaction type, and allowed the correct classification of 80% and 63% of the mixtures in two independent test sets of different similarity to the training set. With random forests (RF), the percentage of correct classifications was increased to 99% and 80% for the same test sets. The RF probability associated to the predictions yielded a robust indication of their reliability. This study demonstrates the possibility of applying machine learning methods to automatically identify types of co-occurring chemical reactions from NMR data. Using no explicit structural information about the reactions participants, reaction elucidation is performed without structure elucidation of

  7. The application of NMR-based milk metabolite analysis in milk authenticity identification.

    Science.gov (United States)

    Li, Qiangqiang; Yu, Zunbo; Zhu, Dan; Meng, Xianghe; Pang, Xiumei; Liu, Yue; Frew, Russell; Chen, He; Chen, Gang

    2017-07-01

    Milk is an important food component in the human diet and is a target for fraud, including many unsafe practices. For example, the unscrupulous adulteration of soymilk into bovine and goat milk or of bovine milk into goat milk in order to gain profit without declaration is a health risk, as the adulterant source and sanitary history are unknown. A robust and fit-for-purpose technique is required to enforce market surveillance and hence protect consumer health. Nuclear magnetic resonance (NMR) is a powerful technique for characterization of food products based on measuring the profile of metabolites. In this study, 1D NMR in conjunction with multivariate chemometrics as well as 2D NMR was applied to differentiate milk types and to identify milk adulteration. Ten metabolites were found which differed among milk types, hence providing characteristic markers for identifying the milk. These metabolites were used to establish mathematical models for milk type differentiation. The limit of quantification (LOQ) of adulteration was 2% (v/v) for soymilk in bovine milk, 2% (v/v) for soymilk in goat milk and 5% (v/v) for bovine milk in goat milk, with relative standard deviation (RSD) less than 10%, which can meet the needs of daily inspection. The NMR method described here is effective for milk authenticity identification, and the study demonstrates that the NMR-based milk metabolite analysis approach provides a means of detecting adulteration at expected levels and can be used for dairy quality monitoring. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  8. Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

    Science.gov (United States)

    Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

    2017-08-10

    To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  9. 1H NMR-based metabolomics of time-dependent responses of Eisenia fetida to sub-lethal phenanthrene exposure

    International Nuclear Information System (INIS)

    Lankadurai, Brian P.; Wolfe, David M.; Simpson, Andre J.; Simpson, Myrna J.

    2011-01-01

    1 H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida after exposure to sub-lethal concentrations of phenanthrene over time. Earthworms were exposed to 0.025 mg/cm 2 of phenanthrene (1/64th of the LC 50 ) via contact tests over four days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, resulting in polar and non-polar fractions that were analyzed by 1 H NMR after one, two, three and four days. NMR-based metabolomic analyses revealed heightened E. fetida responses with longer phenanthrene exposure times. Amino acids alanine and glutamate, the sugar maltose, the lipids cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. The conversion of succinate to fumarate in the Krebs cycle was also interrupted by phenanthrene. Therefore, this study shows that NMR-based metabolomics is a powerful tool for elucidating time-dependent relationships in addition to the mode of toxicity of phenanthrene in earthworm exposure studies. - Highlights: → NMR-based earthworm metabolomic analysis of the mode of action of phenanthrene is presented. → The earthworm species E. fetida were exposed to sub-lethal phenanthrene concentrations. → Both polar and non-polar metabolites of E. fetida tissue extracts were analyzed by 1 H NMR. → Longer phenanthrene exposure times resulted in heightened earthworm responses. → An interruption of the Krebs cycle was also observed due to phenanthrene exposure. - 1 H NMR metabolomics is used to determine the relationship between phenanthrene exposure and the metabolic response of the earthworm E. fetida over time and also to elucidate the phenanthrene mode of toxicity.

  10. (1)H-NMR-based metabolomic analysis of the effect of moderate wine consumption on subjects with cardiovascular risk factors

    OpenAIRE

    Vázquez Fresno, Rosa; Llorach, Rafael; Alcaro, Francesca; Rodríguez Martínez, Miguel Ángel; Vinaixa Crevillent, Maria; Chiva Blanch, Gemma; Estruch Riba, Ramon; Correig Blanchar, Xavier; Andrés Lacueva, Ma. Cristina

    2012-01-01

    Moderate wine consumption is associated with health-promoting activities. An H-NMR-based metabolomic approach was used to identify urinary metabolomic differences of moderate wine intake in the setting of a prospective, randomized, crossover, and controlled trial. Sixty-one male volunteers with high cardiovascular risk factors followed three dietary interventions (28 days): dealcoholized red wine (RWD) (272mL/day, polyphenol control), alcoholized red wine (RWA) (272mL/day) and gin (GIN) (100m...

  11. 1H NMR-based spectroscopy detects metabolic alterations in serum of patients with early-stage ulcerative colitis

    International Nuclear Information System (INIS)

    Zhang, Ying; Lin, Lianjie; Xu, Yanbin; Lin, Yan; Jin, Yu; Zheng, Changqing

    2013-01-01

    Highlights: •Twenty ulcerative colitis patients and nineteen healthy controls were enrolled. •Increased 3-hydroxybutyrate, glucose, phenylalanine, and decreased lipid were found. •We report early stage diagnosis of ulcerative colitis using NMR-based metabolomics. -- Abstract: Ulcerative colitis (UC) has seriously impaired the health of citizens. Accurate diagnosis of UC at an early stage is crucial to improve the efficiency of treatment and prognosis. In this study, proton nuclear magnetic resonance ( 1 H NMR)-based metabolomic analysis was performed on serum samples collected from active UC patients (n = 20) and healthy controls (n = 19), respectively. The obtained spectral profiles were subjected to multivariate data analysis. Our results showed that consistent metabolic alterations were present between the two groups. Compared to healthy controls, UC patients displayed increased 3-hydroxybutyrate, β-glucose, α-glucose, and phenylalanine, but decreased lipid in serum. These findings highlight the possibilities of NMR-based metabolomics as a non-invasive diagnostic tool for UC

  12. MFPred: Rapid and accurate prediction of protein-peptide recognition multispecificity using self-consistent mean field theory.

    Directory of Open Access Journals (Sweden)

    Aliza B Rubenstein

    2017-06-01

    Full Text Available Multispecificity-the ability of a single receptor protein molecule to interact with multiple substrates-is a hallmark of molecular recognition at protein-protein and protein-peptide interfaces, including enzyme-substrate complexes. The ability to perform structure-based prediction of multispecificity would aid in the identification of novel enzyme substrates, protein interaction partners, and enable design of novel enzymes targeted towards alternative substrates. The relatively slow speed of current biophysical, structure-based methods limits their use for prediction and, especially, design of multispecificity. Here, we develop a rapid, flexible-backbone self-consistent mean field theory-based technique, MFPred, for multispecificity modeling at protein-peptide interfaces. We benchmark our method by predicting experimentally determined peptide specificity profiles for a range of receptors: protease and kinase enzymes, and protein recognition modules including SH2, SH3, MHC Class I and PDZ domains. We observe robust recapitulation of known specificities for all receptor-peptide complexes, and comparison with other methods shows that MFPred results in equivalent or better prediction accuracy with a ~10-1000-fold decrease in computational expense. We find that modeling bound peptide backbone flexibility is key to the observed accuracy of the method. We used MFPred for predicting with high accuracy the impact of receptor-side mutations on experimentally determined multispecificity of a protease enzyme. Our approach should enable the design of a wide range of altered receptor proteins with programmed multispecificities.

  13. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

    NARCIS (Netherlands)

    Olijve, L.L.C.; Meister, K.; DeVries, A.L.; Duman, J.G.; Guo, S.; Bakker, H.J.; Voets, I.K.

    2016-01-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of

  14. Peptide fragments induce a more rapid immune response than intact proteins in earthworms.

    Science.gov (United States)

    Hanusová, R; Tucková, L; Halada, P; Bezouska, K; Bilej, M

    1999-01-01

    The effect of in vivo proteolytic processing of protein antigen was studied in Eisenia foetida earthworms. Parenteral administration of the protein antigen induces elevated levels of an antigen-binding protein (ABP) which recognizes the protein used for stimulation. When the protein antigen is administered simultaneously with nontoxic serine proteinase inhibitor, ABP levels remain close to background. On the other hand, the in vivo adaptive response of earthworms to peptide fragments obtained by coelomic fluid digestion of the foreign antigen occurs even in the presence of proteinase inhibitor and, moreover, is significantly faster as compared to the response to intact antigen. These findings confirm the role of proteolytic processing in earthworms. MALDI mass spectrometric analysis of the fragments after coelomic fluid digestion has revealed the presence of the peptide fragments with molecular weights in the mass range 700-1100 Da.

  15. Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Mortensen, Shila; Boye, Mette

    2009-01-01

    The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other...... parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we studied gene expression changes in peripheral lymphoid tissues as compared to hepatic expression changes, 14-18 h after lung...... with measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14-18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase...

  16. Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

    Directory of Open Access Journals (Sweden)

    Rotem Gura Sadovsky

    2017-03-01

    Full Text Available The fluorescence microscopy methods presently used to characterize protein motion in cells infer protein motion from indirect observables, rather than measuring protein motion directly. Operationalizing these methods requires expertise that can constitute a barrier to their broad utilization. Here, we have developed PIPE (photo-converted intensity profile expansion to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We demonstrate PIPE’s success in measuring accurate diffusion coefficients in silico and in vitro and compare effective diffusion coefficients of native cellular proteins and free fluorophores in vivo. We apply PIPE to measure diffusion anomality in the cell and use it to distinguish free fluorophores from native cellular proteins. PIPE’s direct measurement and ease of use make it appealing for cell biologists.

  17. A cost-effective device for the rapid transfer of gel-separated proteins onto membranes.

    Science.gov (United States)

    Tam, Hann W; Huang, Yu-Chen; Tam, Ming F

    2009-03-01

    We describe here the fabrication of a cost-effective semi-dry blotting apparatus for the transfer of proteins onto membranes. Graphite sheets were used as electrodes. Protein mixtures were separated on NuPAGE 4% to 12% polyacrylamide gradient gels. With a Tris-bicine buffer, we demonstrated that close to 80% of the proteins with apparent molecular mass of 80kDa or less were removed from the gels after 8min of blotting. The process is much faster than the techniques reported previously in the literature.

  18. Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

    OpenAIRE

    Wang, Yen-Chih; Dozier, Jonathan K.; Beese, Lorena S.; Distefano, Mark D.

    2014-01-01

    Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequen...

  19. Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs

    Science.gov (United States)

    Bradshaw, Andrew; Salt, Michael; Bell, Ashley; Zeitler, Matt; Litra, Noelle; Smith, Andrew M.

    2011-01-01

    SUMMARY The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (Mr) of 165×103, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20–35% decrease in the amount of soluble protein with a Mr of 40–165 (×103) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a Mr of 15×103 were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue. PMID:21525316

  20. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2014-05-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  1. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2015-08-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  2. Pea fiber and wheat bran fiber show distinct metabolic profiles in rats as investigated by a 1H NMR-based metabolomic approach.

    Directory of Open Access Journals (Sweden)

    Guangmang Liu

    Full Text Available This study aimed to examine the effect of pea fiber (PF and wheat bran fiber (WF supplementation in rat metabolism. Rats were assigned randomly to one of three dietary groups and were given a basal diet containing 15% PF, 15% WF, or no supplemental fiber. Urine and plasma samples were analyzed by NMR-based metabolomics. PF significantly increased the plasma levels of 3-hydroxybutyrate, and myo-inositol as well as the urine levels of alanine, hydroxyphenylacetate, phenylacetyglycine, and α-ketoglutarate. However, PF significantly decreased the plasma levels of isoleucine, leucine, lactate, and pyruvate as well as the urine levels of allantoin, bile acids, and trigonelline. WF significantly increased the plasma levels of acetone, isobutyrate, lactate, myo-inositol, and lipids as well as the urine levels of alanine, lactate, dimethylglycine, N-methylniconamide, and α-ketoglutarate. However, WF significantly decreased the plasma levels of amino acids, and glucose as well as the urine levels of acetate, allantoin, citrate, creatine, hippurate, hydroxyphenylacetate, and trigonelline. Results suggest that PF and WF exposure can promote antioxidant activity and can exhibit common systemic metabolic changes, including lipid metabolism, energy metabolism, glycogenolysis and glycolysis metabolism, protein biosynthesis, and gut microbiota metabolism. PF can also decrease bile acid metabolism. These findings indicate that different fiber diet may cause differences in the biofluid profile in rats.

  3. INVESTIGATING THE ENANTIOSELECTIVE TOXICITY OF CONAZOLE FUNGICIDES IN RAINBOW TROUT THROUGH NMR BASED METABOLOMICS

    Science.gov (United States)

    Recently, metabolomics, or the quantitative measurement of a broad spectrum of metabolic responses of living systems in response to disease onset or genetic modification, has been employed to enable rapid identification of the mechanisms of toxicity for compounds of environmental...

  4. The rapid determination of fat and protein content in fresh raw milk using the laser light scattering technology

    Science.gov (United States)

    Xin, Qi; Zhi Ling, Hou; Jian Long, Tian; Zhu, Yu

    2006-08-01

    The aim was to develop a simple and rapid method for determination of fat and protein content in milk. Based on the laser light scattering theory, the ratio of the scattered light (at 90±0.05° scattering angles) intensity to the transmitted light intensity, which is called scattered-transmitted-ratio method, is adopted as the optical parameter representing the milk fat content and the protein content. In this way, the influence of the fluctuation of the power of the light source is eliminated and the accuracy of determination is improved accordingly. The system we use is real-time and can satisfy the challenging requirements of dairy farming. Results of this study indicate the feasibility of using this technology for fresh milk fat and protein analysis. The fat contents and protein contents of 50 milk samples determined by this method were consistent with the values obtained by the reference methods based on Rose-Gottlieb method and Kjeldahl determination of N method. In this paper, the operating principle of the instrument is introduced and the influence of the environmental conditions, such as the homogenization pressure and homogenization temperature, etc. on the result of the test is analyzed. Through data analysis, the concrete schemes for testing the fat using the curve fitting and testing the protein using the surface fitting technique are determined. Finally, the difference from the reference values of the test is discussed.

  5. A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

    Science.gov (United States)

    Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

    2017-05-01

    Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  6. A Rapid, Onsite, Ultrasensitive Melamine Quantitation Method for Protein Beverages Using Time-Resolved Fluorescence Detection Paper.

    Science.gov (United States)

    Li, Guanghua; Wang, Du; Zhou, Aijun; Sun, Yimin; Zhang, Qi; Poapolathep, Amnart; Zhang, Li; Fan, Zhiyong; Zhang, Zhaowei; Li, Peiwu

    2018-05-02

    To ensure protein beverage safety and prevent illegal melamine use to artificially increase protein content, a rapid, onsite, ultrasensitive detection method for melamine must be developed because melamine is detrimental to human health and life. Herein, an ultrasensitive time-resolved fluorescence detection paper (TFDP) was developed to detect melamine in protein beverages within 15 min using a one-step sample preparation. The lower limits of detection were 0.89, 0.94, and 1.05 ng/mL, and the linear ranges were 2.67-150, 2.82-150, and 3.15-150 ng/mL (R2>0.982) for peanut, walnut, and coconut beverages, respectively. The recovery rates were 85.86-110.60% with a coefficient of variation beverage samples, the TFDP and ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) results were consistent. This method is a promising alternative for rapid, onsite detection of melamine in beverages.

  7. Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates.

    Science.gov (United States)

    Zitterbart, Robert; Krumrey, Michael; Seitz, Oliver

    2017-07-01

    The chemical synthesis of proteins typically involves the solid-phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid-phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography-free synthesis of proteins on the surface of microtiter plates. The method relies on solid-phase synthesis of unprotected peptide fragments, immobilization of the C-terminal fragment and on-surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)-oligohistidine complexes, (ii) Cu-based [2 + 3] alkine-azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on-surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on-surface fluorescence-based saturation binding analysis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  8. Microwave-Assisted Hydrothermal Rapid Synthesis of Calcium Phosphates: Structural Control and Application in Protein Adsorption.

    Science.gov (United States)

    Cai, Zhu-Yun; Peng, Fan; Zi, Yun-Peng; Chen, Feng; Qian, Qi-Rong

    2015-07-31

    Synthetic calcium phosphate (CaP)-based materials have attracted much attention in the biomedical field. In this study, we have investigated the effect of pH values on CaP nanostructures prepared using a microwave-assisted hydrothermal method. The hierarchical nanosheet-assembled hydroxyapatite (HAP) nanostructure was prepared under weak acidic conditions (pH 5), while the HAP nanorod was prepared under neutral (pH 7) and weak alkali (pH 9) condition. However, when the pH value increases to 11, a mixed product of HAP nanorod and tri-calcium phosphate nanoparticle was obtained. The results indicated that the pH value of the initial reaction solution played an important role in the phase and structure of the CaP. Furthermore, the protein adsorption and release performance of the as-prepared CaP nanostructures were investigated by using hemoglobin (Hb) as a model protein. The sample that was prepared at pH = 11 and consisted of mixed morphologies of nanorods and nanoprisms showed a higher Hb protein adsorption capacity than the sample prepared at pH 5, which could be explained by its smaller size and dispersed structure. The results revealed the relatively high protein adsorption capacity of the as-prepared CaP nanostructures, which show promise for applications in various biomedical fields such as drug delivery and protein adsorption.

  9. Microwave-Assisted Hydrothermal Rapid Synthesis of Calcium Phosphates: Structural Control and Application in Protein Adsorption

    Directory of Open Access Journals (Sweden)

    Zhu-Yun Cai

    2015-07-01

    Full Text Available Synthetic calcium phosphate (CaP-based materials have attracted much attention in the biomedical field. In this study, we have investigated the effect of pH values on CaP nanostructures prepared using a microwave-assisted hydrothermal method. The hierarchical nanosheet-assembled hydroxyapatite (HAP nanostructure was prepared under weak acidic conditions (pH 5, while the HAP nanorod was prepared under neutral (pH 7 and weak alkali (pH 9 condition. However, when the pH value increases to 11, a mixed product of HAP nanorod and tri-calcium phosphate nanoparticle was obtained. The results indicated that the pH value of the initial reaction solution played an important role in the phase and structure of the CaP. Furthermore, the protein adsorption and release performance of the as-prepared CaP nanostructures were investigated by using hemoglobin (Hb as a model protein. The sample that was prepared at pH = 11 and consisted of mixed morphologies of nanorods and nanoprisms showed a higher Hb protein adsorption capacity than the sample prepared at pH 5, which could be explained by its smaller size and dispersed structure. The results revealed the relatively high protein adsorption capacity of the as-prepared CaP nanostructures, which show promise for applications in various biomedical fields such as drug delivery and protein adsorption.

  10. Rapid evolution of coral proteins responsible for interaction with the environment.

    Science.gov (United States)

    Voolstra, Christian R; Sunagawa, Shinichi; Matz, Mikhail V; Bayer, Till; Aranda, Manuel; Buschiazzo, Emmanuel; Desalvo, Michael K; Lindquist, Erika; Szmant, Alina M; Coffroth, Mary Alice; Medina, Mónica

    2011-01-01

    Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably. We screened a set of 2,604 putative orthologs from EST-based sequence datasets of the coral species Acropora millepora and Acropora palmata to determine the fraction and identity of proteins that may experience adaptive evolution. 7% of the orthologs show elevated rates of evolution. Taxonomically-restricted (i.e. lineage-specific) genes show a positive selection signature more frequently than genes that are found across many animal phyla. The class of proteins that displayed elevated evolutionary rates was significantly enriched for proteins involved in immunity and defense, reproduction, and sensory perception. We also found elevated rates of evolution in several other functional groups such as management of membrane vesicles, transmembrane transport of ions and organic molecules, cell adhesion, and oxidative stress response. Proteins in these processes might be related to the endosymbiotic relationship corals maintain with dinoflagellates in the genus Symbiodinium. This study provides a birds-eye view of the processes potentially underlying coral adaptation, which will serve as a foundation for future work to elucidate the rates, patterns, and mechanisms of corals' evolutionary response to global climate change.

  11. Modified procedure for rapid labelling of low concentrations of bioactive proteins with indium-111

    Energy Technology Data Exchange (ETDEWEB)

    Zoghbi, S S; Neumann, R D; Gottschalk, A

    1985-01-01

    The authors describe the conjugation of DTPA to 100-500 g of protein in concentrations of 0.6-1.0 mg mL utilizing the mixed anhydride method. Free DTPA is removed by minicolumn gel filtration and centrifugation with minimal protein dilution. Radiolabelling process can be monitored by instant thin layer chromatography. Any radiochemical impurity detected can be eliminated either by additional minicolumn filtration of further chelation with more conjugated protein. In citrate buffer at pH 6 with minicolumn gel chromatography the authors prepared In-DTPA-D3 (3.0 Ci g) monoclonal antibody and used it to image hepatocarcinoma in guinea pigs. 13 references, 5 figures, 2 tables.

  12. Rapid evaluation of seed vigor by the absolute content of protein in seed within the same crop.

    Science.gov (United States)

    Wen, Daxing; Hou, Hongcun; Meng, Aiju; Meng, Jie; Xie, Liuyong; Zhang, Chunqing

    2018-04-03

    Seed vigor, an important index of seed quality, determines the potential for rapid and uniform emergence of plants. The objective of this study was to explore a rapid method for evaluating seed vigor. To analyze the correlation of seed traits and seedling traits related to seed vigor, we designed five experiments including nitrogen fertilizer, irrigation and seed sorting treatments in wheat. The results showed that only the absolute content of protein (ACP) in wheat seed was significantly correlated with plant dry weight in five experiments. Subsequently, another experiment including 30 wheat seed lots was used to validate the above results. Although 100-grain weight was also correlated with plant dry weight (R = 0.799, p vigor and could potentially be used for processing and screening high vigor seeds.

  13. A Force-Activated Trip Switch Triggers Rapid Dissociation of a Colicin from Its Immunity Protein

    Science.gov (United States)

    Farrance, Oliver E.; Hann, Eleanore; Kaminska, Renata; Housden, Nicholas G.; Derrington, Sasha R.; Kleanthous, Colin; Radford, Sheena E.; Brockwell, David J.

    2013-01-01

    Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (Kd≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods. PMID:23431269

  14. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    DEFF Research Database (Denmark)

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most...... diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed...

  15. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    International Nuclear Information System (INIS)

    Kovalev, Valeri I; Bartona, James S; Richardson, Patricia R; Jones, Anita C

    2006-01-01

    There is a risk of contamination of surgical instruments by infectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect ∼10 attomole/cm 2 with a scan speed of ∼3-10 cm 2 /s of the test instrument's surface. A theoretical analysis and experimental measurements will be discussed

  16. Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins.

    Science.gov (United States)

    O'Connor, Marie N; Salles, Isabelle I; Cvejic, Ana; Watkins, Nicholas A; Walker, Adam; Garner, Stephen F; Jones, Chris I; Macaulay, Iain C; Steward, Michael; Zwaginga, Jaap-Jan; Bray, Sarah L; Dudbridge, Frank; de Bono, Bernard; Goodall, Alison H; Deckmyn, Hans; Stemple, Derek L; Ouwehand, Willem H

    2009-05-07

    In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

  17. Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection

    NARCIS (Netherlands)

    Wagemakers, A.; Mason, L. M. K.; Oei, A.; de Wever, B.; van der Poll, T.; Bins, A. D.; Hovius, J. W. R.

    2014-01-01

    Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method

  18. Fabrication of protein microarrays for alpha fetoprotein detection by using a rapid photo-immobilization process

    Directory of Open Access Journals (Sweden)

    Sirasa Yodmongkol

    2016-03-01

    Full Text Available In this study, protein microarrays based on sandwich immunoassays are generated to quantify the amount of alpha fetoprotein (AFP in blood serum. For chip generation a mixture of capture antibody and a photoactive copolymer consisting of N,N-dimethylacrylamide (DMAA, methacryloyloxy benzophenone (MaBP, and Na-4-styrenesulfonate (SSNa was spotted onto unmodified polymethyl methacrylate (PMMA substrates. Subsequently to printing of the microarray, the polymer and protein were photochemically cross-linked and the forming, biofunctionalized hydrogels simultaneously bound to the chip surface by short UV- irradiation. The obtained biochip was incubated with AFP antigen, followed by biotinylated AFP antibody and streptavidin-Cy5 and the fluorescence signal read-out. The developed microarray biochip covers the range of AFP in serum samples such as maternal serum in the range of 5 and 100 ng/ml. The chip production process is based on a fast and simple immobilization process, which can be applied to conventional plastic surfaces. Therefore, this protein microarray production process is a promising method to fabricate biochips for AFP screening processes. Keywords: Photo-immobilization, Protein microarray, Alpha fetoprotein, Hydrogel, 3D surface, Down syndrome

  19. SIMULATING PROTEIN DIGESTION ON TROUT A RAPID AND INEXPENSIVE METHOD FOR DOCUMENTING FISH MEAL QUALITY AND SCREENING NOVEL PROTEIN SOURCES FOR USE IN AQUAFEEDS

    Directory of Open Access Journals (Sweden)

    M Bassompierre

    1997-10-01

    Full Text Available A novel in vitro digestion system, which simulated rainbow trout gastric and intestinal digestion was developed. The method was employed to evaluate the impact of the gastric phase of digestion upon degradation of three fish meals od differing quality. Results illustrated that two-phase gastric-intestinal digestion increased the discriminatory powers of the system when compared to one-step intestinal digestion. A comparison of the system with pH-STAT methods demonstrated that the in vitro technique was superior. The presented method provides an ethical and cost effective means for rapid evaluation of fish meals and potentially, alternative protein sources for aquafeeds.

  20. Rapid evolution of coral proteins responsible for interaction with the environment.

    Directory of Open Access Journals (Sweden)

    Christian R Voolstra

    Full Text Available Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures, pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably.We screened a set of 2,604 putative orthologs from EST-based sequence datasets of the coral species Acropora millepora and Acropora palmata to determine the fraction and identity of proteins that may experience adaptive evolution. 7% of the orthologs show elevated rates of evolution. Taxonomically-restricted (i.e. lineage-specific genes show a positive selection signature more frequently than genes that are found across many animal phyla. The class of proteins that displayed elevated evolutionary rates was significantly enriched for proteins involved in immunity and defense, reproduction, and sensory perception. We also found elevated rates of evolution in several other functional groups such as management of membrane vesicles, transmembrane transport of ions and organic molecules, cell adhesion, and oxidative stress response. Proteins in these processes might be related to the endosymbiotic relationship corals maintain with dinoflagellates in the genus Symbiodinium.This study provides a birds-eye view of the processes potentially underlying coral adaptation, which will serve as a foundation for future work to elucidate the rates, patterns, and mechanisms of corals' evolutionary response to global climate change.

  1. Rapid Evolution of Coral Proteins Responsible for Interaction with the Environment

    Energy Technology Data Exchange (ETDEWEB)

    Voolstra, Christian R.; Sunagawa, Shinichi; Matz, Mikhail V.; Bayer, Till; Aranda, Manuel; Buschiazzo, Emmanuel; DeSalvo, Michael K.; Lindquist, Erika; Szmant, Alina M.; Coffroth, Mary Alice; Medina, Monica

    2011-01-31

    Background: Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably. Methodology/Principal Findings: We screened a set of 2,604 putative orthologs from EST-based sequence datasets of the coral species Acropora millepora and Acropora palmata to determine the fraction and identity of proteins that may experience adaptive evolution. 7percent of the orthologs show elevated rates of evolution. Taxonomically-restricted (i.e. lineagespecific) genes show a positive selection signature more frequently than genes that are found across many animal phyla. The class of proteins that displayed elevated evolutionary rates was significantly enriched for proteins involved in immunity and defense, reproduction, and sensory perception. We also found elevated rates of evolution in several other functional groups such as management of membrane vesicles, transmembrane transport of ions and organic molecules, cell adhesion, and oxidative stress response. Proteins in these processes might be related to the endosymbiotic relationship corals maintain with dinoflagellates in the genus Symbiodinium. Conclusion/Relevance: This study provides a birds-eye view of the processes potentially underlying coral adaptation, which will serve as a foundation for future work to elucidate the rates, patterns, and mechanisms of corals? evolutionary response to global climate change.

  2. Recommendations and Standardization of Biomarker Quantification Using NMR-based Metabolomics with Particular Focus on Urinary Analysis

    KAUST Repository

    Emwas, Abdul-Hamid M.

    2016-01-08

    NMR-based metabolomics has shown considerable promise in disease diagnosis and biomarker discovery because it allows one to non-destructively identify and quantify large numbers of novel metabolite biomarkers in both biofluids and tissues. Indeed, precise metabolite quantification is a necessary prerequisite to move any chemical biomarker or biomarker panel from the lab into the clinic. Among the many biofluids (urine, serum, plasma, cerebrospinal fluid and saliva) commonly used for disease diagnosis and prognosis, urine has several advantages. It is abundant, sterile, easily obtained, needs little sample preparation and does not require any invasive medical procedures for collection. Furthermore, urine captures and concentrates many “unwanted” or “undesirable” compounds throughout the body, thereby providing a rich source of potentially useful disease biomarkers. However, the incredible variation in urine chemical concentrations due to effects such as gender, age, diet, life style, health conditions, and physical activity make the analysis of urine and the identification of useful urinary biomarkers by NMR quite challenging. In this review, we discuss a number of the most significant issues regarding NMR-based urinary metabolomics with a specific emphasis on metabolite quantification for disease biomarker applications. We also propose a number of data collection and instrumental recommendations regarding NMR pulse sequences, acceptable acquisition parameter ranges, relaxation effects on quantitation, proper handling of instrumental differences, as well as recommendations regarding sample preparation and biomarker assessment.

  3. Evaluation of Pacific white shrimp (Litopenaeus vannamei health during a superintensive aquaculture growout using NMR-based metabolomics.

    Directory of Open Access Journals (Sweden)

    Tracey B Schock

    Full Text Available Success of the shrimp aquaculture industry requires technological advances that increase production and environmental sustainability. Indoor, superintensive, aquaculture systems are being developed that permit year-round production of farmed shrimp at high densities. These systems are intended to overcome problems of disease susceptibility and of water quality issues from waste products, by operating as essentially closed systems that promote beneficial microbial communities (biofloc. The resulting biofloc can assimilate and detoxify wastes, may provide nutrition for the farmed organisms resulting in improved growth, and may aid in reducing disease initiated from external sources. Nuclear magnetic resonance (NMR-based metabolomic techniques were used to assess shrimp health during a full growout cycle from the nursery phase through harvest in a minimal-exchange, superintensive, biofloc system. Aberrant shrimp metabolomes were detected from a spike in total ammonia nitrogen in the nursery, from a reduced feeding period that was a consequence of surface scum build-up in the raceway, and from the stocking transition from the nursery to the growout raceway. The biochemical changes in the shrimp that were induced by the stressors were essential for survival and included nitrogen detoxification and energy conservation mechanisms. Inosine and trehalose may be general biomarkers of stress in Litopenaeus vannamei. This study demonstrates one aspect of the practicality of using NMR-based metabolomics to enhance the aquaculture industry by providing physiological insight into common environmental stresses that may limit growth or better explain reduced survival and production.

  4. A Preliminary Investigation of NSCL/P Plasma and Urine in Guizhou Province in China Using NMR-Based Metabonomics.

    Science.gov (United States)

    Lei, Huang Guang; Hong, Luo; Kun, Song Ju; Hai, Yin Xin; Dong, Wang Ya; Ke, Zhao; Ping, Xu; Hao, Chen

    2013-09-01

    Objective : To assess the feasibility of metabonomics in clinical studies. This is a pilot study introducing nuclear magnetic resonance (NMR)-based metabonomics to elucidate and compare the metabolism of patients with nonsyndromic cleft lip and/or palate (NSCL/P) and children without orofacial clefts. Methods : High-resolution (1)H NMR spectroscopy was performed on plasma and urine samples obtained from NSCL/P and healthy children. The (1)H NMR spectra were further analyzed with principal component analysis. Results : Compared to the control group, the level of low-molecular-weight metabolites in plasma such as asparagine was higher in NSCL/P patients, while arginine, lysine, acetate, lactate, proline, glutamine, pyruvate, creatinine, choline, and β-glucose were lower. The carnitine, citrate, and formate excretion in urine appeared to be higher in the healthy children, while the NSCL/P group excreted higher concentrations of aspartic acid and phenylalanine in urine. Conclusion : The present study clearly demonstrated the great potential of NMR-based metabonomics in elucidating NSCL/P plasma metabolism and the possible application of this technology in clinical diagnosis and screening.

  5. Metabolic Profiling and Classification of Propolis Samples from Southern Brazil: An NMR-Based Platform Coupled with Machine Learning.

    Science.gov (United States)

    Maraschin, Marcelo; Somensi-Zeggio, Amélia; Oliveira, Simone K; Kuhnen, Shirley; Tomazzoli, Maíra M; Raguzzoni, Josiane C; Zeri, Ana C M; Carreira, Rafael; Correia, Sara; Costa, Christopher; Rocha, Miguel

    2016-01-22

    The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching ∼90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.

  6. Recommendations and Standardization of Biomarker Quantification Using NMR-based Metabolomics with Particular Focus on Urinary Analysis

    KAUST Repository

    Emwas, Abdul-Hamid M.; Roy, Raja; McKay, Ryan T.; Ryan, Danielle; Brennan, Lorraine; Tenori, Leonardo; Luchinat, Claudio; Gao, Xin; Zeri, Ana Carolina; Gowda, G. A. Nagana; Raftery, Daniel; Steinbeck, Christoph; Salek, Reza M; Wishart, David S.

    2016-01-01

    NMR-based metabolomics has shown considerable promise in disease diagnosis and biomarker discovery because it allows one to non-destructively identify and quantify large numbers of novel metabolite biomarkers in both biofluids and tissues. Indeed, precise metabolite quantification is a necessary prerequisite to move any chemical biomarker or biomarker panel from the lab into the clinic. Among the many biofluids (urine, serum, plasma, cerebrospinal fluid and saliva) commonly used for disease diagnosis and prognosis, urine has several advantages. It is abundant, sterile, easily obtained, needs little sample preparation and does not require any invasive medical procedures for collection. Furthermore, urine captures and concentrates many “unwanted” or “undesirable” compounds throughout the body, thereby providing a rich source of potentially useful disease biomarkers. However, the incredible variation in urine chemical concentrations due to effects such as gender, age, diet, life style, health conditions, and physical activity make the analysis of urine and the identification of useful urinary biomarkers by NMR quite challenging. In this review, we discuss a number of the most significant issues regarding NMR-based urinary metabolomics with a specific emphasis on metabolite quantification for disease biomarker applications. We also propose a number of data collection and instrumental recommendations regarding NMR pulse sequences, acceptable acquisition parameter ranges, relaxation effects on quantitation, proper handling of instrumental differences, as well as recommendations regarding sample preparation and biomarker assessment.

  7. Evaluation of Pacific White Shrimp (Litopenaeus vannamei) Health during a Superintensive Aquaculture Growout Using NMR-Based Metabolomics

    Science.gov (United States)

    Schock, Tracey B.; Duke, Jessica; Goodson, Abby; Weldon, Daryl; Brunson, Jeff; Leffler, John W.; Bearden, Daniel W.

    2013-01-01

    Success of the shrimp aquaculture industry requires technological advances that increase production and environmental sustainability. Indoor, superintensive, aquaculture systems are being developed that permit year-round production of farmed shrimp at high densities. These systems are intended to overcome problems of disease susceptibility and of water quality issues from waste products, by operating as essentially closed systems that promote beneficial microbial communities (biofloc). The resulting biofloc can assimilate and detoxify wastes, may provide nutrition for the farmed organisms resulting in improved growth, and may aid in reducing disease initiated from external sources. Nuclear magnetic resonance (NMR)-based metabolomic techniques were used to assess shrimp health during a full growout cycle from the nursery phase through harvest in a minimal-exchange, superintensive, biofloc system. Aberrant shrimp metabolomes were detected from a spike in total ammonia nitrogen in the nursery, from a reduced feeding period that was a consequence of surface scum build-up in the raceway, and from the stocking transition from the nursery to the growout raceway. The biochemical changes in the shrimp that were induced by the stressors were essential for survival and included nitrogen detoxification and energy conservation mechanisms. Inosine and trehalose may be general biomarkers of stress in Litopenaeus vannamei. This study demonstrates one aspect of the practicality of using NMR-based metabolomics to enhance the aquaculture industry by providing physiological insight into common environmental stresses that may limit growth or better explain reduced survival and production. PMID:23555690

  8. Rapid concentration and dialysis of proteins with single hollow fibers: possible applications in analysis of protein secretion by isolated cells and steroid radioimmunoassays

    International Nuclear Information System (INIS)

    Rommerts, F.F.G.; Clotscher, W.F.; Van der Molen, H.J.

    1977-01-01

    Single hollow fibers were used in specially made cells for fast concentration and dialysis of solutions containing macromolecules. Volumes on the order of 5 ml of diluted protein solutions could be concentrated to 50--100 μl or less within 7 min with a protein recovery of 60--80%. More than 99% of the molecules with a molecular weight less than 500 could be removed in less than 1 hr. A possible application of the rapid dialysis method for the mechanization of radioimmunoassays is indicated. It was shown that in the radioimmunoassay of steriods the unbound steroids could be removed after incubation with antiserum, within 10 min and without a change in volume

  9. Population Divergence in Venom Bioactivities of Elapid Snake Pseudonaja textilis: Role of Procoagulant Proteins in Rapid Rodent Prey Incapacitation

    Science.gov (United States)

    Skejić, Jure; Hodgson, Wayne C.

    2013-01-01

    This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD) venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality) and Queensland (Mackay locality) populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver. PMID:23691135

  10. Population divergence in venom bioactivities of elapid snake Pseudonaja textilis: role of procoagulant proteins in rapid rodent prey incapacitation.

    Directory of Open Access Journals (Sweden)

    Jure Skejić

    Full Text Available This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality and Queensland (Mackay locality populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver.

  11. Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains: implications for rapid diagnostic tests.

    Directory of Open Access Journals (Sweden)

    Joanne Baker

    Full Text Available BACKGROUND: Although rapid diagnostic tests (RDTs for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2 are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. METHODS: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. RESULTS: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. CONCLUSIONS: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

  12. A Lateral Flow Protein Microarray for Rapid and Sensitive Antibody Assays

    Directory of Open Access Journals (Sweden)

    Helene Andersson-Svahn

    2011-11-01

    Full Text Available Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (< 30 ng/mL determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.

  13. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    Energy Technology Data Exchange (ETDEWEB)

    Kovalev, Valeri I [School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); Bartona, James S [School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); Richardson, Patricia R [School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ (United Kingdom); Jones, Anita C [School of Chemistry, University of Edinburgh, Edinburgh, EH9 3JJ (United Kingdom)

    2006-07-15

    There is a risk of contamination of surgical instruments by infectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect {approx}10 attomole/cm{sup 2} with a scan speed of {approx}3-10 cm{sup 2}/s of the test instrument's surface. A theoretical analysis and experimental measurements will be discussed.

  14. Rapid evolution of coral proteins responsible for interaction with the environment.

    KAUST Repository

    Voolstra, Christian R.; Sunagawa, Shinichi; Matz, Mikhail V; Bayer, Till; Aranda, Manuel; Buschiazzo, Emmanuel; Desalvo, Michael K; Lindquist, Erika; Szmant, Alina M; Coffroth, Mary Alice; Medina, Mó nica

    2011-01-01

    Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably.

  15. Rapid detection, classification and accurate alignment of up to a million or more related protein sequences.

    Science.gov (United States)

    Neuwald, Andrew F

    2009-08-01

    The patterns of sequence similarity and divergence present within functionally diverse, evolutionarily related proteins contain implicit information about corresponding biochemical similarities and differences. A first step toward accessing such information is to statistically analyze these patterns, which, in turn, requires that one first identify and accurately align a very large set of protein sequences. Ideally, the set should include many distantly related, functionally divergent subgroups. Because it is extremely difficult, if not impossible for fully automated methods to align such sequences correctly, researchers often resort to manual curation based on detailed structural and biochemical information. However, multiply-aligning vast numbers of sequences in this way is clearly impractical. This problem is addressed using Multiply-Aligned Profiles for Global Alignment of Protein Sequences (MAPGAPS). The MAPGAPS program uses a set of multiply-aligned profiles both as a query to detect and classify related sequences and as a template to multiply-align the sequences. It relies on Karlin-Altschul statistics for sensitivity and on PSI-BLAST (and other) heuristics for speed. Using as input a carefully curated multiple-profile alignment for P-loop GTPases, MAPGAPS correctly aligned weakly conserved sequence motifs within 33 distantly related GTPases of known structure. By comparison, the sequence- and structurally based alignment methods hmmalign and PROMALS3D misaligned at least 11 and 23 of these regions, respectively. When applied to a dataset of 65 million protein sequences, MAPGAPS identified, classified and aligned (with comparable accuracy) nearly half a million putative P-loop GTPase sequences. A C++ implementation of MAPGAPS is available at http://mapgaps.igs.umaryland.edu. Supplementary data are available at Bioinformatics online.

  16. Rapid evolution of coral proteins responsible for interaction with the environment.

    KAUST Repository

    Voolstra, Christian R.

    2011-05-25

    Corals worldwide are in decline due to climate change effects (e.g., rising seawater temperatures), pollution, and exploitation. The ability of corals to cope with these stressors in the long run depends on the evolvability of the underlying genetic networks and proteins, which remain largely unknown. A genome-wide scan for positively selected genes between related coral species can help to narrow down the search space considerably.

  17. Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

    International Nuclear Information System (INIS)

    Blowers, D.P.; Boss, W.F.; Trewavas, A.J.

    1988-01-01

    Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ- 32 P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M r 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic 32 P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses

  18. Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

    Science.gov (United States)

    Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

    2017-11-01

    Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

  19. Development of new probes for NMR based analysis of biomolecules' cellular functions

    International Nuclear Information System (INIS)

    Fernandes, Laetitia

    2015-01-01

    Most NMR studies are carried out in vitro, but the structure and dynamics of some biomolecules inside cells differ from those in vitro. It thus becomes interesting to analyze biomolecules such as proteins in their natural environment: the cell. Recent progress of in cell NMR allowed to better understand the behaviour of proteins: their dynamics and their interactions with other biomolecules in the cell. But the low concentration of proteins leads to low signal intensity. Moreover, the viscosity of the environment induces faster transverse relaxation, resulting in line broadening for proteins signals. The use of the Long-Lived States and Coherencies (LLS and LLC, respectively) as well as dissolution Dynamic Nuclear Polarization (dissolution-DNP) can improve NMR observations in cells. LLS were used to understand and characterize the structure of the N-terminal domain of c-Src, which is intrinsically disordered. To follow the phosphorylation of proteins, a first preliminary study of a 21-aa peptides derived from IKBa electroporated into HepG2 cell lines was carried out. (author)

  20. Radioiododestannylation. Convenient synthesis of a stable penicillin derivative for rapid penicillin binding protein (PBP) assay

    International Nuclear Information System (INIS)

    Blaszczak, L.C.; Halligan, N.G.; Seitz, D.E.

    1989-01-01

    Radioiodination of p-(trimethylstannyl)penicillin V with [ 125 I]Na using a modification of the chloramine-T method is simple, high yielding, and site-specific. The structure and penicillin binding protein (PBP) affinity of p-[ 125 I]-penicillin V (IPV) are similar to penicillin G and the product can be used directly without purification in the PBP assay. Because of the high degree of stability toward autoradiolysis and equivalent PBP binding affinity, IPV can be used in place of [ 3 H]-penicillin G or [ 14 C]-penicillin G for these experiments. (author)

  1. Rapid and accurate prediction and scoring of water molecules in protein binding sites.

    Directory of Open Access Journals (Sweden)

    Gregory A Ross

    Full Text Available Water plays a critical role in ligand-protein interactions. However, it is still challenging to predict accurately not only where water molecules prefer to bind, but also which of those water molecules might be displaceable. The latter is often seen as a route to optimizing affinity of potential drug candidates. Using a protocol we call WaterDock, we show that the freely available AutoDock Vina tool can be used to predict accurately the binding sites of water molecules. WaterDock was validated using data from X-ray crystallography, neutron diffraction and molecular dynamics simulations and correctly predicted 97% of the water molecules in the test set. In addition, we combined data-mining, heuristic and machine learning techniques to develop probabilistic water molecule classifiers. When applied to WaterDock predictions in the Astex Diverse Set of protein ligand complexes, we could identify whether a water molecule was conserved or displaced to an accuracy of 75%. A second model predicted whether water molecules were displaced by polar groups or by non-polar groups to an accuracy of 80%. These results should prove useful for anyone wishing to undertake rational design of new compounds where the displacement of water molecules is being considered as a route to improved affinity.

  2. [A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

    Science.gov (United States)

    Mo, Qianzhen; Mai, Rongjia; Yang, Zhixiao; Chen, Minfang; Yang, Tiezhao; Lai, Huafang; Yang, Peiliang; Chen, Qiang; Zhou, Xiaohong

    2012-06-01

    To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

  3. Guinea Pig Prion Protein Supports Rapid Propagation of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease Prions.

    Science.gov (United States)

    Watts, Joel C; Giles, Kurt; Saltzberg, Daniel J; Dugger, Brittany N; Patel, Smita; Oehler, Abby; Bhardwaj, Sumita; Sali, Andrej; Prusiner, Stanley B

    2016-11-01

    The biochemical and neuropathological properties of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) prions are faithfully maintained upon transmission to guinea pigs. However, primary and secondary transmissions of BSE and vCJD in guinea pigs result in long incubation periods of ∼450 and ∼350 days, respectively. To determine if the incubation periods of BSE and vCJD prions could be shortened, we generated transgenic (Tg) mice expressing guinea pig prion protein (GPPrP). Inoculation of Tg(GPPrP) mice with BSE and vCJD prions resulted in mean incubation periods of 210 and 199 days, respectively, which shortened to 137 and 122 days upon serial transmission. In contrast, three different isolates of sporadic CJD prions failed to transmit disease to Tg(GPPrP) mice. Many of the strain-specified biochemical and neuropathological properties of BSE and vCJD prions, including the presence of type 2 protease-resistant PrP Sc , were preserved upon propagation in Tg(GPPrP) mice. Structural modeling revealed that two residues near the N-terminal region of α-helix 1 in GPPrP might mediate its susceptibility to BSE and vCJD prions. Our results demonstrate that expression of GPPrP in Tg mice supports the rapid propagation of BSE and vCJD prions and suggest that Tg(GPPrP) mice may serve as a useful paradigm for bioassaying these prion isolates. Variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the prion strains most relevant to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult because of prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for

  4. Rapid RNase L-driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery.

    Science.gov (United States)

    Donovan, Jesse; Rath, Sneha; Kolet-Mandrikov, David; Korennykh, Alexei

    2017-11-01

    Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest. © 2017 Donovan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  5. Protein tethering enables rapid and label-free SERS platform for screening drugs of abuse (Conference Presentation)

    Science.gov (United States)

    Siddhanta, Soumik; Wróbel, Maciej S.; Barman, Ishan

    2017-02-01

    A quick, cost-effective method for detection of drugs of abuse in biological fluids would be of great value in healthcare, law enforcement, and home testing applications. The alarming rise in narcotics abuse has led to considerable focus on developing potent and versatile analytical tools that can address this societal problem. While laboratory testing plays a key role in the current detection of drug misuse and the evaluation of patients with drug induced intoxication, these typically require expensive reagents and trained personnel, and may take hours to complete. Thus, a significant unmet need is to engineer a facile method that can rapidly detect drugs with little sample preparation, especially the bound fraction that is typically dominant in the blood stream. Here we report an approach that combines the exquisite sensitivity of surface enhanced Raman spectroscopy (SERS) and a facile protein tethering mechanism to reliably detect four different classes of drugs, barbiturate, benzodiazepine, amphetamine and benzoylecgonine. The proposed approach harnesses the reliable and specific attachment of proteins to both drugs and nanoparticle to facilitate the enhancement of spectral markers that are sensitive to the presence of the drugs. In conjunction with chemometric tools, we have shown the ability to quantify these drugs lower than levels achievable by existing clinical immunoassays. Through molecular docking simulations, we also probe the mechanistic underpinnings of the protein tethering approach, opening the door to detection of a broad class of narcotics in biological fluids within a few minutes as well as for groundwater analysis and toxin detection.

  6. Effect of Rapid Chilling on Beef Quality and Cytoskeletal Protein Degradation in of Chinese Yellow Crossbred Bulls

    Directory of Open Access Journals (Sweden)

    Yanwei Mao

    2012-08-01

    Full Text Available The objective of this study was to investigate the effect of rapid chilling (RC on beef quality and the degradation of cytoskeletal proteins. Twenty Chinese Yellow crossbred bulls were selected and randomly divided into two groups. RC and conventional chilling (CC were applied to left and right sides of the carcasses respectively after slaughtering. To determine whether electrical stimulation (ES treatment can alleviate the potential hazard of RC on meat quality, ES was applied to one group. The effects of RC and ES were determined by meat color, shear force and cytoskeletal protein degradation postmortem (PM. The results showed that RC decreased beef tenderness at 1 d and 3 d postmortem, but had no detrimental effect on meat color. Western blotting showed that RC decreased the degradation rate of desmin and troponin-T, but the effects weakened gradually as postmortem aging extended. Degradation rates of both desmin and troponin-T were accelerated by ES. The combination of RC and ES could improve beef color, accelerate degradation rate of cytoskeletal protein and improve beef tenderness.

  7. Fast Identification of Radical Scavengers from Securigera varia by Combining 13C-NMR-Based Dereplication to Bioactivity-Guided Fractionation.

    Science.gov (United States)

    Sientzoff, Pacôme; Hubert, Jane; Janin, Coralie; Voutquenne-Nazabadioko, Laurence; Renault, Jean-Hugues; Nuzillard, Jean-Marc; Harakat, Dominique; Magid, Abdulmagid Alabdul

    2015-08-14

    Securigera varia (Fabaceae) is a common herbaceous perennial plant widely growing in Europe and Asia and purposely established for erosion control, roadside planting, and soil rehabilitation. The aim of this study was to determine the radical scavenging activity of a crude methanol extract of S. varia aerial parts by using the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl) and to rapidly identify the compounds involved in this activity. The crude extract was initially separated in five fractions on Diaion HP20 resin and the most active part was fractionated by Centrifugal Partition Extraction (CPE). Known compounds were directly identified by a (13)C-NMR-based dereplication method. Semi-preparative high performance liquid chromatography purification experiments were further performed to identify unknown or minor active compounds. As a result, one new (13) and twelve known flavonoid glycosides together with three nitropropanoylglucopyranoses were isolated, including astragalin (1), kaempferol-3-O-(6-O-acetyl)-β-D-glucopyranoside (2), kaempferol-3,4'-di-O-β-D-glucopyranoside (3), trifolin (4), isoquercitrin (5), hyperoside (6), isovitexin (7), isoorientin (8), isovitexin 4'-O-β-D-glucopyranoside (9), apigenin 7-O-β-D-glucuronopyranoside (10), luteolin 7-O-β-D-glucuronopyranoside (11), apigenin 7-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranoside (12), apigenin 7-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranoside (13), 6-O-(3-nitropropanoyl)-β-D-glucopyranoside (14), coronillin (16) and coronarian (15). 120 mg of the most active compound isoorientin against the free radical DPPH was recovered by CPE with an HPLC purity of 99%.

  8. Fast Identification of Radical Scavengers from Securigera varia by Combining 13C-NMR-Based Dereplication to Bioactivity-Guided Fractionation

    Directory of Open Access Journals (Sweden)

    Pacôme Sientzoff

    2015-08-01

    Full Text Available Securigera varia (Fabaceae is a common herbaceous perennial plant widely growing in Europe and Asia and purposely established for erosion control, roadside planting, and soil rehabilitation. The aim of this study was to determine the radical scavenging activity of a crude methanol extract of S. varia aerial parts by using the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl and to rapidly identify the compounds involved in this activity. The crude extract was initially separated in five fractions on Diaion HP20 resin and the most active part was fractionated by Centrifugal Partition Extraction (CPE. Known compounds were directly identified by a 13C-NMR-based dereplication method. Semi-preparative high performance liquid chromatography purification experiments were further performed to identify unknown or minor active compounds. As a result, one new (13 and twelve known flavonoid glycosides together with three nitropropanoylglucopyranoses were isolated, including astragalin (1, kaempferol-3-O-(6-O-acetyl-β-D-glucopyranoside (2, kaempferol-3,4′-di-O-β-D-glucopyranoside (3, trifolin (4, isoquercitrin (5, hyperoside (6, isovitexin (7, isoorientin (8, isovitexin 4′-O-β-D-glucopyranoside (9, apigenin 7-O-β-D-glucuronopyranoside (10, luteolin 7-O-β-D-glucuronopyranoside (11, apigenin 7-O-α-L-rhamnopyranosyl-(1→2-β-D-glucuronopyranoside (12, apigenin 7-O-β-D-glucopyranosyl-(1→2-β-D-glucuronopyranoside (13, 6-O-(3-nitropropanoyl-β-D-glucopyranoside (14, coronillin (16 and coronarian (15. 120 mg of the most active compound isoorientin against the free radical DPPH was recovered by CPE with an HPLC purity of 99%.

  9. A rapid screening method to monitor expression of various recombinant proteins from prokaryotic and eukaryotic expression systems using MALDI-TOF mass spectrometry

    DEFF Research Database (Denmark)

    Jebanathirajah, J.A.; Andersen, S.; Blagoev, B.

    2002-01-01

    Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive...

  10. Rapid bursts of androgen-binding protein (Abp) gene duplication occurred independently in diverse mammals.

    Science.gov (United States)

    Laukaitis, Christina M; Heger, Andreas; Blakley, Tyler D; Munclinger, Pavel; Ponting, Chris P; Karn, Robert C

    2008-02-12

    The draft mouse (Mus musculus) genome sequence revealed an unexpected proliferation of gene duplicates encoding a family of secretoglobin proteins including the androgen-binding protein (ABP) alpha, beta and gamma subunits. Further investigation of 14 alpha-like (Abpa) and 13 beta- or gamma-like (Abpbg) undisrupted gene sequences revealed a rich diversity of developmental stage-, sex- and tissue-specific expression. Despite these studies, our understanding of the evolution of this gene family remains incomplete. Questions arise from imperfections in the initial mouse genome assembly and a dearth of information about the gene family structure in other rodents and mammals. Here, we interrogate the latest 'finished' mouse (Mus musculus) genome sequence assembly to show that the Abp gene repertoire is, in fact, twice as large as reported previously, with 30 Abpa and 34 Abpbg genes and pseudogenes. All of these have arisen since the last common ancestor with rat (Rattus norvegicus). We then demonstrate, by sequencing homologs from species within the Mus genus, that this burst of gene duplication occurred very recently, within the past seven million years. Finally, we survey Abp orthologs in genomes from across the mammalian clade and show that bursts of Abp gene duplications are not specific to the murid rodents; they also occurred recently in the lagomorph (rabbit, Oryctolagus cuniculus) and ruminant (cattle, Bos taurus) lineages, although not in other mammalian taxa. We conclude that Abp genes have undergone repeated bursts of gene duplication and adaptive sequence diversification driven by these genes' participation in chemosensation and/or sexual identification.

  11. NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions.

    Science.gov (United States)

    Harris, Kimberly A; Bobay, Benjamin G; Sarachan, Kathryn L; Sims, Alexis F; Bilbille, Yann; Deutsch, Christopher; Iwata-Reuyl, Dirk; Agris, Paul F

    2015-08-14

    The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. A rapid generation of adenovirus vector with a genetic modification in hexon protein.

    Science.gov (United States)

    Di, Bingyan; Mao, Qinwen; Zhao, Junli; Li, Xing; Wang, Dongyang; Xia, Haibin

    2012-02-10

    The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. NMR-Based Metabonomic Investigation of Heat Stress in Myotubes Reveals a Time-Dependent Change in the Metabolites

    DEFF Research Database (Denmark)

    Straadt, Ida K; Young, Jette F; Bross, Peter

    2010-01-01

    NMR-based metabonomics was applied to elucidate the time-dependent stress responses in mouse myotubes after heat exposure of either 42 or 45 degrees C for 1 h. Principal component analysis (PCA) revealed that the gradual time-dependent changes in metabolites contributing to the clustering...... and separation of the control samples from the different time points after heat stress primarily are in the metabolites glucose, leucine, lysine, phenylalanine, creatine, glutamine, and acetate. In addition, PC scores revealed a maximum change in metabolite composition 4 h after the stress exposure; thereafter......, samples returned toward control samples, however, without reaching the control samples even 10 h after stress. The results also indicate that the myotubes efficiently regulate the pH level by release of lactate to the culture medium at a heat stress level of 42 degrees C, which is a temperature level...

  14. NMR-based metabolomics approach to study the toxicity of lambda-cyhalothrin to goldfish (Carassius auratus)

    International Nuclear Information System (INIS)

    Li, Minghui; Wang, Junsong; Lu, Zhaoguang; Wei, Dandan; Yang, Minghua; Kong, Lingyi

    2014-01-01

    Highlights: •A goldfish model was established to investigate the toxicity of lambda-cyhalothrin (LCT) exposure on multiple organs. •NMR based metabolomics approach were firstly used to provide a global view of the toxicity of LCT. •LCT induced neurotransmitters and osmoregulatory imbalances, oxidative stress, energy and amino acid metabolic disorders. •Glutamate–glutamine–GABA axis as a potential target for LCT toxicity was first found. -- Abstract: In this study, a 1 H nuclear magnetic resonance (NMR) based metabolomics approach was applied to investigate the toxicity of lambda-cyhalothrin (LCT) in goldfish (Carassius auratus). LCT showed tissue-specific damage to gill, heart, liver and kidney tissues of goldfish. NMR profiling combined with statistical methods such as orthogonal partial least squares discriminant analysis (OPLS-DA) and two-dimensional statistical total correlation spectroscopy (2D-STOCSY) was developed to discern metabolite changes occurring after one week LCT exposure in brain, heart and kidney tissues of goldfish. LCT exposure influenced levels of many metabolites (e.g., leucine, isoleucine and valine in brain and kidney; lactate in brain, heart and kidney; alanine in brain and kidney; choline in brain, heart and kidney; taurine in brain, heart and kidney; N-acetylaspartate in brain; myo-inositol in brain; phosphocreatine in brain and heart; 2-oxoglutarate in brain; cis-aconitate in brain, and etc.), and broke the balance of neurotransmitters and osmoregulators, evoked oxidative stress, disturbed metabolisms of energy and amino acids. The implication of glutamate–glutamine–gamma-aminobutyric axis in LCT induced toxicity was demonstrated for the first time. Our findings demonstrated the applicability and potential of metabolomics approach for the elucidation of toxicological effects of pesticides and the underlying mechanisms, and the discovery of biomarkers for pesticide pollution in aquatic environment

  15. 1H NMR-based serum metabolomics reveals erythromycin-induced liver toxicity in albino Wistar rats

    Directory of Open Access Journals (Sweden)

    Atul Rawat

    2016-01-01

    Full Text Available Introduction: Erythromycin (ERY is known to induce hepatic toxicity which mimics other liver diseases. Thus, ERY is often used to produce experimental models of drug-induced liver-toxicity. The serum metabolic profiles can be used to evaluate the liver-toxicity and to further improve the understanding of underlying mechanism. Objective: To establish the serum metabolic patterns of Erythromycin induced hepatotoxicity in albino wistar rats using 1H NMR based serum metabolomics. Experimental: Fourteen male rats were randomly divided into two groups (n = 7 in each group: control and ERY treated. After 28 days of intervention, the metabolic profiles of sera obtained from ERY and control groups were analyzed using high-resolution 1D 1H CPMG and diffusion-edited nuclear magnetic resonance (NMR spectra. The histopathological and SEM examinations were employed to evaluate the liver toxicity in ERY treated group. Results: The serum metabolic profiles of control and ERY treated rats were compared using multivariate statistical analysis and the metabolic patterns specific to ERY-induced liver toxicity were established. The toxic response of ERY was characterized with: (a increased serum levels of Glucose, glutamine, dimethylamine, malonate, choline, phosphocholine and phospholipids and (b decreased levels of isoleucine, leucine, valine, alanine, glutamate, citrate, glycerol, lactate, threonine, circulating lipoproteins, N-acetyl glycoproteins, and poly-unsaturated lipids. These metabolic alterations were found to be associated with (a decreased TCA cycle activity and enhanced fatty acid oxidation, (b dysfunction of lipid and amino acid metabolism and (c oxidative stress. Conclusion and Recommendations: Erythromycin is often used to produce experimental models of liver toxicity; therefore, the established NMR-based metabolic patterns will form the basis for future studies aiming to evaluate the efficacy of anti-hepatotoxic agents or the hepatotoxicity of new

  16. Diagnosis of coinfection by schistosomiasis and viral hepatitis B or C using 1H NMR-based metabonomics.

    Science.gov (United States)

    Gouveia, Liana Ribeiro; Santos, Joelma Carvalho; Silva, Ronaldo Dionísio; Batista, Andrea Dória; Domingues, Ana Lúcia Coutinho; Lopes, Edmundo Pessoa de Almeida; Silva, Ricardo Oliveira

    2017-01-01

    Diagnosis of liver involvement due to schistosomiasis in asymptomatic patients from endemic areas previously diagnosed with chronic hepatitis B (HBV) or C (HCV) and periportal fibrosis is challenging. H-1 Nuclear Magnetic Resonance (NMR)-based metabonomics strategy is a powerful tool for providing a profile of endogenous metabolites of low molecular weight in biofluids in a non-invasive way. The aim of this study was to diagnose periportal fibrosis due to schistosomiasis mansoni in patients with chronic HBV or HCV infection through NMR-based metabonomics models. The study included 40 patients divided into two groups: (i) 18 coinfected patients with schistosomiasis mansoni and HBV or HCV; and (ii) 22 HBV or HCV monoinfected patients. The serum samples were analyzed through H-1 NMR spectroscopy and the models were based on Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA). Ultrasonography examination was used to ascertain the diagnosis of periportal fibrosis. Exploratory analysis showed a clear separation between coinfected and monoinfected samples. The supervised model built from PLS-DA showed accuracy, R2 and Q2 values equal to 100%, 98.1% and 97.5%, respectively. According to the variable importance in the projection plot, lactate serum levels were higher in the coinfected group, while the signals attributed to HDL serum cholesterol were more intense in the monoinfected group. The metabonomics models constructed in this study are promising as an alternative tool for diagnosis of periportal fibrosis by schistosomiasis in patients with chronic HBV or HCV infection from endemic areas for Schistosoma mansoni.

  17. NMR-based metabolomics approach to study the toxicity of lambda-cyhalothrin to goldfish (Carassius auratus)

    Energy Technology Data Exchange (ETDEWEB)

    Li, Minghui [State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009 (China); Wang, Junsong, E-mail: wang.junsong@gmail.com [Center for Molecular Metabolism, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, 200 Xiao Ling Wei Street, Nanjing 210094 (China); Lu, Zhaoguang; Wei, Dandan; Yang, Minghua [State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009 (China); Kong, Lingyi, E-mail: cpu_lykong@126.com [State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009 (China)

    2014-01-15

    Highlights: •A goldfish model was established to investigate the toxicity of lambda-cyhalothrin (LCT) exposure on multiple organs. •NMR based metabolomics approach were firstly used to provide a global view of the toxicity of LCT. •LCT induced neurotransmitters and osmoregulatory imbalances, oxidative stress, energy and amino acid metabolic disorders. •Glutamate–glutamine–GABA axis as a potential target for LCT toxicity was first found. -- Abstract: In this study, a {sup 1}H nuclear magnetic resonance (NMR) based metabolomics approach was applied to investigate the toxicity of lambda-cyhalothrin (LCT) in goldfish (Carassius auratus). LCT showed tissue-specific damage to gill, heart, liver and kidney tissues of goldfish. NMR profiling combined with statistical methods such as orthogonal partial least squares discriminant analysis (OPLS-DA) and two-dimensional statistical total correlation spectroscopy (2D-STOCSY) was developed to discern metabolite changes occurring after one week LCT exposure in brain, heart and kidney tissues of goldfish. LCT exposure influenced levels of many metabolites (e.g., leucine, isoleucine and valine in brain and kidney; lactate in brain, heart and kidney; alanine in brain and kidney; choline in brain, heart and kidney; taurine in brain, heart and kidney; N-acetylaspartate in brain; myo-inositol in brain; phosphocreatine in brain and heart; 2-oxoglutarate in brain; cis-aconitate in brain, and etc.), and broke the balance of neurotransmitters and osmoregulators, evoked oxidative stress, disturbed metabolisms of energy and amino acids. The implication of glutamate–glutamine–gamma-aminobutyric axis in LCT induced toxicity was demonstrated for the first time. Our findings demonstrated the applicability and potential of metabolomics approach for the elucidation of toxicological effects of pesticides and the underlying mechanisms, and the discovery of biomarkers for pesticide pollution in aquatic environment.

  18. Application of NMR-based metabolomics for environmental assessment in the Great Lakes using zebra mussel (Dreissena polymorpha).

    Science.gov (United States)

    Watanabe, Miki; Meyer, Kathryn A; Jackson, Tyler M; Schock, Tracey B; Johnson, W Edward; Bearden, Daniel W

    Zebra mussel, Dreissena polymorpha , in the Great Lakes is being monitored as a bio-indicator organism for environmental health effects by the National Oceanic and Atmospheric Administration's Mussel Watch program. In order to monitor the environmental effects of industrial pollution on the ecosystem, invasive zebra mussels were collected from four stations-three inner harbor sites (LMMB4, LMMB1, and LMMB) in Milwaukee Estuary, and one reference site (LMMB5) in Lake Michigan, Wisconsin. Nuclear magnetic resonance (NMR)-based metabolomics was used to evaluate the metabolic profiles of the mussels from these four sites. The objective was to observe whether there were differences in metabolite profiles between impacted sites and the reference site; and if there were metabolic profile differences among the impacted sites. Principal component analyses indicated there was no significant difference between two impacted sites: north Milwaukee harbor (LMMB and LMMB4) and the LMMB5 reference site. However, significant metabolic differences were observed between the impacted site on the south Milwaukee harbor (LMMB1) and the LMMB5 reference site, a finding that correlates with preliminary sediment toxicity results. A total of 26 altered metabolites (including two unidentified peaks) were successfully identified in a comparison of zebra mussels from the LMMB1 site and LMMB5 reference site. The application of both uni- and multivariate analysis not only confirmed the variability of altered metabolites but also ensured that these metabolites were identified via unbiased analysis. This study has demonstrated the feasibility of the NMR-based metabolomics approach to assess whole-body metabolomics of zebra mussels to study the physiological impact of toxicant exposure at field sites.

  19. Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

    Directory of Open Access Journals (Sweden)

    Maxime Belondrade

    Full Text Available The prevalence of variant Creutzfeldt-Jakob disease (vCJD in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA. This assay allowed the specific detection of minute quantities (10-8 brain dilution of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

  20. A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

    Directory of Open Access Journals (Sweden)

    Ingrid Kikkas

    Full Text Available Type 1 diabetes (T1D results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

  1. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

    Science.gov (United States)

    Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

  2. Efficiency of histidine rich protein II-based rapid diagnostic tests for monitoring malaria transmission intensities in an endemic area

    Science.gov (United States)

    Modupe, Dokunmu Titilope; Iyabo, Olasehinde Grace; Oladoke, Oladejo David; Oladeji, Olanrewaju; Abisola, Akinbobola; Ufuoma, Adjekukor Cynthia; Faith, Yakubu Omolara; Humphrey, Adebayo Abiodun

    2018-04-01

    In recent years there has been a global decrease in the prevalence of malaria due to scaling up of control measures, hence global control efforts now target elimination and eradication of the disease. However, a major problem associated with elimination is asymptomatic reservoir of infection especially in endemic areas. This study aims to determine the efficiency of histidine rich protein II (HRP-2) based rapid diagnostic tests (RDT) for monitoring transmission intensities in an endemic community in Nigeria during the pre-elimination stage. Plasmodium falciparum asymptomatic malaria infection in healthy individuals and symptomatic cases were detected using HRP-2. RDT negative tests were re-checked by microscopy and by primer specific PCR amplification of merozoite surface protein 2 (msp-2) for asexual parasites and Pfs25 gene for gametocytes in selected samples to detect low level parasitemia undetectable by microscopy. The mean age of the study population (n=280) was 6.12 years [95% CI 5.16 - 7.08, range 0.5 - 55], parasite prevalence was 44.6% and 36.3% by microscopy and RDT respectively (p =0.056). The parasite prevalence of 61.5% in children aged >2 - 10 years was significantly higher than 3.7% rate in adults >18years (p malaria in endemic areas.

  3. Effect of magnetic field strength on NMR-based metabonomic human urine data. Comparative study of 250, 400, 500, and 800 MHz

    DEFF Research Database (Denmark)

    Bertram, Hanne Christine; Malmendal, Anders; Petersen, Bent O.

    2007-01-01

    Metabonomic analysis of urine utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to an intervention. To assess the effect of magnetic field strength on information contained in NMR-based metabonomic data sets, 1H NMR...

  4. NMR-based metabonomics study on the effect of Gancao in the attenuation of toxicity in rats induced by Fuzi.

    Science.gov (United States)

    Sun, Bo; Wang, Xubin; Cao, Ruili; Zhang, Qi; Liu, Qiao; Xu, Meifeng; Zhang, Ming; Du, Xiangbo; Dong, Fangting; Yan, Xianzhong

    2016-12-04

    Fuzi, the processed lateral root of Aconitum carmichaelii Debeaux, is a traditional Chinese medicine used for its analgesic, antipyretic, anti-rheumatoid arthritis and anti-inflammation effects; however, it is also well known for its toxicity. Gancao, the root of Glycyrrhiza uralensis Fisch., is often used concurrently with Fuzi to alleviate its toxicity. However, the mechanism of detoxication is still not well clear. In this study, the effect of Gancao on the metabolic changes induced by Fuzi was investigated by NMR-based metabonomic approaches. Fifty male Wistar rats were randomly divided into five groups (group A: control, group B: Fuzi decoction alone, group C: Gancao decoction alone, group D: Fuzi decoction and Gancao decoction simultaneously, group E: Fuzi decoction 5h after Gancao decoction) and urine samples were collected for NMR-based metabolic profiling analysis. Statistical analyses such as unsupervised PCA, t-test, hierarchical cluster, and pathway analysis were used to detect the effects of Gancao on the metabolic changes induced by Fuzi. The behavioral and biochemical characteristics showed that Fuzi exhibited toxic effects on treated rats (group B) and statistical analyses showed that their metabolic profiles were in contrast to those in groups A and C. However, when Fuzi was administered with Gancao, the metabolic profiles became similar to controls, whereby Gancao reduced the levels of trimethylamine N-oxide, betaine, dimethylglycine, valine, acetoacetate, citrate, fumarate, 2-ketoglutarate and hippurate, and regulated the concentrations of taurine and 3-hydroxybutyrate, resulting in a decrease in toxicity. Furthermore, important pathways that are known to be involved in the effect of Gancao on Fuzi, including phenylalanine, tyrosine and tryptophan biosynthesis, the synthesis and degradation of ketone bodies, and the TCA cycle, were altered in co-treated rats. Gancao treatment mitigated the metabolic changes altered by Fuzi administration in rats

  5. Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

    Science.gov (United States)

    Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

    2015-10-29

    HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

  6. Efficacy of Corrected Rapid Turnover Protein Increment Index (CRII) for Early Detection of Improvement of Nutrition Status in Patients with Malnutrition

    OpenAIRE

    Suyama, Yohji; Adachi, Kyoichi; Notsu, Yoshitomo; Satoh, Emi; Nariai, Yukiko; Hashimoto, Yohko; Sumi, Asako; Kawaguchi, Mikiko; Ishimura, Norihisa

    2009-01-01

    Serum prealbumin level is useful for assessment of changes in nutritional status but it is markedly affected by the inflammation. In this study, we examined the efficacy of the corrected rapid turnover protein increment index (CRII) for prealbumin, which is calculated as [prealbumin level/C-reactive protein (CRP) level on the assessment day]/[prealbumin level/CRP level on the day of starting nutritional care], for prediction of improvement of nutritional status in patients with malnutrition. ...

  7. NMR-based metabolomic studies on the toxicological effects of cadmium and copper on green mussels Perna viridis

    International Nuclear Information System (INIS)

    Wu Huifeng; Wang Wenxiong

    2010-01-01

    Traditional toxicology studies have focused on selected biomarkers to characterize the biological stress induced by metals in marine organisms. In this study, a system biology tool, metabolomics, was applied to the marine mussel Perna viridis to investigate changes in the metabolic profiles of soft tissue as a response to copper (Cu) and cadmium (Cd), both as single metal and as a mixture. The major metabolite changes corresponding to metal exposure are related to amino acids, osmolytes, and energy metabolites. Following metal exposure for 1 week, there was a significant increase in the levels of branched chain amino acids, histidine, glutamate, glutamine, hypotaurine, dimethylglycine, arginine and ATP/ADP. For the Cu + Cd co-exposed mussels, the levels of lactate, branched chain amino acid, succinate, and NAD increased, whereas the levels of glucose, glycogen, and ATP/ADP decreased, indicating a different metabolic profile for the single metal exposure groups. After 2 weeks of exposure, the mussels showed acclimatization to Cd exposure based on the recovery of some metabolites. However, the metabolic profile induced by the metal mixture was very similar to that from Cu exposure, suggesting that Cu dominantly induced the metabolic disturbances. Both Cu and Cd may lead to neurotoxicity, disturbances in energy metabolism, and osmoregulation changes. These results demonstrate the high applicability and reliability of NMR-based metabolomics in interpreting the toxicological mechanisms of metals using global metabolic biomarkers.

  8. 1H NMR-based metabolic profiling reveals inherent biological variation in yeast and nematode model systems

    International Nuclear Information System (INIS)

    Szeto, Samuel S. W.; Reinke, Stacey N.; Lemire, Bernard D.

    2011-01-01

    The application of metabolomics to human and animal model systems is poised to provide great insight into our understanding of disease etiology and the metabolic changes that are associated with these conditions. However, metabolomic studies have also revealed that there is significant, inherent biological variation in human samples and even in samples from animal model systems where the animals are housed under carefully controlled conditions. This inherent biological variability is an important consideration for all metabolomics analyses. In this study, we examined the biological variation in 1 H NMR-based metabolic profiling of two model systems, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Using relative standard deviations (RSD) as a measure of variability, our results reveal that both model systems have significant amounts of biological variation. The C. elegans metabolome possesses greater metabolic variance with average RSD values of 29 and 39%, depending on the food source that was used. The S. cerevisiae exometabolome RSD values ranged from 8% to 12% for the four strains examined. We also determined whether biological variation occurs between pairs of phenotypically identical yeast strains. Multivariate statistical analysis allowed us to discriminate between pair members based on their metabolic phenotypes. Our results highlight the variability of the metabolome that exists even for less complex model systems cultured under defined conditions. We also highlight the efficacy of metabolic profiling for defining these subtle metabolic alterations.

  9. (1)H-NMR-based metabolomic analysis of the effect of moderate wine consumption on subjects with cardiovascular risk factors.

    Science.gov (United States)

    Vázquez-Fresno, Rosa; Llorach, Rafael; Alcaro, Francesca; Rodríguez, Miguel Ángel; Vinaixa, Maria; Chiva-Blanch, Gemma; Estruch, Ramon; Correig, Xavier; Andrés-Lacueva, Cristina

    2012-08-01

    Moderate wine consumption is associated with health-promoting activities. An H-NMR-based metabolomic approach was used to identify urinary metabolomic differences of moderate wine intake in the setting of a prospective, randomized, crossover, and controlled trial. Sixty-one male volunteers with high cardiovascular risk factors followed three dietary interventions (28 days): dealcoholized red wine (RWD) (272mL/day, polyphenol control), alcoholized red wine (RWA) (272mL/day) and gin (GIN) (100mL/day, alcohol control). After each period, 24-h urine samples were collected and analyzed by (1) H-NMR. According to the results of a one-way ANOVA, significant markers were grouped in four categories: alcohol-related markers (ethanol); gin-related markers; wine-related markers; and gut microbiota markers (hippurate and 4-hydroxphenylacetic acid). Wine metabolites were classified into two groups; first, metabolites of food metabolome: tartrate (RWA and RWD), ethanol, and mannitol (RWA); and second, biomarkers that relates to endogenous modifications after wine consumption, comprising branched-chain amino acid (BCAA) metabolite (3-methyl-oxovalerate). Additionally, a possible interaction between alcohol and gut-related biomarkers has been identified. To our knowledge, this is the first time that this approach has been applied in a nutritional intervention with red wine. The results show the capacity of this approach to obtain a comprehensive metabolome picture including food metabolome and endogenous biomarkers of moderate wine intake. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. NMR-based metabonomic analyses of the effects of ultrasmall superparamagnetic particles of iron oxide (USPIO) on macrophage metabolism

    Science.gov (United States)

    Feng, Jianghua; Zhao, Jing; Hao, Fuhua; Chen, Chang; Bhakoo, Kishore; Tang, Huiru

    2011-05-01

    The metabonomic changes in murine RAW264.7 macrophage-like cell line induced by ultrasmall superparamagnetic particles of iron oxides (USPIO) have been investigated, by analyzing both the cells and culture media, using high-resolution NMR in conjunction with multivariate statistical methods. Upon treatment with USPIO, macrophage cells showed a significant decrease in the levels of triglycerides, essential amino acids such as valine, isoleucine, and choline metabolites together with an increase of glycerophospholipids, tyrosine, phenylalanine, lysine, glycine, and glutamate. Such cellular responses to USPIO were also detectable in compositional changes of cell media, showing an obvious depletion of the primary nutrition molecules, such as glucose and amino acids and the production of end-products of glycolysis, such as pyruvate, acetate, and lactate and intermediates of TCA cycle such as succinate and citrate. At 48 h treatment, there was a differential response to incubation with USPIO in both cell metabonome and medium components, indicating that USPIO are phagocytosed and released by macrophages. Furthermore, information on cell membrane modification can be derived from the changes in choline-like metabolites. These results not only suggest that NMR-based metabonomic methods have sufficient sensitivity to identify the metabolic consequences of murine RAW264.7 macrophage-like cell line response to USPIO in vitro, but also provide useful information on the effects of USPIO on cellular metabolism.

  11. Comparison of Chemical Compositions in Pseudostellariae Radix from Different Cultivated Fields and Germplasms by NMR-Based Metabolomics

    Directory of Open Access Journals (Sweden)

    Yujiao Hua

    2016-11-01

    Full Text Available Pseudostellariae Radix (PR is an important traditional Chinese medicine (TCM, which is consumed commonly for its positive health effects. However, the chemical differences of PR from different cultivated fields and germplasms are still unknown. In order to comprehensively compare the chemical compositions of PR from different cultivated fields, in this study, 1H-NMR-based metabolomics coupled with high performance liquid chromatography (HPLC were used to investigate the different metabolites in PR from five germplasms (jr, zs1, zs2, sb, and xc cultivated in traditional fields (Jurong, Jiangsu, JSJR and cultivated fields (Zherong, Fujian, FJZR. A total of 34 metabolites were identified based on 1H-NMR data, and fourteen of them were found to be different in PR from JSJR and FJZR. The relative contents of alanine, lactate, lysine, taurine, sucrose, tyrosine, linolenic acid, γ-aminobutyrate, and hyperoside in PR from JSJR were higher than that in PR from FJZR, while PR from FJZR contained higher levels of glutamine, raffinose, xylose, unsaturated fatty acid, and formic acid. The contents of Heterophyllin A and Heterophyllin B were higher in PR from FJZR. This study will provide the basic information for exploring the influence law of ecological environment and germplasm genetic variation on metabolite biosynthesis of PR and its quality formation mechanism.

  12. {sup 1}H NMR-based metabolic profiling reveals inherent biological variation in yeast and nematode model systems

    Energy Technology Data Exchange (ETDEWEB)

    Szeto, Samuel S. W.; Reinke, Stacey N.; Lemire, Bernard D., E-mail: bernard.lemire@ualberta.ca [University of Alberta, Department of Biochemistry, School of Molecular and Systems Medicine (Canada)

    2011-04-15

    The application of metabolomics to human and animal model systems is poised to provide great insight into our understanding of disease etiology and the metabolic changes that are associated with these conditions. However, metabolomic studies have also revealed that there is significant, inherent biological variation in human samples and even in samples from animal model systems where the animals are housed under carefully controlled conditions. This inherent biological variability is an important consideration for all metabolomics analyses. In this study, we examined the biological variation in {sup 1}H NMR-based metabolic profiling of two model systems, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Using relative standard deviations (RSD) as a measure of variability, our results reveal that both model systems have significant amounts of biological variation. The C. elegans metabolome possesses greater metabolic variance with average RSD values of 29 and 39%, depending on the food source that was used. The S. cerevisiae exometabolome RSD values ranged from 8% to 12% for the four strains examined. We also determined whether biological variation occurs between pairs of phenotypically identical yeast strains. Multivariate statistical analysis allowed us to discriminate between pair members based on their metabolic phenotypes. Our results highlight the variability of the metabolome that exists even for less complex model systems cultured under defined conditions. We also highlight the efficacy of metabolic profiling for defining these subtle metabolic alterations.

  13. Toxicological effects induced by cadmium in gills of Manila clam ruditapes philippinarum using NMR-based metabolomics

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Linbao; Liu, Xiaoli; You, Liping; Zhou, Di [Key Laboratory of Coastal Zone Environment Processes, CAS, Shandong Provincial Key Laboratory of Coastal Zone Environment Processes, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai (China); The Graduate School of Chinese Academy of Sciences, Beijing (China); Yu, Junbao; Zhao, Jianmin; Wu, Huifeng [Key Laboratory of Coastal Zone Environment Processes, CAS, Shandong Provincial Key Laboratory of Coastal Zone Environment Processes, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai (China); Feng, Jianghua [Department of Electronic Science, Fujian Key Laboratory of Plasma and Magnetic Resonance, State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen (China)

    2011-11-15

    Cadmium (Cd) has become an important heavy metal contaminant in the sediment and seawater along the Bohai Sea and been of great ecological risk due to its toxic effects to marine organisms. In this work, the toxicological effects caused by environmentally relevant concentrations (10 and 40 {mu}g L{sup -1}) of Cd were studied in the gill tissues of Manila clam Ruditapes philippinarum after exposure for 24, 48, and 96 h. Both low (10 {mu}g L{sup -1}) and high (40 {mu}g L{sup -1}) doses of Cd caused the disturbances in energy metabolism and osmotic regulation and neurotoxicity based on the metabolic biomarkers such as succinate, alanine, branched chain amino acids, betaine, hypotaurine, and glutamate in clam gills after 24 h of exposure. However, the recovery of toxicological effects of Cd after exposure for 96 h was obviously observed in clam to Cd exposures. Overall, these results indicated that NMR-based metabolomics was applicable to elucidate the toxicological effects of heavy metal contaminants in the marine bioindicator. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  14. NMR-based metabonomic analyses of the effects of ultrasmall superparamagnetic particles of iron oxide (USPIO) on macrophage metabolism

    International Nuclear Information System (INIS)

    Feng Jianghua; Zhao Jing; Hao Fuhua; Chen Chang; Bhakoo, Kishore; Tang, Huiru

    2011-01-01

    The metabonomic changes in murine RAW264.7 macrophage-like cell line induced by ultrasmall superparamagnetic particles of iron oxides (USPIO) have been investigated, by analyzing both the cells and culture media, using high-resolution NMR in conjunction with multivariate statistical methods. Upon treatment with USPIO, macrophage cells showed a significant decrease in the levels of triglycerides, essential amino acids such as valine, isoleucine, and choline metabolites together with an increase of glycerophospholipids, tyrosine, phenylalanine, lysine, glycine, and glutamate. Such cellular responses to USPIO were also detectable in compositional changes of cell media, showing an obvious depletion of the primary nutrition molecules, such as glucose and amino acids and the production of end-products of glycolysis, such as pyruvate, acetate, and lactate and intermediates of TCA cycle such as succinate and citrate. At 48 h treatment, there was a differential response to incubation with USPIO in both cell metabonome and medium components, indicating that USPIO are phagocytosed and released by macrophages. Furthermore, information on cell membrane modification can be derived from the changes in choline-like metabolites. These results not only suggest that NMR-based metabonomic methods have sufficient sensitivity to identify the metabolic consequences of murine RAW264.7 macrophage-like cell line response to USPIO in vitro, but also provide useful information on the effects of USPIO on cellular metabolism.

  15. A rapid radioimmunoassay using 125I-labeled staphylococcal protein A for antibody to varicella-zoster virus

    International Nuclear Information System (INIS)

    Richman, D.D.; Cleveland, P.H.; Oxman, M.N.; Zaia, J.A.

    1981-01-01

    A sensitive radioimmunoassay for serum antibody to varicella-zoster virus is described; it uses 125I-labeled staphylococcal protein A and a specially designed immunofiltration apparatus. The assay accurately distinguishes between individuals who are susceptible and those who are immune to infection with varicella-zoster virus. In addition, it can detect passive antibody in recipients of varicella-zoster immune globulin. This radioimmunoassay also detects the heterologous antibody responses that occasionally occur in patients infected with herpes simplex virus, which also have been detected by other antibody assays. The particular advantages of this assay are the use of noninfectious reagents, the speed of execution (less than 3 hr), the requirement for only small quantities of serum (30 microliters), the objectivity of end-point determination, and the capability of screening large numbers of sera. Consequently, this radioimmunoassay is especially useful for the rapid identification of susceptible individuals, which is essential for the appropriate management of patients and hospital personnel after exposure to varicella

  16. Development of an automated on-line pepsin digestion-liquid chromatography-tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents

    NARCIS (Netherlands)

    Carol-Visser, J.; van der Schans, M.; Fidder, A.; Huist, A.G.; van Baar, B.L.M.; Irth, H.; Noort, D.

    2008-01-01

    Rapid monitoring and retrospective verification are key issues in protection against and non-proliferation of chemical warfare agents (CWA). Such monitoring and verification are adequately accomplished by the analysis of persistent protein adducts of these agents. Liquid chromatography-mass

  17. Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

    Science.gov (United States)

    Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

    2012-04-01

    Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

  18. Organic conjugated small molecule materials based optical probe for rapid, colorimetric and UV-vis spectral detection of phosphorylated protein in placental tissue.

    Science.gov (United States)

    Wang, Yanfang; Yang, Na; Liu, Yi

    2018-04-05

    A novel organic small molecule with D-Pi-A structure was prepared, which was found to be a promising colorimetric and ratiometric UV-vis spetral probe for detection of phosphorylated proteins with the help of tetravalent zirconium ion. Such optical probe based on chromophore WYF-1 shows a rapid response (within 10s) and high selectivity and sensitivity for phosphorylated proteins, giving distinct colorimetric and ratiometric UV-vis changes at 720 and 560nm. The detection limit for phosphorylated proteins was estimated to be 100nM. In addition, detection of phosphorylated proteins in placental tissue samples with this probe was successfully applied, which indicates that this probe holds great potential for phosphorylated proteins detection. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. NMR-based Metabolomics Analysis of Liver from C57BL/6 Mouse Exposed to Ionizing Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Xiongjie [Pacific Northwest National Laboratory, Richland, Washington 99352; State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, Wuhan, 430071, PR China; University of Chinese Academy of Sciences, Beijing 100049, China; Hu, Mary [Pacific Northwest National Laboratory, Richland, Washington 99352; Zhang, Xu [State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, the Chinese Academy of Sciences, Wuhan, 430071, PR China; Hu, Jian Zhi [Pacific Northwest National Laboratory, Richland, Washington 99352

    2017-07-01

    The health effects of exposing to ionizing radiation are attracting great interest in the space exploration community and patients considering radiotherapy. However, the impact to metabolism after exposure to high dose radiation has not yet been clearly defined in livers. In the present study, 1H nuclear magnetic resonance (NMR) based metabolomics combined with multivariate data analysis are applied to study the changes of metabolism in the liver of C57BL/6 mouse after whole body exposure to either gamma (3.0 and 7.8 Gy) or proton (3.0 Gy) radiation. Principal component analysis (PCA) and orthogonal projection to latent structures analysis (OPLS) are employed for classification and identification of potential biomarkers associated with gamma and proton irradiation. The results show that the radiation exposed groups can be well separated from the control group. At the same radiation dosage, the group exposed to proton radiation is well separated from the group exposed to gamma radiation, indicating different radiation sources induce different alterations based on metabolic profiling. Common to both gamma and proton radiation at the high radiation doses studied in this work, compared with the control groups the concentrations of choline, O-phosphocholine and trimethylamine N-oxide are decreased statistically, while those of glutamine, glutathione, malate, creatinine, phosphate, betaine and 4-hydroxyphenylacetate are statistically and significantly elevated after exposure to radiation. Since these altered metabolites are associated with multiple biological pathways, the changes suggest that the exposure to radiation induce abnormality in multiple biological pathways. In particular, metabolites such as 4-hydroxyphenylacetate, betaine, glutamine, choline and trimethylamine N-oxide may be good candidates of pre-diagnose biomarkers for ionizing radiation in liver.

  20. NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats after oral administration

    Science.gov (United States)

    Bu, Qian; Yan, Guangyan; Deng, Pengchi; Peng, Feng; Lin, Hongjun; Xu, Youzhi; Cao, Zhixing; Zhou, Tian; Xue, Aiqin; Wang, Yanli; Cen, Xiaobo; Zhao, Ying-Lan

    2010-03-01

    As titanium dioxide nanoparticles (TiO2 NPs) are widely used commercially, their potential toxicity on human health has attracted particular attention. In the present study, the oral toxicological effects of TiO2 NPs (dosed at 0.16, 0.4 and 1 g kg - 1, respectively) were investigated using conventional approaches and metabonomic analysis in Wistar rats. Serum chemistry, hematology and histopathology examinations were performed. The urine and serum were investigated by 1H nuclear magnetic resonance (NMR) using principal components and partial least squares discriminant analysis. The metabolic signature of urinalysis in TiO2 NP-treated rats showed increases in the levels of taurine, citrate, hippurate, histidine, trimethylamine-N-oxide (TMAO), citrulline, α-ketoglutarate, phenylacetylglycine (PAG) and acetate; moreover, decreases in the levels of lactate, betaine, methionine, threonine, pyruvate, 3-D-hydroxybutyrate (3-D-HB), choline and leucine were observed. The metabonomics analysis of serum showed increases in TMAO, choline, creatine, phosphocholine and 3-D-HB as well as decreases in glutamine, pyruvate, glutamate, acetoacetate, glutathione and methionine after TiO2 NP treatment. Aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated and mitochondrial swelling in heart tissue was observed in TiO2 NP-treated rats. These findings indicate that disturbances in energy and amino acid metabolism and the gut microflora environment may be attributable to the slight injury to the liver and heart caused by TiO2 NPs. Moreover, the NMR-based metabolomic approach is a reliable and sensitive method to study the biochemical effects of nanomaterials.

  1. NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats

    International Nuclear Information System (INIS)

    Lu Chunfeng; Wang Yimei; Sheng Zhiguo; Liu Gang; Fu Ze; Zhao Jing; Zhao Jun; Yan Xianzhong; Zhu Benzhan; Peng Shuangqing

    2010-01-01

    A metabonomic approach using 1 H NMR spectroscopy was adopted to investigate the metabonomic pattern of rat urine after oral administration of environmental endocrine disruptors (EDs) polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone or in combination and to explore the possible hepatotoxic mechanisms of combined exposure to PCBs and TCDD. 1 H NMR spectra of urines collected 24 h before and after exposure were analyzed via pattern recognition by using principal component analysis (PCA). Serum biochemistry and liver histopathology indicated significant hepatotoxicity in the rats of the combined group. The PCA scores plots of urinary 1 H NMR data showed that all the treatment groups could be easily distinguished from the control group, so could the PCBs or TCDD group and the combined group. The loadings plots of the PCA revealed remarkable increases in the levels of lactate, glucose, taurine, creatine, and 2-hydroxy-isovaleric acid and reductions in the levels of 2-oxoglutarate, citrate, succinate, hippurate, and trimethylamine-N-oxide in rat urine after exposure. These changes were more striking in the combined group. The changed metabolites may be considered possible biomarker for the hepatotoxicity. The present study demonstrates that combined exposure to PCBs and TCDD induced significant hepatotoxicity in rats, and mitochondrial dysfunction and fatty acid metabolism perturbations might contribute to the hepatotoxicity. There was good conformity between changes in the urine metabonomic pattern and those in serum biochemistry and liver histopathology. These results showed that the NMR-based metabonomic approach may provide a promising technique for the evaluation of the combined toxicity of EDs.

  2. (1)H NMR-based metabonomics revealed protective effect of Naodesheng bioactive extract on ischemic stroke rats.

    Science.gov (United States)

    Luo, Lan; Zhen, Lifeng; Xu, Yatao; Yang, Yongxia; Feng, Suxiang; Wang, Shumei; Liang, Shengwang

    2016-06-20

    Stroke is a leading cause of death and disability in the world. However, current therapies are limited. Naodesheng, a widely used traditional Chinese medicine prescription, has shown a good clinical curative effect on ischemic stroke. Also, Naodesheng has been suggested to have neuroprotective effect on focal cerebral ischemia rats, but the underlying molecular mechanism remains unclear. The present study was designed to evaluate the effect of Naodesheng bioactive extract on the metabolic changes in brain tissue, plasma and urine induced by cerebral ischemia perfusion injury, and explore the possible metabolic mechanisms by using a (1)H NMR-based metabonomics approach. A middle cerebral artery occlusion rat model was established and confirmed by the experiments of neurobehavioral abnormality evaluation, brain tissue TTC staining and pathological examination. The metabolic changes in brain tissue, plasma and urine were then assessed by a (1)H NMR technique combined with multivariate statistical analysis method. These NMR data showed that cerebral ischemia reperfusion induced great metabolic disorders in brain tissue, plasma and urine metabolisms. However, Naodesheng bioactive extract could reverse most of the imbalanced metabolites. Meanwhile, it was found that both the medium and high dosages of Naodesheng bioactive extract were more effective on the metabolic changes than the low dosage, consistent with histopathological assessments. These results revealed that Naodesheng had protective effect on ischemic stroke rats and the underlying mechanisms involved multiple metabolic pathways, including energy metabolism, amino acid metabolism, oxidative stress and inflammatory injury. The present study could provide evidence that metabonomics revealed its capacity to evaluate the holistic efficacy of traditional Chinese medicine and explore the underlying mechanisms. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats after oral administration

    International Nuclear Information System (INIS)

    Bu Qian; Lin Hongjun; Xu Youzhi; Cao Zhixing; Zhou Tian; Zhao Yinglan; Yan Guangyan; Cen Xiaobo; Deng Pengchi; Peng Feng; Xue Aiqin; Wang Yanli

    2010-01-01

    As titanium dioxide nanoparticles (TiO 2 NPs) are widely used commercially, their potential toxicity on human health has attracted particular attention. In the present study, the oral toxicological effects of TiO 2 NPs (dosed at 0.16, 0.4 and 1 g kg -1 , respectively) were investigated using conventional approaches and metabonomic analysis in Wistar rats. Serum chemistry, hematology and histopathology examinations were performed. The urine and serum were investigated by 1 H nuclear magnetic resonance (NMR) using principal components and partial least squares discriminant analysis. The metabolic signature of urinalysis in TiO 2 NP-treated rats showed increases in the levels of taurine, citrate, hippurate, histidine, trimethylamine-N-oxide (TMAO), citrulline, α-ketoglutarate, phenylacetylglycine (PAG) and acetate; moreover, decreases in the levels of lactate, betaine, methionine, threonine, pyruvate, 3-D-hydroxybutyrate (3-D-HB), choline and leucine were observed. The metabonomics analysis of serum showed increases in TMAO, choline, creatine, phosphocholine and 3-D-HB as well as decreases in glutamine, pyruvate, glutamate, acetoacetate, glutathione and methionine after TiO 2 NP treatment. Aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated and mitochondrial swelling in heart tissue was observed in TiO 2 NP-treated rats. These findings indicate that disturbances in energy and amino acid metabolism and the gut microflora environment may be attributable to the slight injury to the liver and heart caused by TiO 2 NPs. Moreover, the NMR-based metabolomic approach is a reliable and sensitive method to study the biochemical effects of nanomaterials.

  4. An NMR-based metabolomic approach to investigate the effects of supplementation with glutamic acid in piglets challenged with deoxynivalenol.

    Science.gov (United States)

    Wu, Miaomiao; Xiao, Hao; Ren, Wenkai; Yin, Jie; Hu, Jiayu; Duan, Jielin; Liu, Gang; Tan, Bie; Xiong, Xia; Oso, Abimbola Oladele; Adeola, Olayiwola; Yao, Kang; Yin, Yulong; Li, Tiejun

    2014-01-01

    Deoxynivalenol (DON) has various toxicological effects in humans and pigs that result from the ingestion of contaminated cereal products. This study was conducted to investigate the protective effects of dietary supplementation with glutamic acid on piglets challenged with DON. A total of 20 piglets weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (5 piglets/treatment): 1) basal diet, negative control (NC); 2) basal diet +4 mg/kg DON (DON); 3) basal diet +2% (g/g) glutamic acid (GLU); 4) basal diet +4 mg/kg DON +2% glutamic acid (DG). A 7-d adaptation period was followed by 30 days of treatment. A metabolite analysis using nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic technology and the determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities for plasma, as well as the activity of Caspase-3 and the proliferation of epithelial cells were conducted. The results showed that contents of low-density lipoprotein, alanine, arginine, acetate, glycoprotein, trimethylamine-N-oxide (TMAO), glycine, lactate, and urea, as well as the glutamate/creatinine ratio were higher but high-density lipoprotein, proline, citrate, choline, unsaturated lipids and fumarate were lower in piglets of DON treatment than that of NC treatment (Pglutamic acid increased the plasma concentrations of proline, citrate, creatinine, unsaturated lipids, and fumarate, and decreased the concentrations of alanine, glycoprotein, TMAO, glycine, and lactate, as well as the glutamate/creatinine ratio (Pglutamic acid to DON treatment increased the plasma activities of SOD and GSH-Px and the proliferating cell nuclear antigen (PCNA) labeling indexes for the jejunum and ileum (Pglutamic acid has the potential to repair the injuries associated with oxidative stress as well as the disturbances of energy and amino acid metabolism induced by DON.

  5. NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats after oral administration

    Energy Technology Data Exchange (ETDEWEB)

    Bu Qian; Lin Hongjun; Xu Youzhi; Cao Zhixing; Zhou Tian; Zhao Yinglan [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Yan Guangyan; Cen Xiaobo [National Chengdu Center for Safety Evaluation of Drugs, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Deng Pengchi [Analytical and Testing Center, Sichuan University, Chengdu 610041 (China); Peng Feng [Department of Thoracic Oncology of Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Xue Aiqin [Institute of Bioengineering, Zhejiang Sci-Tech University Road 2, Xiasha, Hangzhou 310018 (China); Wang Yanli, E-mail: alancenxb@sina.com [Tianjin Children' s Hospital, Tianjin 300074 (China)

    2010-03-26

    As titanium dioxide nanoparticles (TiO{sub 2} NPs) are widely used commercially, their potential toxicity on human health has attracted particular attention. In the present study, the oral toxicological effects of TiO{sub 2} NPs (dosed at 0.16, 0.4 and 1 g kg{sup -1}, respectively) were investigated using conventional approaches and metabonomic analysis in Wistar rats. Serum chemistry, hematology and histopathology examinations were performed. The urine and serum were investigated by {sup 1}H nuclear magnetic resonance (NMR) using principal components and partial least squares discriminant analysis. The metabolic signature of urinalysis in TiO{sub 2} NP-treated rats showed increases in the levels of taurine, citrate, hippurate, histidine, trimethylamine-N-oxide (TMAO), citrulline, {alpha}-ketoglutarate, phenylacetylglycine (PAG) and acetate; moreover, decreases in the levels of lactate, betaine, methionine, threonine, pyruvate, 3-D-hydroxybutyrate (3-D-HB), choline and leucine were observed. The metabonomics analysis of serum showed increases in TMAO, choline, creatine, phosphocholine and 3-D-HB as well as decreases in glutamine, pyruvate, glutamate, acetoacetate, glutathione and methionine after TiO{sub 2} NP treatment. Aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated and mitochondrial swelling in heart tissue was observed in TiO{sub 2} NP-treated rats. These findings indicate that disturbances in energy and amino acid metabolism and the gut microflora environment may be attributable to the slight injury to the liver and heart caused by TiO{sub 2} NPs. Moreover, the NMR-based metabolomic approach is a reliable and sensitive method to study the biochemical effects of nanomaterials.

  6. Identification of anti-HIV active dicaffeoylquinic- and tricaffeoylquinic acids in Helichrysum populifolium by NMR-based metabolomic guided fractionation.

    Science.gov (United States)

    Heyman, Heino Martin; Senejoux, François; Seibert, Isabell; Klimkait, Thomas; Maharaj, Vinesh Jaichand; Meyer, Jacobus Johannes Marion

    2015-06-01

    South Africa being home to more than 35% of the world's Helichrysum species (c.a. 244) of which many are used in traditional medicine, is seen potentially as a significant resource in the search of new anti-HIV chemical entities. It was established that five of the 30 Helichrysum species selected for this study had significant anti-HIV activity ranging between 12 and 21 μg/mL (IC50) by using an in-house developed DeCIPhR method on a full virus model. Subsequent toxicity tests also revealed little or no toxicity for these active extracts. With the use of NMR-based metabolomics, the search for common chemical characteristics within the plant extract was conducted, which resulted in specific chemical shift areas identified that could be linked to the anti-HIV activity of the extracts. The NMR chemical shifts associated with the activity were identified to be 2.56-3.08 ppm, 5.24-6.28 ppm, 6.44-7.04 ppm and 7.24-8.04 ppm. This activity profile was then used to guide the fractionation process by narrowing down and focusing the fractionation and purification processes to speed up the putative identification of five compounds with anti-HIV activity in the most active species, Helichrysum populifolium. The anti-HIV compounds identified for the first time from H. populifolium were three dicaffeoylquinic acid derivatives, i.e. 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid as well as two tricaffeoylquinic acid derivatives i.e. 1,3,5-tricaffeoylquinic acid and either 5-malonyl-1,3,4-tricaffeoylquinic or 3-malonyl-1,4,5-tricaffeoylquinic acid, with the latter being identified for the first time in the genus. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. {sup 1}H NMR-based metabolomics of time-dependent responses of Eisenia fetida to sub-lethal phenanthrene exposure

    Energy Technology Data Exchange (ETDEWEB)

    Lankadurai, Brian P.; Wolfe, David M.; Simpson, Andre J. [Department of Chemistry, University of Toronto, 1265 Military Trail, Toronto, Ontario M1C 1A4 Canada (Canada); Simpson, Myrna J., E-mail: myrna.simpson@utoronto.ca [Department of Chemistry, University of Toronto, 1265 Military Trail, Toronto, Ontario M1C 1A4 Canada (Canada)

    2011-10-15

    {sup 1}H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida after exposure to sub-lethal concentrations of phenanthrene over time. Earthworms were exposed to 0.025 mg/cm{sup 2} of phenanthrene (1/64th of the LC{sub 50}) via contact tests over four days. Earthworm tissues were extracted using a mixture of chloroform, methanol and water, resulting in polar and non-polar fractions that were analyzed by {sup 1}H NMR after one, two, three and four days. NMR-based metabolomic analyses revealed heightened E. fetida responses with longer phenanthrene exposure times. Amino acids alanine and glutamate, the sugar maltose, the lipids cholesterol and phosphatidylcholine emerged as potential indicators of phenanthrene exposure. The conversion of succinate to fumarate in the Krebs cycle was also interrupted by phenanthrene. Therefore, this study shows that NMR-based metabolomics is a powerful tool for elucidating time-dependent relationships in addition to the mode of toxicity of phenanthrene in earthworm exposure studies. - Highlights: > NMR-based earthworm metabolomic analysis of the mode of action of phenanthrene is presented. > The earthworm species E. fetida were exposed to sub-lethal phenanthrene concentrations. > Both polar and non-polar metabolites of E. fetida tissue extracts were analyzed by {sup 1}H NMR. > Longer phenanthrene exposure times resulted in heightened earthworm responses. > An interruption of the Krebs cycle was also observed due to phenanthrene exposure. - {sup 1}H NMR metabolomics is used to determine the relationship between phenanthrene exposure and the metabolic response of the earthworm E. fetida over time and also to elucidate the phenanthrene mode of toxicity.

  8. 1H NMR-Based Metabolomic Analysis of Sub-Lethal Perfluorooctane Sulfonate Exposure to the Earthworm, Eisenia fetida, in Soil

    Directory of Open Access Journals (Sweden)

    Myrna J. Simpson

    2013-08-01

    Full Text Available 1H NMR-based metabolomics was used to measure the response of Eisenia fetida earthworms after exposure to sub-lethal concentrations of perfluorooctane sulfonate (PFOS in soil. Earthworms were exposed to a range of PFOS concentrations (five, 10, 25, 50, 100 or 150 mg/kg for two, seven and fourteen days. Earthworm tissues were extracted and analyzed by 1H NMR. Multivariate statistical analysis of the metabolic response of E. fetida to PFOS exposure identified time-dependent responses that were comprised of two separate modes of action: a non-polar narcosis type mechanism after two days of exposure and increased fatty acid oxidation after seven and fourteen days of exposure. Univariate statistical analysis revealed that 2-hexyl-5-ethyl-3-furansulfonate (HEFS, betaine, leucine, arginine, glutamate, maltose and ATP are potential indicators of PFOS exposure, as the concentrations of these metabolites fluctuated significantly. Overall, NMR-based metabolomic analysis suggests elevated fatty acid oxidation, disruption in energy metabolism and biological membrane structure and a possible interruption of ATP synthesis. These conclusions obtained from analysis of the metabolic profile in response to sub-lethal PFOS exposure indicates that NMR-based metabolomics is an excellent discovery tool when the mode of action (MOA of contaminants is not clearly defined.

  9. Rapid protein disappearance rates along the small intestine advantage poultry performance and influence the post-enteral availability of amino acids.

    Science.gov (United States)

    Truong, Ha H; Chrystal, Peter V; Moss, Amy F; Selle, Peter H; Liu, Sonia Yun

    2017-12-01

    A foundation diet, an intermediate blend and a summit diet were formulated with different levels of soyabean meal, casein and crystalline amino acids to compare 'slow' and 'rapid' protein diets. The diets were offered to male Ross 308 chicks from 7 to 28 d post-hatch and assessed parameters included growth performance, nutrient utilisation, apparent digestibility coefficients and disappearance rates of starch and protein (N) in four small intestinal segments. Digestibility coefficients and disappearance rates of sixteen amino acids in three small intestinal segments and amino acid concentrations in plasma from portal and systemic circulations from the foundation and summit diets were determined. The dietary transition significantly accelerated protein (N) disappearance rates in the distal jejunum and ileum. The transition from foundation to summit diets significantly increased starch digestibility coefficients in the ileum and disappearance rates in all four small intestinal segments. These starch responses were associated with significant enhancements in nutrient utilisation. The dietary transition linearly increased digestibility coefficients and disappearance rates of amino acids in the majority of cases. The summit diet increased plasma concentrations of five amino acids but decreased those of four amino acids relative to the foundation diet to significant extents. Plasma concentrations of free amino acids were higher in the portal than systemic circulations. Rapid protein disappearance rates advantaged poultry performance and influenced post-enteral availability of amino acids. If the underlying mechanisms are to be identified, further research into the impact of protein digestive dynamics on broiler performance is required but appears justified.

  10. Quantification of whey proteins by reversed phase-HPLC and effectiveness of mid-infrared spectroscopy for their rapid prediction in sweet whey.

    Science.gov (United States)

    Sturaro, Alba; De Marchi, Massimo; Masi, Antonio; Cassandro, Martino

    2016-01-01

    In the dairy industry, membrane filtration is used to reduce the amount of whey waste and, simultaneously, to recover whey proteins (WP). The composition of WP can strongly affect the filtration treatment of whey, and rapid determination of WP fractions would be of interest for dairy producers to monitor WP recovery. This study aimed to develop mid-infrared spectroscopy (MIRS) prediction models for the rapid quantification of protein in sweet whey, using a validated rapid reversed phase (RP)-HPLC as a reference method. Quantified WP included α-lactalbumin (α-LA), β-lactoglobulin (β-LG) A and B, bovine serum albumin, caseinomacropeptides, and proteose peptone. Validation of RP-HPLC was performed by calculating the relative standard deviation (RSD) in repeatability and reproducibility tests for WP retention time and peak areas. Samples of liquid whey (n=187) were analyzed by RP-HPLC and scanned through MIRS to collect spectral information (900 to 4,000 cm(-1)); statistical analysis was carried out through partial least squares regression and random cross-validation procedure. Retention times in RP-HPLC method were stable (RSD between 0.03 and 0.80%), whereas the RSD of peak area (from 0.25 to 8.48%) was affected by WP relative abundance. Higher coefficients of determination in validation for MIRS model were obtained for protein fractions present in whey in large amounts, such as β-LG (0.58), total identified WP (0.58), and α-LA (0.56). Results of this study suggest that MIRS is an easy method for rapid quantification of detail protein in sweet whey, even if better resolution was achieved with the method based on RP-HPLC. The prediction of WP in sweet whey by MIRS might be used for screening and for classifying sweet whey according to its total and individual WP contents. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  11. Biosynthesis of intestinal microvillar proteins. Rapid expression of cytoskeletal components in microvilli of pig small intestinal mucosal explants

    DEFF Research Database (Denmark)

    Cowell, G M; Danielsen, E M

    1984-01-01

    Using alkaline extraction to separate cytoskeletal and membrane proteins of intestinal microvilli, the kinetics of assembly of these two microvillar protein compartments was studied by pulse-chase labelling of pig small intestinal mucosal explants, kept in organ culture. Following a 10 min pulse...... of [35S]methionine, the membrane proteins did not appear in the microvillar fraction until after 40-60 min of chase. In contrast, the cytoskeletal components, of which the 110-kDa protein and villin were immunologically identified, were expressed in the microvillar fraction immediately after the 10 min...

  12. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    Directory of Open Access Journals (Sweden)

    Christy Catherine

    Full Text Available Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

  13. Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

    Science.gov (United States)

    Otero, Fátima; Tamayo, María; Santiso, Rebeca; Gosálvez, Jaime; Bou, Germán; Fernández, José Luis

    2017-04-01

    A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

  14. Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain.

    Science.gov (United States)

    Suzuki, Yoshio; Takagi, Nobuyuki; Chimuro, Tomoyuki; Shinohara, Atsushi; Sakaguchi, Nao; Hiratsuka, Atsunori; Yokoyama, Kenji

    2011-06-01

    A new fluorescent molecular probe, 2,2'-(1E,1'E)-2,2'-(4-(dicyanomethylene)-4H-pyrane-2,6-diyl)bis(ethene-2,1-diyl)bis(sodium benzenesulfonate) salt (1), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high-throughput SDS-PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in-gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60-90 min under optimum conditions much shorter than that required by the less-sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in-gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1, which is a modified protocol of blue native PAGE (BN-PAGE). Thus, 1 may facilitate high-sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Sex and estrous cycle-dependent rapid protein kinase signaling actions of estrogen in distal colonic cells.

    LENUS (Irish Health Repository)

    O'Mahony, Fiona

    2008-10-01

    Previous studies from our laboratory demonstrated that 17beta-estradiol (E2) rapidly inhibits Cl(-) secretion in rat and human distal colonic epithelium. The inhibition has been shown to occur via targeting of a basolateral K(+) channel identified as the KCNQ1 (KvLQT1) channel. E2 indirectly modulates the channel activity via a cascade of second messengers which are rapidly phosphorylated in response to E2. The anti-secretory mechanism may be the manner by which E2 induces fluid retention in the intestine during periods of high circulating plasma E2. Here we review the sex-dependent and estrous cycle regulation of this novel rapid response to E2. The inhibition of KCNQ1 channel activity and Cl(-) secretion will be of interest in the future in the investigation of the retentive effects of estrogen in female tissue and also in the study of secretory disorders and drugable targets of the intestine.

  16. Metabolic effects of basic fibroblast growth factor in streptozotocin-induced diabetic rats: A 1H NMR-based metabolomics investigation

    OpenAIRE

    Lin, Xiaodong; Zhao, Liangcai; Tang, Shengli; Zhou, Qi; Lin, Qiuting; Li, Xiaokun; Zheng, Hong; Gao, Hongchang

    2016-01-01

    The fibroblast growth factors (FGFs) family shows a great potential in the treatment of diabetes, but little attention is paid to basic FGF (bFGF). In this study, to explore the metabolic effects of bFGF on diabetes, metabolic changes in serum and feces were analyzed in the normal rats, the streptozocin (STZ)-induced diabetic rats and the bFGF-treated diabetic rats using a 1H nuclear magnetic resonance (NMR)-based metabolomic approach. Interestingly, bFGF treatment significantly decreased glu...

  17. NMR-based metabonomic studies reveal changes in the biochemical profile of plasma and urine from pigs fed high fibre rye bread

    DEFF Research Database (Denmark)

    Bertram, Hanne C.; Bach Knudsen, Knud E.; Serena, Anja

    2006-01-01

    This study presents an NMR-based metabonomic approach to elucidate the overall endogenous biochemical effects of a wholegrain diet. Two diets with similar levels of dietary fibre and macronutrients, but with contrasting levels of wholegrain ingredients, were prepared from wholegrain rye (wholegrain...... diet (WGD)) and non-wholegrain wheat (non-wholegrain diet (NWD)) and fed to four pigs in a crossover design. Plasma samples were collected after 7 d on each diet, and 1H NMR spectra were acquired on these. Partial least squares regression discriminant analysis (PLS-DA) on spectra obtained for plasma...

  18. Rapid, large-scale purification and characterization of Ada protein (O sup 6 methylguanine-DNA methyltransferase) of E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharyya, D.; Tano, K.; Bunick, G.J.; Uberbacher, E.C.; Mitra, S. (Oak Ridge National Laboratory, TN (USA)); Behnke, W.D. (Univ. of Cincinnati College of Medicine, OH (USA))

    1988-07-25

    The E. coli Ada protein (O{sup 6}-methylguanine-DNA methyltransferase) has been purified using a high-level expression vector with a yield of about 3 mg per liter of E. coli culture. The 39-kDa protein has an extinction coefficient (E{sup 280nm}{sub 1%}) of 5.3. Its isoelectric point of 7.1 is lower than that predicted from the amino acid content. The homogeneous Ada protein is fully active as a methyl acceptor from O{sup 6}-methylguanine in DNA. Its reaction with O{sup 6}-methylguanine in a synthetic DNA has a second-order rate constant of 1.1 {times} 10{sup 9} M{sup {minus}1} min{sup {minus}1} at 0{degree}C. Both the native form and the protein methylated at Cys-69 are monomeric. The CD spectrum suggests a low {alpha}-helical content and the radius of gyration of 23 {angstrom} indicates a compact, globular shape. The middle region of the protein is sensitive to a variety of proteases, including an endogenous activity in E. coli, suggesting that the protein is composed of N-terminal and C-terminal domains connected by a hinge region. E. coli B has a higher level of this protease than does K12.

  19. INVESTIGATING THE ENANTIOSELECTIVE TOXICITY OF CONAZOLE FUNGICIDES IN RAINBOW TROUT THROUGH THE USE OF NMR BASED METABONOMICS

    Science.gov (United States)

    In support of the Environmental Protection Agency's Computational Toxicology Program, metabonomics, the quantitative measurement of a broad spectrum of metabolic responses of living systems in response to disease onset or genetic modification, is being employed to enable rapid id...

  20. SynPAnal: software for rapid quantification of the density and intensity of protein puncta from fluorescence microscopy images of neurons.

    Directory of Open Access Journals (Sweden)

    Eric Danielson

    Full Text Available Continuous modification of the protein composition at synapses is a driving force for the plastic changes of synaptic strength, and provides the fundamental molecular mechanism of synaptic plasticity and information storage in the brain. Studying synaptic protein turnover is not only important for understanding learning and memory, but also has direct implication for understanding pathological conditions like aging, neurodegenerative diseases, and psychiatric disorders. Proteins involved in synaptic transmission and synaptic plasticity are typically concentrated at synapses of neurons and thus appear as puncta (clusters in immunofluorescence microscopy images. Quantitative measurement of the changes in puncta density, intensity, and sizes of specific proteins provide valuable information on their function in synaptic transmission, circuit development, synaptic plasticity, and synaptopathy. Unfortunately, puncta quantification is very labor intensive and time consuming. In this article, we describe a software tool designed for the rapid semi-automatic detection and quantification of synaptic protein puncta from 2D immunofluorescence images generated by confocal laser scanning microscopy. The software, dubbed as SynPAnal (for Synaptic Puncta Analysis, streamlines data quantification for puncta density and average intensity, thereby increases data analysis throughput compared to a manual method. SynPAnal is stand-alone software written using the JAVA programming language, and thus is portable and platform-free.

  1. Rapid Diminution in the Level and Activity of DNA-Dependent Protein Kinase in Cancer Cells by a Reactive Nitro-Benzoxadiazole Compound

    Directory of Open Access Journals (Sweden)

    Viviane A. O. Silva

    2016-05-01

    Full Text Available The expression and activity of DNA-dependent protein kinase (DNA-PK is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-ylsulfanyl]-2,1,3-benzoxadiazole (NSC, bearing a nitro-benzoxadiazole (NBD scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells.

  2. Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells

    DEFF Research Database (Denmark)

    Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla

    2009-01-01

    : rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression...... for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents...... agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates...

  3. A protein microarray for the rapid screening of patients suspected of infection with various food-borne helminthiases.

    Directory of Open Access Journals (Sweden)

    Jia-Xu Chen

    Full Text Available BACKGROUND: Food-borne helminthiases (FBHs have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches. METHODOLOGY/PRINCIPAL FINDINGS: In this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA. The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI: 95.3-98.7% to 100.0% (95% CI: 100.0% in the protein microarray and from 97.7% (95% CI: 96.2-99.2% to 100.0% (95% CI: 100.0% in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1-96.3% to 92.1% (95% CI: 83.5-100.0% in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4-92.6% to 92.1% (95% CI: 83.5-100.0%. Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used. CONCLUSIONS/SIGNIFICANCE: The protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

  4. [Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

    Science.gov (United States)

    Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

    2011-08-01

    We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

  5. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    International Nuclear Information System (INIS)

    Semaan, Sheila J; Nickells, Robert W

    2010-01-01

    Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116 BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. HCT116 BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the

  6. The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

    Directory of Open Access Journals (Sweden)

    Semaan Sheila J

    2010-10-01

    Full Text Available Abstract Background Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Methods Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. Results HCT116BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Conclusion Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of

  7. Rapid heating of Alaska pollock and chicken breast myofibrillar protein gels as affecting water-holding properties.

    Science.gov (United States)

    Stevenson, Clinton D; Liu, Wenjie; Lanier, Tyre C

    2012-10-10

    The gelation response of salted muscle minces to rapid versus slow heating rates is thought to differ between homeotherm and poikilotherm species. This study investigated water-holding (WH) properties of pastes prepared from refined myofibrils, at equal pH, of chicken breast versus Alaska pollock both during [cook loss (CL)] and following [expressible water (EW)] their cooking by rapid [microwave (MW)] versus slow [water bath (WB)] heating and whether such properties were related to gel matrix structure parameters and water mobility. Results did not confirm the industrial experience that pastes of meat from homeotherms benefit from slower cooking. Gels of equally high WH ability (low CL or EW) were made by rapid heating when the holding time did not exceed 5 min prior to cooling, which was sufficient for completion of gelation. Reduced CL and EW correlated with larger and smaller amplitudes of T21 and T22 water pools, respectively, measured by time-domain nuclear magnetic resonance (TD-NMR).

  8. Rapid detection of DNA-interstrand and DNA-protein cross-links in mammalian cells by gravity-flow alkaline elution

    International Nuclear Information System (INIS)

    Hincks, J.R.; Coulombe, R.A. Jr.

    1989-01-01

    Alkaline elution is a sensitive and commonly used technique to detect cellular DNA damage in the form of DNA strand breaks and DNA cross-links. Conventional alkaline elution procedures have extensive equipment requirements and are tedious to perform. Our laboratory recently presented a rapid, simplified, and sensitive modification of the alkaline elution technique to detect carcinogen-induced DNA strand breaks. In the present study, we have further modified this technique to enable the rapid characterization of chemically induced DNA-interstrand and DNA-protein associated cross-links in cultured epithelial cells. Cells were exposed to three known DNA cross-linking agents, nitrogen mustard (HN 2 ), mitomycin C (MMC), or ultraviolet irradiation (UV). One hour exposures of HN 2 at 0.25, 1.0, and 4.0 microM or of MMC at 20, 40, and 60 microM produced a dose-dependent induction of total DNA cross-links by these agents. Digestion with proteinase K revealed that HN 2 and MMC induced both DNA-protein cross-links and DNA-interstrand cross-links. Ultraviolet irradiation induced both DNA cross-links and DNA strand breaks, the latter of which were either protein and nonprotein associated. The results demonstrate that gravity-flow alkaline elution is a sensitive and accurate method to characterize the molecular events of DNA cross-linking. Using this procedure, elution of DNA from treated cells is completed in 1 hr, and only three fractions per sample are analyzed. This method may be useful as a rapid screening assay for genotoxicity and/or as an adjunct to other predictive assays for potential mutagenic or carcinogenic agents

  9. Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: a review

    KAUST Repository

    Emwas, Abdul-Hamid M.

    2014-11-21

    The metabolic composition of human biofluids can provide important diagnostic and prognostic information. Among the biofluids most commonly analyzed in metabolomic studies, urine appears to be particularly useful. It is abundant, readily available, easily stored and can be collected by simple, noninvasive techniques. Moreover, given its chemical complexity, urine is particularly rich in potential disease biomarkers. This makes it an ideal biofluid for detecting or monitoring disease processes. Among the metabolomic tools available for urine analysis, NMR spectroscopy has proven to be particularly well-suited, because the technique is highly reproducible and requires minimal sample handling. As it permits the identification and quantification of a wide range of compounds, independent of their chemical properties, NMR spectroscopy has been frequently used to detect or discover disease fingerprints and biomarkers in urine. Although protocols for NMR data acquisition and processing have been standardized, no consensus on protocols for urine sample selection, collection, storage and preparation in NMR-based metabolomic studies have been developed. This lack of consensus may be leading to spurious biomarkers being reported and may account for a general lack of reproducibility between laboratories. Here, we review a large number of published studies on NMR-based urine metabolic profiling with the aim of identifying key variables that may affect the results of metabolomics studies. From this survey, we identify a number of issues that require either standardization or careful accounting in experimental design and provide some recommendations for urine collection, sample preparation and data acquisition.

  10. Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: a review.

    Science.gov (United States)

    Emwas, Abdul-Hamid; Luchinat, Claudio; Turano, Paola; Tenori, Leonardo; Roy, Raja; Salek, Reza M; Ryan, Danielle; Merzaban, Jasmeen S; Kaddurah-Daouk, Rima; Zeri, Ana Carolina; Nagana Gowda, G A; Raftery, Daniel; Wang, Yulan; Brennan, Lorraine; Wishart, David S

    The metabolic composition of human biofluids can provide important diagnostic and prognostic information. Among the biofluids most commonly analyzed in metabolomic studies, urine appears to be particularly useful. It is abundant, readily available, easily stored and can be collected by simple, noninvasive techniques. Moreover, given its chemical complexity, urine is particularly rich in potential disease biomarkers. This makes it an ideal biofluid for detecting or monitoring disease processes. Among the metabolomic tools available for urine analysis, NMR spectroscopy has proven to be particularly well-suited, because the technique is highly reproducible and requires minimal sample handling. As it permits the identification and quantification of a wide range of compounds, independent of their chemical properties, NMR spectroscopy has been frequently used to detect or discover disease fingerprints and biomarkers in urine. Although protocols for NMR data acquisition and processing have been standardized, no consensus on protocols for urine sample selection, collection, storage and preparation in NMR-based metabolomic studies have been developed. This lack of consensus may be leading to spurious biomarkers being reported and may account for a general lack of reproducibility between laboratories. Here, we review a large number of published studies on NMR-based urine metabolic profiling with the aim of identifying key variables that may affect the results of metabolomics studies. From this survey, we identify a number of issues that require either standardization or careful accounting in experimental design and provide some recommendations for urine collection, sample preparation and data acquisition.

  11. Standardizing the experimental conditions for using urine in NMR-based metabolomic studies with a particular focus on diagnostic studies: a review

    KAUST Repository

    Emwas, Abdul-Hamid M.; Luchinat, Claudio; Turano, Paola; Tenori, Leonardo; Roy, Raja; Salek, Reza M.; Ryan, Danielle; Merzaban, Jasmeen; Kaddurah-Daouk, Rima; Zeri, Ana Carolina; Nagana Gowda, G. A.; Raftery, Daniel; Wang, Yulan; Brennan, Lorraine; Wishart, David S.

    2014-01-01

    The metabolic composition of human biofluids can provide important diagnostic and prognostic information. Among the biofluids most commonly analyzed in metabolomic studies, urine appears to be particularly useful. It is abundant, readily available, easily stored and can be collected by simple, noninvasive techniques. Moreover, given its chemical complexity, urine is particularly rich in potential disease biomarkers. This makes it an ideal biofluid for detecting or monitoring disease processes. Among the metabolomic tools available for urine analysis, NMR spectroscopy has proven to be particularly well-suited, because the technique is highly reproducible and requires minimal sample handling. As it permits the identification and quantification of a wide range of compounds, independent of their chemical properties, NMR spectroscopy has been frequently used to detect or discover disease fingerprints and biomarkers in urine. Although protocols for NMR data acquisition and processing have been standardized, no consensus on protocols for urine sample selection, collection, storage and preparation in NMR-based metabolomic studies have been developed. This lack of consensus may be leading to spurious biomarkers being reported and may account for a general lack of reproducibility between laboratories. Here, we review a large number of published studies on NMR-based urine metabolic profiling with the aim of identifying key variables that may affect the results of metabolomics studies. From this survey, we identify a number of issues that require either standardization or careful accounting in experimental design and provide some recommendations for urine collection, sample preparation and data acquisition.

  12. A 1H-NMR-Based Metabonomic Study on the Anti-Depressive Effect of the Total Alkaloid of Corydalis Rhizoma

    Directory of Open Access Journals (Sweden)

    Hongwei Wu

    2015-05-01

    Full Text Available Corydalis Rhizoma, named YuanHu in China, is the dried tuber of Corydalis yanhusuo W.T. Wang which is used in Traditional Chinese Medicine for pain relief and blood activation. Previous pharmacological studies showed that apart from analgesics, the alkaloids from YuanHu may be useful in the therapy of depression by acting on the GABA, dopamine and benzodiazepine receptors. In this study, the antidepressive effect of the total alkaloid of YuanHu (YHTA was investigated in a chronic unpredictable mild stress (CUMS rat model using 1H-NMR-based metabonomics. Plasma metabolic profiles were analyzed and multivariate data analysis was applied to discover the metabolic biomarkers in CUMS rats. Thirteen biomarkers of CUMS-introduced depression were identified, which are myo-inositol, glycerol, glycine, creatine, glutamine, glutamate, β-glucose, α-glucose, acetoacetate, 3-hydroxybutyrate, leucine and unsaturated lipids (L7, L9. Moreover, a metabolic network of the potential biomarkers in plasma perturbed by CUMS was detected. After YHTA treatment, clear separation between the model group and YHTA-treated group was achieved. The levels of all the abnormal metabolites mentioned above showed a tendency of restoration to normal levels. The results demonstrated the therapeutic efficacy of YHTA against depression and suggested that NMR-based metabolomics can provide a simple and easy tool for the evaluation of herbal therapeutics.

  13. Discriminative Analysis of Different Grades of Gaharu (Aquilaria malaccensis Lamk. via 1H-NMR-Based Metabolomics Using PLS-DA and Random Forests Classification Models

    Directory of Open Access Journals (Sweden)

    Siti Nazirah Ismail

    2017-09-01

    Full Text Available Gaharu (agarwood, Aquilaria malaccensis Lamk. is a valuable tropical rainforest product traded internationally for its distinctive fragrance. It is not only popular as incense and in perfumery, but also favored in traditional medicine due to its sedative, carminative, cardioprotective and analgesic effects. The current study addresses the chemical differences and similarities between gaharu samples of different grades, obtained commercially, using 1H-NMR-based metabolomics. Two classification models: partial least squares-discriminant analysis (PLS-DA and Random Forests were developed to classify the gaharu samples on the basis of their chemical constituents. The gaharu samples could be reclassified into a ‘high grade’ group (samples A, B and D, characterized by high contents of kusunol, jinkohol, and 10-epi-γ-eudesmol; an ‘intermediate grade’ group (samples C, F and G, dominated by fatty acid and vanillic acid; and a ‘low grade’ group (sample E and H, which had higher contents of aquilarone derivatives and phenylethyl chromones. The results showed that 1H- NMR-based metabolomics can be a potential method to grade the quality of gaharu samples on the basis of their chemical constituents.

  14. Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors

    NARCIS (Netherlands)

    Jongsma, Maikel; Florczyk, Urszula M.; Hendriks-Balk, Marieelle C.; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2007-01-01

    Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative

  15. Rapid capillary coating by epoxy-poly-(dimethylacrylamide): Performance in capillary zone electrophoresis of protein and polystyrene carboxylate

    Czech Academy of Sciences Publication Activity Database

    Chiari, M.; Cretich, M.; Šťastná, Miroslava; Radko, S. P.; Chrambach, A.

    2001-01-01

    Roč. 22, č. 4 (2001), s. 656-659 ISSN 0173-0835 Institutional research plan: CEZ:AV0Z4031919 Keywords : capillary coating * capillary zone electrophoresis * proteins Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 4.282, year: 2001

  16. An emergency brake for protein synthesis The integrated stress response is able to rapidly shut down the synthesis of proteins in eukaryotic cells.

    Czech Academy of Sciences Publication Activity Database

    Hronová, Vladislava; Valášek, Leoš

    2017-01-01

    Roč. 6, APR 25 (2017), s. 1-3, č. článku e27085. ISSN 2050-084X Institutional support: RVO:61388971 Keywords : synthesis of proteins * eukaryotic cells * eIF2 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 7.725, year: 2016

  17. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    International Nuclear Information System (INIS)

    Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with [ 32 P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa β-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor β-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32 P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission

  18. Labelling of pneumococcal penicillin-binding proteins with [3H]propionyl-ampicillin. A rapid method for monitoring penicillin-binding activity

    International Nuclear Information System (INIS)

    Hakenbeck, R.; Kohiyama, M.

    1982-01-01

    Penicillin-binding proteins (PBPs) are membrane components ubiquitous to all bacteria examined so far. Some of them are present in only a few copies per cell. The conventional method of visualizing these proteins consists in binding of radioactive penicillin to the fractions containing PBPs followed by SDS-PAGE and finally fluorography. Although this procedure is laborious, it is necessary for the determination of the identity as well as for the quantification of each PBP. On the other hand, when penicillin-binding conditions are to be examined or binding activity has to be followed through fractionation and purification of PBPs, no fast monitoring device for these proteins has been available. The authors developed a rapid and easy assay for penicillin-binding activity with a filter-binding technique using [ 3 H]propionyl ampicillin ( 3 H-PA) of high specific activity. As little 2μg of crude membranes obtained from the highly penicillin-sensitive, β-lactamase-negative organism Streptococcus pneumoniae, are sufficient to detect binding activity. In this paper they describe optimum conditions for the assay of PBPs and show that this binding activity correlates with the presence of native penicillin-binding proteins. (Auth.)

  19. Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

    DEFF Research Database (Denmark)

    Esmail Nazari, Zeinab; van de Weert, Marco; Bou-Assaf, George

    2016-01-01

    Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) has become an established method for analysis of protein higher-order structure. Here, we use HDX-MS methodology based on manual Solid-Phase Extraction (SPE) to allow fast and simplified conformational analysis of proteins under...... pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In Mode 1, we used DMSO-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable...... in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that the SPE-HDX-MS system can...

  20. A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum

    Directory of Open Access Journals (Sweden)

    Lorena Vázquez-Iglesias

    2017-06-01

    Full Text Available Clostridium septicum produces a number of diseases in human and farm animals which, in most of the cases, are fatal without clinical intervention. Alpha toxin is an important agent and the unique lethal virulent factor produced by Clostridium septicum. This toxin is haemolytic, highly lethal and necrotizing activities but is being used as an antigen to develop animal vaccines. The aim of this study was to isolate the alpha toxin of Clostridium septicum and produce highly specific antibodies against it. In this work, we have developed a simple and efficient method for alpha toxin purification, based on electroelution that can be used as a time-saving method for purifying proteins. This technique avoids contamination by other proteins that could appear during other protein purification techniques such chromatography. The highly purified toxin was used to produce polyclonal antibodies. The specificity of the antibodies was tested by western blot and these antibodies can be applied to the quantitative determination of alpha toxin by slot blot.

  1. Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells

    International Nuclear Information System (INIS)

    Vara, F.; Schneider, J.A.; Rozengurt, E.

    1985-01-01

    Diacylglycerol and phorbol esters activate protein kinase C in intact cells. The authors report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive 86 Rb + uptake, a measure of the activity of the Na + /K + pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na + influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt 2 ) also enhanced ouabain sensitive 86 Rb + uptake and amiloride-sensitive 22 Na + influx. Prolonged treatment (40 hr) of 3T3 cells with PBt 2 at a saturating dose, which reduces the number of PBt 2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt 2 . They suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na + /H + antiport activity, which, in turn, leads to Na + influx, intracellular pH modulation, and stimulation of the Na + /K + pump

  2. ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

    Science.gov (United States)

    Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

    2016-06-15

    Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Rapid changes in the serum total protein and globulin levels in complications caused by facultatively pathogenic Gram-negative bacteria.

    Science.gov (United States)

    Petrás, G; Kiss, S; Juraszek, J; Merétey, K

    1978-01-01

    The changes in the levels of total protein and four globulin fractions were followed up throughout the entire course of complications caused by Gram-negative facultative pathogens in 37 acute cases of respiratory insufficiency accompanying different underlying illnesses and in 9 chronic, bedridden patients given artificial ventilation. At the onset of the infectious complications, in the first place in septic shock, the levels of various globulin fractions showed a decrease corresponding to a half-life of 2 to 4 days. Neither the increased catabolism, nor the protein losses by the urine and tracheal secretions offer a sufficient explanation for the escape of globulins of this extent from the plasma. It seems that this is a consequence of the increase in capillary permeability due to the effect of antigen-antibody reactions and that of endotoxin. As a result, in the critical phase of the infectious complications, at the point of culmination, e.g. in septic shock, diminished amount of different globulins is transported to the site of utilization, that is, to the inflammatory area.

  4. CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.

    Science.gov (United States)

    Park, Arnold; Won, Sohui T; Pentecost, Mickey; Bartkowski, Wojciech; Lee, Benhur

    2014-01-01

    Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to

  5. Ameliorating effects of Mango (Mangifera indica L.) fruit on plasma ethanol level in a mouse model assessed with 1H-NMR based metabolic profiling

    Science.gov (United States)

    Kim, So-Hyun; K. Cho, Somi; Min, Tae-Sun; Kim, Yujin; Yang, Seung-Ok; Kim, Hee-Su; Hyun, Sun-Hee; Kim, Hana; Kim, Young-Suk; Choi, Hyung-Kyoon

    2011-01-01

    The ameliorating effects of Mango (Mangifera indica L.) flesh and peel samples on plasma ethanol level were investigated using a mouse model. Mango fruit samples remarkably decreased mouse plasma ethanol levels and increased the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase. The 1H-NMR-based metabolomic technique was employed to investigate the differences in metabolic profiles of mango fruits, and mouse plasma samples fed with mango fruit samples. The partial least squares-discriminate analysis of 1H-NMR spectral data of mouse plasma demonstrated that there were clear separations among plasma samples from mice fed with buffer, mango flesh and peel. A loading plot demonstrated that metabolites from mango fruit, such as fructose and aspartate, might stimulate alcohol degradation enzymes. This study suggests that mango flesh and peel could be used as resources for functional foods intended to decrease plasma ethanol level after ethanol uptake. PMID:21562641

  6. Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

    Directory of Open Access Journals (Sweden)

    Nielsen Niels

    2010-02-01

    Full Text Available Abstract Background Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. Methods Differentiated mouse myotube cultures (C2C12 were exposed to 5 mM creatine monohydrate (CMH for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH. Results The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the

  7. Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

    Science.gov (United States)

    Zhang, Ming; Sun, Bo; Zhang, Qi; Gao, Rong; Liu, Qiao; Dong, Fangting; Fang, Haiqin; Peng, Shuangqing; Li, Famei; Yan, Xianzhong

    2017-01-15

    A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Feasibility Study of NMR Based Serum Metabolomic Profiling to Animal Health Monitoring: A Case Study on Iron Storage Disease in Captive Sumatran Rhinoceros (Dicerorhinus sumatrensis).

    Science.gov (United States)

    Watanabe, Miki; Roth, Terri L; Bauer, Stuart J; Lane, Adam; Romick-Rosendale, Lindsey E

    2016-01-01

    A variety of wildlife species maintained in captivity are susceptible to iron storage disease (ISD), or hemochromatosis, a disease resulting from the deposition of excess iron into insoluble iron clusters in soft tissue. Sumatran rhinoceros (Dicerorhinus sumatrensis) is one of the rhinoceros species that has evolutionarily adapted to a low-iron diet and is susceptible to iron overload. Hemosiderosis is reported at necropsy in many African black and Sumatran rhinoceroses but only a small number of animals reportedly die from hemochromatosis. The underlying cause and reasons for differences in susceptibility to hemochromatosis within the taxon remains unclear. Although serum ferritin concentrations have been useful in monitoring the progression of ISD in many species, there is some question regarding their value in diagnosing hemochromatosis in the Sumatran rhino. To investigate the metabolic changes during the development of hemochromatosis and possibly increase our understanding of its progression and individual susceptibility differences, the serum metabolome from a Sumatran rhinoceros was investigated by nuclear magnetic resonance (NMR)-based metabolomics. The study involved samples from female rhinoceros at the Cincinnati Zoo (n = 3), including two animals that died from liver failure caused by ISD, and the Sungai Dusun Rhinoceros Conservation Centre in Peninsular Malaysia (n = 4). Principal component analysis was performed to visually and statistically compare the metabolic profiles of the healthy animals. The results indicated that significant differences were present between the animals at the zoo and the animals in the conservation center. A comparison of the 43 serum metabolomes of three zoo rhinoceros showed two distinct groupings, healthy (n = 30) and unhealthy (n = 13). A total of eighteen altered metabolites were identified in healthy versus unhealthy samples. Results strongly suggest that NMR-based metabolomics is a valuable tool for animal health

  9. Feasibility Study of NMR Based Serum Metabolomic Profiling to Animal Health Monitoring: A Case Study on Iron Storage Disease in Captive Sumatran Rhinoceros (Dicerorhinus sumatrensis.

    Directory of Open Access Journals (Sweden)

    Miki Watanabe

    Full Text Available A variety of wildlife species maintained in captivity are susceptible to iron storage disease (ISD, or hemochromatosis, a disease resulting from the deposition of excess iron into insoluble iron clusters in soft tissue. Sumatran rhinoceros (Dicerorhinus sumatrensis is one of the rhinoceros species that has evolutionarily adapted to a low-iron diet and is susceptible to iron overload. Hemosiderosis is reported at necropsy in many African black and Sumatran rhinoceroses but only a small number of animals reportedly die from hemochromatosis. The underlying cause and reasons for differences in susceptibility to hemochromatosis within the taxon remains unclear. Although serum ferritin concentrations have been useful in monitoring the progression of ISD in many species, there is some question regarding their value in diagnosing hemochromatosis in the Sumatran rhino. To investigate the metabolic changes during the development of hemochromatosis and possibly increase our understanding of its progression and individual susceptibility differences, the serum metabolome from a Sumatran rhinoceros was investigated by nuclear magnetic resonance (NMR-based metabolomics. The study involved samples from female rhinoceros at the Cincinnati Zoo (n = 3, including two animals that died from liver failure caused by ISD, and the Sungai Dusun Rhinoceros Conservation Centre in Peninsular Malaysia (n = 4. Principal component analysis was performed to visually and statistically compare the metabolic profiles of the healthy animals. The results indicated that significant differences were present between the animals at the zoo and the animals in the conservation center. A comparison of the 43 serum metabolomes of three zoo rhinoceros showed two distinct groupings, healthy (n = 30 and unhealthy (n = 13. A total of eighteen altered metabolites were identified in healthy versus unhealthy samples. Results strongly suggest that NMR-based metabolomics is a valuable tool for

  10. A (1H NMR-Based Metabonomic Investigation of Time-Related Metabolic Trajectories of the Plasma, Urine and Liver Extracts of Hyperlipidemic Hamsters.

    Directory of Open Access Journals (Sweden)

    Chun-Ying Jiang

    Full Text Available The hamster has been previously found to be a suitable model to study the changes associated with diet-induced hyperlipidemia in humans. Traditionally, studies of hyperlipidemia utilize serum- or plasma-based biochemical assays and histopathological evaluation. However, unbiased metabonomic technologies have the potential to identify novel biomarkers of disease. Thus, to obtain a better understanding of the progression of hyperlipidemia and discover potential biomarkers, we have used a proton nuclear magnetic resonance spectroscopy ((1H-NMR-based metabonomics approach to study the metabolic changes occurring in the plasma, urine and liver extracts of hamsters fed a high-fat/high-cholesterol diet. Samples were collected at different time points during the progression of hyperlipidemia, and individual proton NMR spectra were visually and statistically assessed using two multivariate analyses (MVA: principal component analysis (PCA and orthogonal partial least squares-discriminant analysis (OPLS-DA. Using the commercial software package Chenomx NMR suite, 40 endogenous metabolites in the plasma, 80 in the urine and 60 in the water-soluble fraction of liver extracts were quantified. NMR analysis of all samples showed a time-dependent transition from a physiological to a pathophysiological state during the progression of hyperlipidemia. Analysis of the identified biomarkers of hyperlipidemia suggests that significant perturbations of lipid and amino acid metabolism, as well as inflammation, oxidative stress and changes in gut microbiota metabolites, occurred following cholesterol overloading. The results of this study substantially broaden the metabonomic coverage of hyperlipidemia, enhance our understanding of the mechanism of hyperlipidemia and demonstrate the effectiveness of the NMR-based metabonomics approach to study a complex disease.

  11. Formalin-induced behavioural hypersensitivity and neuronal hyperexcitability are mediated by rapid protein synthesis at the spinal level

    Science.gov (United States)

    Asante, Curtis O; Wallace, Victoria C; Dickenson, Anthony H

    2009-01-01

    Background The mammalian target of rapamycin (mTOR) is a key regulator of mRNA translation whose action can be inhibited by the drug rapamycin. Forms of long-term plasticity require protein synthesis and evidence indicates that mRNA in dendrites, axon terminals and cell bodies is essential for long-term synaptic plasticity. Specific to pain, shifts in pain thresholds and responsiveness are an expression of neuronal plasticity and this likely contributes to persistent pain. We investigated this by inhibiting the activity of mTOR with rapamycin at the spinal level, of rats that were subjected to the formalin test, using both behavioural and electrophysiological techniques. Results For in vivo electrophysiology, Sprague Dawley rats were fully anaesthetised and single-unit extracellular recordings were obtained from lamina V wide dynamic range (WDR) dorsal horn spinal neurones at the region where input is received from the hind paw. Neuronal responses from naive rats showed that rapamycin-sensitive pathways were important in nociceptive-specific C-fibre mediated transmission onto WDR neurones as well mechanically-evoked responses since rapamycin was effective in attenuating these measures. Formalin solution was injected into the hind paw prior to which, rapamycin or vehicle was applied directly onto the exposed spinal cord. When rapamycin was applied to the spinal cord prior to hind paw formalin injection, there was a significant attenuation of the prolonged second phase of the formalin test, which comprises continuing afferent input to the spinal cord, neuronal hyperexcitability and an activated descending facilitatory drive from the brainstem acting on spinal neurones. In accordance with electrophysiological data, behavioural studies showed that rapamycin attenuated behavioural hypersensitivity elicited by formalin injection into the hind paw. Conclusion We conclude that mTOR has a role in maintaining persistent pain states via mRNA translation and thus protein

  12. Formalin-induced behavioural hypersensitivity and neuronal hyperexcitability are mediated by rapid protein synthesis at the spinal level

    Directory of Open Access Journals (Sweden)

    Wallace Victoria C

    2009-06-01

    Full Text Available Abstract Background The mammalian target of rapamycin (mTOR is a key regulator of mRNA translation whose action can be inhibited by the drug rapamycin. Forms of long-term plasticity require protein synthesis and evidence indicates that mRNA in dendrites, axon terminals and cell bodies is essential for long-term synaptic plasticity. Specific to pain, shifts in pain thresholds and responsiveness are an expression of neuronal plasticity and this likely contributes to persistent pain. We investigated this by inhibiting the activity of mTOR with rapamycin at the spinal level, of rats that were subjected to the formalin test, using both behavioural and electrophysiological techniques. Results For in vivo electrophysiology, Sprague Dawley rats were fully anaesthetised and single-unit extracellular recordings were obtained from lamina V wide dynamic range (WDR dorsal horn spinal neurones at the region where input is received from the hind paw. Neuronal responses from naive rats showed that rapamycin-sensitive pathways were important in nociceptive-specific C-fibre mediated transmission onto WDR neurones as well mechanically-evoked responses since rapamycin was effective in attenuating these measures. Formalin solution was injected into the hind paw prior to which, rapamycin or vehicle was applied directly onto the exposed spinal cord. When rapamycin was applied to the spinal cord prior to hind paw formalin injection, there was a significant attenuation of the prolonged second phase of the formalin test, which comprises continuing afferent input to the spinal cord, neuronal hyperexcitability and an activated descending facilitatory drive from the brainstem acting on spinal neurones. In accordance with electrophysiological data, behavioural studies showed that rapamycin attenuated behavioural hypersensitivity elicited by formalin injection into the hind paw. Conclusion We conclude that mTOR has a role in maintaining persistent pain states via m

  13. Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

    Science.gov (United States)

    Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang

    2017-12-01

    Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.

  14. Spontaneous generation of rapidly transmissible prions in transgenic mice expressing wild-type bank vole prion protein.

    Science.gov (United States)

    Watts, Joel C; Giles, Kurt; Stöhr, Jan; Oehler, Abby; Bhardwaj, Sumita; Grillo, Sunny K; Patel, Smita; DeArmond, Stephen J; Prusiner, Stanley B

    2012-02-28

    Currently, there are no animal models of the most common human prion disorder, sporadic Creutzfeldt-Jakob disease (CJD), in which prions are formed spontaneously from wild-type (WT) prion protein (PrP). Interestingly, bank voles (BV) exhibit an unprecedented promiscuity for diverse prion isolates, arguing that bank vole PrP (BVPrP) may be inherently prone to adopting misfolded conformations. Therefore, we constructed transgenic (Tg) mice expressing WT BVPrP. Tg(BVPrP) mice developed spontaneous CNS dysfunction between 108 and 340 d of age and recapitulated the hallmarks of prion disease, including spongiform degeneration, pronounced astrogliosis, and deposition of alternatively folded PrP in the brain. Brain homogenates of ill Tg(BVPrP) mice transmitted disease to Tg(BVPrP) mice in ∼35 d, to Tg mice overexpressing mouse PrP in under 100 d, and to WT mice in ∼185 d. Our studies demonstrate experimentally that WT PrP can spontaneously form infectious prions in vivo. Thus, Tg(BVPrP) mice may be useful for studying the spontaneous formation of prions, and thus may provide insight into the etiology of sporadic CJD.

  15. Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors.

    Science.gov (United States)

    Jongsma, Maikel; Florczyk, Urszula M; Hendriks-Balk, Mariëlle C; Michel, Martin C; Peters, Stephan L M; Alewijnse, Astrid E

    2007-07-01

    Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P(1) receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands.

  16. Rapid colorimetric detection of p53 protein function using DNA-gold nanoconjugates with applications for drug discovery and cancer diagnostics.

    Science.gov (United States)

    Assah, Enock; Goh, Walter; Zheng, Xin Ting; Lim, Ting Xiang; Li, Jun; Lane, David; Ghadessy, Farid; Tan, Yen Nee

    2018-05-05

    The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. In this paper, we report a rapid, label-free and versatile colorimetric assay to detect wildtype p53 DNA-binding function in complex solutions. The assay design is based on a concept that alters interparticle-distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wildtype p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution color change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no color change was observed for the binding-deficient p53 mutants and non-specific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, we further demonstrate its utility for fast detection of drug-induced DNA binding function to cancer-associated Y220C mutant p53 protein using well-established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this assay is configurable to detect low numbers of mutant p53 expressing cells in miniscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanogold enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with great promises for clinically pertinent applications. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Rapid determination of collagen in meat-based foods by microwave hydrolysis of proteins and HPAEC-PAD analysis of 4-hydroxyproline.

    Science.gov (United States)

    Messia, M C; Di Falco, T; Panfili, G; Marconi, E

    2008-10-01

    A rapid microwave procedure for protein hydrolysis coupled with High Performance Anion Exchange Chromatography and Pulsed Amperometric Detection (HPAEC-PAD) was developed to quantify the amino acid 4-hydroxyproline in meat and meat-based products. This innovative approach was successfully applied to determine collagen content (4-hydroxyproline×8) as the index quality of meat material employed in the preparation of typical meat sausages ("Mortadella di Bologna PGI" and "Salamini italiani alla cacciatora PDO") and fresh filled pastas. Microwave hydrolysis showed a precision and accuracy similar to traditional hydrolysis (RSD% from 0.0 to 6.4; relative error 1.4-10.0%) with a reduction in the hydrolysis time from 24h to 20min. HPAEC-PAD allowed detection of 4-hydroxyproline without pre or post-column derivatization and the use of non-toxic eluents.

  18. Tamoxifen and ICI 182,780 activate hypothalamic G protein-coupled estrogen receptor 1 to rapidly facilitate lordosis in female rats.

    Science.gov (United States)

    Long, Nathan; Long, Bertha; Mana, Asma; Le, Dream; Nguyen, Lam; Chokr, Sima; Sinchak, Kevin

    2017-03-01

    In the female rat, sexual receptivity (lordosis) can be facilitated by sequential activation of estrogen receptor (ER) α and G protein-coupled estrogen receptor 1 (GPER) by estradiol. In the estradiol benzoate (EB) primed ovariectomized (OVX) rat, EB initially binds to ERα in the plasma membrane that complexes with and transactivates metabotropic glutamate receptor 1a to activate β-endorphin neurons in the arcuate nucleus of the hypothalamus (ARH) that project to the medial preoptic nucleus (MPN). This activates MPN μ-opioid receptors (MOP), inhibiting lordosis. Infusion of non-esterified 17β-estradiol into the ARH rapidly reduces MPN MOP activation and facilitates lordosis via GPER. Tamoxifen (TAM) and ICI 182,780 (ICI) are selective estrogen receptor modulators that activate GPER. Therefore, we tested the hypothesis that TAM and ICI rapidly facilitate lordosis via activation of GPER in the ARH. Our first experiment demonstrated that injection of TAM intraperitoneal, or ICI into the lateral ventricle, deactivated MPN MOP and facilitated lordosis in EB-primed rats. We then tested whether TAM and ICI were acting rapidly through a GPER dependent pathway in the ARH. In EB-primed rats, ARH infusion of either TAM or ICI facilitated lordosis and reduced MPN MOP activation within 30min compared to controls. These effects were blocked by pretreatment with the GPER antagonist, G15. Our findings demonstrate that TAM and ICI deactivate MPN MOP and facilitate lordosis in a GPER dependent manner. Thus, TAM and ICI may activate GPER in the CNS to produce estrogenic actions in neural circuits that modulate physiology and behavior. Published by Elsevier Inc.

  19. Rapid reduction of hepatitis C virus-Core protein in the peripheral blood improve the immunological response in chronic hepatitis C patients.

    Science.gov (United States)

    Kondo, Yasuteru; Ueno, Yoshiyuki; Wakui, Yuta; Ninomiya, Masashi; Kakazu, Eiji; Inoue, Jun; Kobayashi, Koju; Obara, Noriyuki; Shimosegawa, Tooru

    2011-12-01

      The extracellular hepatitis C virus (HCV)-antigen, including HCV-Core protein, can suppress immune cells. Recently, the efficacy of double filtration plasmapheresis (DFPP) for chronic hepatitis C (CHC) was reported. However, the mechanism of efficacy of DFPP might not be only the reduction of HCV but also the effect of immune cells via direct and/or indirect mechanisms. The aim of this study is to analyze the virological and immunological parameters of difficult-to-treat HCV patients treated with DFPP combined with Peg-interferon and RBV (DFPP/Peg-IFN/RBV) therapy.   Twelve CHC patients were enrolled and treated with DFPP/Peg-IFN/RBV therapy. The immunological, virological and genetic parameters were studied.   All patients (4/4) treated with the major IL28B allele (T/T) could achieve complete early virological response (EVR). The amounts of HCV-Core antigen in the peripheral blood of EVR patients treated with DFPP/Peg-IFN/RBV rapidly declined in comparison to those of late virological response (LVR) patients treated with DFPP/Peg-IFN/RBV and EVR patients treated with Peg-IFN and RBV (Peg-IFN/RBV). The amount of IFN-γ produced from peripheral blood gradually increased. On the other hand, the amount of IL10 gradually decreased in the EVR patients. The frequencies of HCV-Core binding on CD3+ T cells rapidly declined in EVR patients treated with DFPP/Peg-IFN/RBV therapy. Moreover, the distributions of activated CD4(+) and CD8(+) T cells and CD16-CD56 high natural killer cells were significantly changed between before and after DFPP.   The rapid reduction of HCV-Core antigens and changes in the distribution of lymphoid cells could contribute to the favorable immunological response during DFPP/Peg-IFN/RBV therapy. © 2011 The Japan Society of Hepatology.

  20. An NMR-based scoring function improves the accuracy of binding pose predictions by docking by two orders of magnitude

    Energy Technology Data Exchange (ETDEWEB)

    Orts, Julien [EMBL, Structure and Computational Biology Unit (Germany); Bartoschek, Stefan [Industriepark Hoechst, Sanofi-Aventis Deutschland GmbH, R and D LGCR/Parallel Synthesis and Natural Products (Germany); Griesinger, Christian [Max Planck Institute for Biophysical Chemistry (Germany); Monecke, Peter [Industriepark Hoechst, Sanofi-Aventis Deutschland GmbH, R and D LGCR/Structure, Design and Informatics (Germany); Carlomagno, Teresa, E-mail: teresa.carlomagno@embl.de [EMBL, Structure and Computational Biology Unit (Germany)

    2012-01-15

    Low-affinity ligands can be efficiently optimized into high-affinity drug leads by structure based drug design when atomic-resolution structural information on the protein/ligand complexes is available. In this work we show that the use of a few, easily obtainable, experimental restraints improves the accuracy of the docking experiments by two orders of magnitude. The experimental data are measured in nuclear magnetic resonance spectra and consist of protein-mediated NOEs between two competitively binding ligands. The methodology can be widely applied as the data are readily obtained for low-affinity ligands in the presence of non-labelled receptor at low concentration. The experimental inter-ligand NOEs are efficiently used to filter and rank complex model structures that have been pre-selected by docking protocols. This approach dramatically reduces the degeneracy and inaccuracy of the chosen model in docking experiments, is robust with respect to inaccuracy of the structural model used to represent the free receptor and is suitable for high-throughput docking campaigns.

  1. A rapid and accurate method for determining protein content in dairy products based on asynchronous-injection alternating merging zone flow-injection spectrophotometry.

    Science.gov (United States)

    Liang, Qin-Qin; Li, Yong-Sheng

    2013-12-01

    An accurate and rapid method and a system to determine protein content using asynchronous-injection alternating merging zone flow-injection spectrophotometry based on reaction between coomassie brilliant blue G250 (CBBG) and protein was established. Main merit of our approach is that it can avoid interferences of other nitric-compounds in samples, such as melamine and urea. Optimized conditions are as follows: Concentrations of CBBG, polyvinyl alcohol (PVA), NaCl and HCl are 150 mg/l, 30 mg/l, 0.1 mol/l and 1.0% (v/v), respectively; volumes of the sample and reagent are 150 μl and 30 μl, respectively; length of a reaction coil is 200 cm; total flow rate is 2.65 ml/min. The linear range of the method is 0.5-15 mg/l (BSA), its detection limit is 0.05 mg/l, relative standard deviation is less than 1.87% (n=11), and analytical speed is 60 samples per hour. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

    DEFF Research Database (Denmark)

    Young, Jette Feveile; Larsen, Lotte Bach; Malmendal, Anders

    2010-01-01

    Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat......-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating...... the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. METHODS: Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension...

  3. NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation

    International Nuclear Information System (INIS)

    Wu, Junfang; Domellöf, Magnus; Zivkovic, Angela M.; Larsson, Göran; Öhman, Anders; Nording, Malin L.

    2016-01-01

    Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H_2O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H_2O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9–24 days after delivery) and late (31–87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant. - Highlights: • 36 metabolites were simultaneously quantified in human milk by NMR. • Ultrafiltration more efficiently reduces interferences than MeOH/H_2O extraction. • Compositional changes of the human milk exist during the matured lactation stage.

  4. NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Junfang [Department of Chemistry, Umeå University (Sweden); Domellöf, Magnus [Department of Clinical Sciences, Pediatrics, Umeå University (Sweden); Zivkovic, Angela M. [Foods for Health Institute, University of California, Davis, CA (United States); Department of Nutrition, University of California, Davis, CA (United States); Larsson, Göran [Department of Medical Biochemistry and Biophysics, Unit of Research, Education and Development-Östersund, Umeå University (Sweden); Öhman, Anders, E-mail: anders.ohman01@umu.se [Department of Pharmacology and Clinical Neuroscience, Umeå University (Sweden); Nording, Malin L., E-mail: malin.nording@umu.se [Department of Chemistry, Umeå University (Sweden)

    2016-01-15

    Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H{sub 2}O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H{sub 2}O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9–24 days after delivery) and late (31–87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant. - Highlights: • 36 metabolites were simultaneously quantified in human milk by NMR. • Ultrafiltration more efficiently reduces interferences than MeOH/H{sub 2}O extraction. • Compositional changes of the human milk exist during the matured lactation stage.

  5. Rapid detection of hypoxia-inducible factor-1-active tumours: pretargeted imaging with a protein degrading in a mechanism similar to hypoxia-inducible factor-1{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Masashi [Kyoto University, Radioisotopes Research Laboratory, Kyoto University Hospital, Faculty of Medicine, Kyoto (Japan); Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Kudo, Takashi; Konishi, Hiroaki; Miyano, Azusa; Ono, Masahiro; Saji, Hideo [Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Kuge, Yuji [Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Hokkaido University, Central Institute of Isotope Science, Sapporo (Japan); Mukai, Takahiro [Kyushu University, Department of Biomolecular Recognition Chemistry, Graduate School of Pharmaceutical Sciences, Fukuoka (Japan); Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Hiraoka, Masahiro [Kyoto University, Department of Radiation Oncology and Image-applied Therapy, Graduate School of Medicine, Kyoto (Japan)

    2010-08-15

    Hypoxia-inducible factor-1 (HIF-1) plays an important role in malignant tumour progression. For the imaging of HIF-1-active tumours, we previously developed a protein, POS, which is effectively delivered to and selectively stabilized in HIF-1-active cells, and a radioiodinated biotin derivative, (3-{sup 123}I-iodobenzoyl)norbiotinamide ({sup 123}I-IBB), which can bind to the streptavidin moiety of POS. In this study, we aimed to investigate the feasibility of the pretargeting method using POS and {sup 123}I-IBB for rapid imaging of HIF-1-active tumours. Tumour-implanted mice were pretargeted with POS. After 24 h, {sup 125}I-IBB was administered and subsequently, the biodistribution of radioactivity was investigated at several time points. In vivo planar imaging, comparison between {sup 125}I-IBB accumulation and HIF-1 transcriptional activity, and autoradiography were performed at 6 h after the administration of {sup 125}I-IBB. The same sections that were used in autoradiographic analysis were subjected to HIF-1{alpha} immunohistochemistry. {sup 125}I-IBB accumulation was observed in tumours of mice pretargeted with POS (1.6%ID/g at 6 h). This result is comparable to the data derived from {sup 125}I-IBB-conjugated POS-treated mice (1.4%ID/g at 24 h). In vivo planar imaging provided clear tumour images. The tumoral accumulation of {sup 125}I-IBB significantly correlated with HIF-1-dependent luciferase bioluminescence (R=0.84, p<0.01). The intratumoral distribution of {sup 125}I-IBB was heterogeneous and was significantly correlated with HIF-1{alpha}-positive regions (R=0.58, p<0.0001). POS pretargeting with {sup 123}I-IBB is a useful technique in the rapid imaging and detection of HIF-1-active regions in tumours. (orig.)

  6. Rapid and Specific Detection of Acidovorax avenae subsp. citrulli Using SYBR Green-Based Real-Time PCR Amplification of the YD-Repeat Protein Gene.

    Science.gov (United States)

    Cho, Min Seok; Park, Duck Hwan; Ahn, Tae-Young; Park, Dong Suk

    2015-09-01

    The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10(0) fg/μl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

  7. Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

    Science.gov (United States)

    Khan, Mateen A; Ma, Jia; Walden, William E; Merrick, William C; Theil, Elizabeth C; Goss, Dixie J

    2014-06-01

    Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

    Directory of Open Access Journals (Sweden)

    Steiner Jennifer A

    2009-08-01

    Full Text Available Abstract Background The Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT transporter (SERT is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. Results In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting

  9. NMR-Based Metabolomic Investigations on the Differential Responses in Adductor Muscles from Two Pedigrees of Manila Clam Ruditapes philippinarum to Cadmium and Zinc

    Directory of Open Access Journals (Sweden)

    Junbao Yu

    2011-09-01

    Full Text Available Manila clam Ruditapes philippinarum is one of the most important economic species in shellfishery in China due to its wide geographic distribution and high tolerance to environmental changes (e.g., salinity, temperature. In addition, Manila clam is a good biomonitor/bioindicator in “Mussel Watch Programs” and marine environmental toxicology. However, there are several pedigrees of R. philippinarum distributed in the marine environment in China. No attention has been paid to the biological differences between various pedigrees of Manila clams, which may introduce undesirable biological variation in toxicology studies. In this study, we applied NMR-based metabolomics to detect the biological differences in two main pedigrees (White and Zebra of R. philippinarum and their differential responses to heavy metal exposures (Cadmium and Zinc using adductor muscle as a target tissue to define one sensitive pedigree of R. philippinarum as biomonitor for heavy metals. Our results indicated that there were significant metabolic differences in adductor muscle tissues between White and Zebra clams, including higher levels of alanine, glutamine, hypotaurine, phosphocholine and homarine in White clam muscles and higher levels of branched chain amino acids (valine, leucine and isoleucine, succinate and 4-aminobutyrate in Zebra clam muscles, respectively. Differential metabolic responses to heavy metals between White and Zebra clams were also found. Overall, we concluded that White pedigree of clam could be a preferable bioindicator/biomonitor in marine toxicology studies and for marine heavy metals based on the relatively high sensitivity to heavy metals.

  10. Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR.

    Science.gov (United States)

    Happyana, Nizar; Kayser, Oliver

    2016-08-01

    Cannabis sativa trichomes are glandular structures predominantly responsible for the biosynthesis of cannabinoids, the biologically active compounds unique to this plant. To the best of our knowledge, most metabolomic works on C. sativa that have been reported previously focused their investigations on the flowers and leaves of this plant. In this study, (1)H NMR-based metabolomics and real-time PCR analysis were applied for monitoring the metabolite profiles of C. sativa trichomes, variety Bediol, during the last 4 weeks of the flowering period. Partial least squares discriminant analysis models successfully classified metabolites of the trichomes based on the harvest time. Δ (9)-Tetrahydrocannabinolic acid (1) and cannabidiolic acid (2) constituted the vital differential components of the organic preparations, while asparagine, glutamine, fructose, and glucose proved to be their water-extracted counterparts. According to RT-PCR analysis, gene expression levels of olivetol synthase and olivetolic acid cyclase influenced the accumulation of cannabinoids in the Cannabis trichomes during the monitoring time. Moreover, quantitative (1)H NMR and RT-PCR analysis of the Cannabis trichomes suggested that the gene regulation of cannabinoid biosynthesis in the C. sativa variety Bediol is unique when compared with other C. sativa varieties. Georg Thieme Verlag KG Stuttgart · New York.

  11. A Systematic, Integrated Study on the Neuroprotective Effects of Hydroxysafflor Yellow A Revealed by H1 NMR-Based Metabonomics and the NF-κB Pathway

    Directory of Open Access Journals (Sweden)

    Yuanyan Liu

    2013-01-01

    Full Text Available Hydroxysafflor yellow A (HSYA is the main active component of the Chinese herb Carthamus tinctorius L.. Purified HSYA is used as a neuroprotective agent to prevent cerebral ischemia. Injectable safflor yellow (50 mg, containing 35 mg HSYA is widely used to treat patients with ischemic cardiocerebrovascular disease. However, it is unknown how HSYA exerts a protective effect on cerebral ischemia at the molecular level. A systematical integrated study, including histopathological examination, neurological evaluation, blood-brain barrier (BBB, metabonomics, and the nuclear factor-κB (NF-κB pathway, was applied to elucidate the pathophysiological mechanisms of HSYA neuroprotection at the molecular level. HSYA could travel across the BBB, significantly reducing the infarct volume and improving the neurological functions of rats with ischemia. Treatment with HSYA could lead to relative corrections of the impaired metabolic pathways through energy metabolism disruption, excitatory amino acid toxicity, oxidative stress, and membrane disruption revealed by 1H NMR-based metabonomics. Meanwhile, HSYA treatment inhibits the NF-κB pathway via suppressing proinflammatory cytokine expression and p65 translocation and binding activity while upregulating an anti-inflammatory cytokine.

  12. Characterization of a rapid, blue light-mediated change in detectable phosphorylation of a plasma membrane protein from etiolated pea (Pisum sativum L.) seedlings

    International Nuclear Information System (INIS)

    Short, T.W.; Briggs, W.R.

    1990-01-01

    When crude microsomal membranes from apical stem segments of etiolated Pisum sativum L. cv Alaska are mixed in vitro with γ-[ 32 P]ATP, a phosphorylated band of apparent molecular mass 120 kilodaltons can be detected on autoradiographs of sodium dodecyl sulfate electrophoresis gels. If the stem sections are exposed to blue light immediately prior to membrane isolation, this band is not evident. Comparisons of the kinetics, tissue distribution, and dark recovery of the phosphorylation response with those published for blue light mediated phototropism or rapid growth inhibition indicate that the phosphorylation could be linked to one or both of the reactions described. However, the fluence-response relationships for the change in detectable phosphorylation match quite closely those reported for phototropism but not those for growth inhibition. Blue light has also been found to regulate the capacity for in vitro phosphorylation of a second protein. It has an apparent molecular mass of 84 kilodaltons and is localized primarily in basal stem sections

  13. Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot

    Science.gov (United States)

    Markwalter, Christine F.; Gibson, Lauren E.; Mudenda, Lwiindi; Kimmel, Danielle W.; Mbambara, Saidon; Thuma, Philip E.; Wright, David W.

    2018-01-01

    Abstract. A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM (pLDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify pLDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; pLDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35–52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria. PMID:29557342

  14. Direct detection of methicillin-resistant Staphylococcus aureus from blood cultures using an immunochromatographic immunoassay-based MRSA rapid kit for the detection of penicillin-binding protein 2a.

    Science.gov (United States)

    Shin, Kyeong Seob; Song, Hyung Geun; Kim, Haejung; Yoon, Sangsun; Hong, Seung Bok; Koo, Sun Hoe; Kim, Jimyung; Kim, Jongwan; Roh, Kyoung Ho

    2010-07-01

    Using an EZ-Step MRSA rapid kit, a novel screening test for methicillin-resistant Staphylococcus aureus (MRSA) that detects penicillin-binding protein 2a, 34 of 36 MRSA-positive clinical blood culture samples were positive on direct testing (sensitivity, 94.4%), whereas 21 of 21 methicillin-susceptible S. aureus-positive samples were negative (specificity, 100%).

  15. NMR-based metabolomics applications

    DEFF Research Database (Denmark)

    Iaccarino, Nunzia

    Metabolomics is the scientific discipline that identifies and quantifies endogenous and exogenous metabolites in different biological samples. Metabolites are crucial components of a biological system and they are highly informative about its functional state, due to their closeness to the organism...... focused on the analysis of various samples covering a wide range of fields, namely, food and nutraceutical sciences, cell metabolomics and medicine using a metabolomics approach. Indeed, the first part of the thesis describes two exploratory studies performed on Algerian extra virgin olive oil and apple...... juice from ancient Danish apple cultivars. Both studies revealed variety-related peculiarities that would have been difficult to detect by means of traditional analysis. The second part of the project includes four metabolomics studies performed on samples of biological origin. In particular, the first...

  16. High performance of histidine-rich protein 2 based rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum.

    Directory of Open Access Journals (Sweden)

    Mélanie Trouvay

    Full Text Available BACKGROUND: Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs. Most RDTs target the histidine-rich protein-2 antigen (PfHRP2 to detect P. falciparum, as it is abundant and shows great heat stability. However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered. METHODS: The performance study compared the SD Malaria Ag test P.f/Pan® kit with the current gold standard diagnosis by microscopy. The prevalence of pfhrp2 and pfhrp3 deletions were evaluated from 221 P. falciparum isolates collected between 2009 and 2011 in French Guiana. RESULTS: Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of P. falciparum was 96.8% (95% CI: 90.9-99.3, and 86.0% (95% CI: 78.9-91.5 for the detection of P. vivax. No isolates (95% CI: 0-4.5 lacking either exon of the pfhrp2 gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8-15.4 lacked the exon 2 part of the pfhrp3 gene. CONCLUSIONS: Field isolates lacking either exon of the pfhrp2 gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment.

  17. NMR-Based Metabolic Profiling of Field-Grown Leaves from Sugar Beet Plants Harbouring Different Levels of Resistance to Cercospora Leaf Spot Disease

    Directory of Open Access Journals (Sweden)

    Yasuyo Sekiyama

    2017-01-01

    Full Text Available Cercospora leaf spot (CLS is one of the most serious leaf diseases for sugar beet (Beta vulgaris L. worldwide. The breeding of sugar beet cultivars with both high CLS resistance and high yield is a major challenge for breeders. In this study, we report the nuclear magnetic resonance (NMR-based metabolic profiling of field-grown leaves for a subset of sugar beet genotypes harbouring different levels of CLS resistance. Leaves were collected from 12 sugar beet genotypes at four time points: seedling, early growth, root enlargement, and disease development stages. 1H-NMR spectra of foliar metabolites soluble in a deuterium-oxide (D2O-based buffer were acquired and subjected to multivariate analyses. A principal component analysis (PCA of the NMR data from the sugar beet leaves shows clear differences among the growth stages. At the later time points, the sugar and glycine betaine contents were increased, whereas the choline content was decreased. The relationship between the foliar metabolite profiles and resistance level to CLS was examined by combining partial least squares projection to latent structure (PLS or orthogonal PLS (OPLS analysis and univariate analyses. It was difficult to build a robust model for predicting precisely the disease severity indices (DSIs of each genotype; however, GABA and Gln differentiated susceptible genotypes (genotypes with weak resistance from resistant genotypes (genotypes with resistance greater than a moderate level before inoculation tests. The results suggested that breeders might exclude susceptible genotypes from breeding programs based on foliar metabolites profiled without inoculation tests, which require an enormous amount of time and effort.

  18. 1H-NMR-based metabolic analysis of human serum reveals novel markers of myocardial energy expenditure in heart failure patients.

    Directory of Open Access Journals (Sweden)

    Zhiyong Du

    Full Text Available OBJECTIVE: Elevated myocardial energy expenditure (MEE is related with reduced left ventricular ejection fraction, and has also been documented as an independent predictor of cardiovascular mortality. However, the serum small-molecule metabolite profiles and pathophysiological mechanisms of elevated MEE in heart failure (HF are still lacking. Herein, we used 1H-NMR-based metabolomics analysis to screen for potential biomarkers of MEE in HF. METHODS: A total of 61 subjects were enrolled, including 46 patients with heart failure and 15 age-matched controls. Venous serum samples were collected from subjects after an 8-hour fast. An INOVA 600 MHz nuclear magnetic resonance spectrometer with Carr-Purcell-Melboom-Gill (CPMG pulse sequence was employed for the metabolomics analysis and MEE was calculated using colored Doppler echocardiography. Metabolomics data were processed using orthogonal signal correction and regression analysis was performed using the partial least squares method. RESULTS: The mean MEE levels of HF patients and controls were 139.61±58.18 cal/min and 61.09±23.54 cal/min, respectively. Serum metabolomics varied with MEE changed, and 3-hydroxybutyrate, acetone and succinate were significantly elevated with the increasing MEE. Importantly, these three metabolites were independent of administration of angiotensin converting enzyme inhibitor, β-receptor blockers, diuretics and statins (P>0.05. CONCLUSIONS: These results suggested that in patients with heart failure, MEE elevation was associated with significant changes in serum metabolomics profiles, especially the concentration of 3-hydroxybutyrate, acetone and succinate. These compounds could be used as potential serum biomarkers to study myocardial energy mechanism in HF patients.

  19. A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

    Science.gov (United States)

    Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N; Fujiwara, Yuko; Rajewsky, Klaus; Zhang, Baochun; Alt, Frederick W

    2015-06-01

    The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. ©2015 American Association for Cancer Research.

  20. {sup 1}H NMR-based metabolomics reveals sub-lethal toxicity of a mixture of diabetic and lipid-regulating pharmaceuticals on amphibian larvae

    Energy Technology Data Exchange (ETDEWEB)

    Melvin, Steven D., E-mail: s.melvin@griffith.edu.au [Australian Rivers Institute, Griffith University, Southport, QLD 4222 (Australia); Habener, Leesa J. [Griffith School of Environment, Griffith University, Southport, QLD 4222 (Australia); Leusch, Frederic D.L. [Australian Rivers Institute, Griffith University, Southport, QLD 4222 (Australia); Griffith School of Environment, Griffith University, Southport, QLD 4222 (Australia); Carroll, Anthony R. [Griffith School of Environment, Griffith University, Southport, QLD 4222 (Australia)

    2017-03-15

    Highlights: • Pharmaceutical pollutants are a concern for eliciting adverse effects in wildlife. • Diabetes and lipid regulating drugs are widely used and poorly removed from sewage. • We explored the toxicity of a mixture of metformin, atorvastatin and bezafibrate on tadpoles. • Exposure caused increased growth and development but no effects on lipids or cholesterol. • {sup 1}H NMR-based metabolomics reveal increased lactic acid and BCAAs in exposed animals. - Abstract: Pharmaceuticals are widely used for the treatment of various physical and psychological ailments. Due to incomplete removal during sewage treatment many pharmaceuticals are frequently detected in aquatic waterways at trace concentrations. The diversity of pharmaceutical contaminants and potential for complex mixtures to occur makes it very difficult to predict the toxicity of these compounds on wildlife, and robust methods are therefore needed to explore sub-lethal effects. Metabolic syndrome is one of the most widespread health concerns currently facing the human population, and various drugs, including anti-diabetic medications and lipid- and cholesterol-lowering fibrates and statins, are widely prescribed as treatment. In this study, we exposed striped marsh frog (Limnodynastes peronii) tadpoles to a mixture of the drugs metformin, atorvastatin and bezafibrate at 0.5, 5, 50 and 500 μg/L to explore possible effects on growth and development, energy reserves (triglycerides and cholesterol), and profiles of small polar metabolites extracted from hepatic tissues. It was hypothesised that exposure would result in a general reduction in energy reserves, and that this would subsequently correspond with reduced growth and development. Responses differed from expected outcomes based on the known mechanisms of these compounds in humans, with no changes to hepatic triglycerides or cholesterol and a general increase in mass and condition with increasing exposure concentration. Deviation from the

  1. "1H NMR-based metabolomics reveals sub-lethal toxicity of a mixture of diabetic and lipid-regulating pharmaceuticals on amphibian larvae

    International Nuclear Information System (INIS)

    Melvin, Steven D.; Habener, Leesa J.; Leusch, Frederic D.L.; Carroll, Anthony R.

    2017-01-01

    Highlights: • Pharmaceutical pollutants are a concern for eliciting adverse effects in wildlife. • Diabetes and lipid regulating drugs are widely used and poorly removed from sewage. • We explored the toxicity of a mixture of metformin, atorvastatin and bezafibrate on tadpoles. • Exposure caused increased growth and development but no effects on lipids or cholesterol. • "1H NMR-based metabolomics reveal increased lactic acid and BCAAs in exposed animals. - Abstract: Pharmaceuticals are widely used for the treatment of various physical and psychological ailments. Due to incomplete removal during sewage treatment many pharmaceuticals are frequently detected in aquatic waterways at trace concentrations. The diversity of pharmaceutical contaminants and potential for complex mixtures to occur makes it very difficult to predict the toxicity of these compounds on wildlife, and robust methods are therefore needed to explore sub-lethal effects. Metabolic syndrome is one of the most widespread health concerns currently facing the human population, and various drugs, including anti-diabetic medications and lipid- and cholesterol-lowering fibrates and statins, are widely prescribed as treatment. In this study, we exposed striped marsh frog (Limnodynastes peronii) tadpoles to a mixture of the drugs metformin, atorvastatin and bezafibrate at 0.5, 5, 50 and 500 μg/L to explore possible effects on growth and development, energy reserves (triglycerides and cholesterol), and profiles of small polar metabolites extracted from hepatic tissues. It was hypothesised that exposure would result in a general reduction in energy reserves, and that this would subsequently correspond with reduced growth and development. Responses differed from expected outcomes based on the known mechanisms of these compounds in humans, with no changes to hepatic triglycerides or cholesterol and a general increase in mass and condition with increasing exposure concentration. Deviation from the

  2. A rapid chemical method of labelling human plasma proteins with sup(99m)Tc-pertechnetate at pH 7.4

    International Nuclear Information System (INIS)

    Wong, D.W.; Mishkin, F.; Lee, T.

    1978-01-01

    A successful method for labelling human plasma proteins with sup(99m)Tc-pertechnetate by chemical means is described. The labelling methodology involves the production of Sup(99m)Tc-(Sn)citrate complex species with high protein binding capacity at pH 7.4 condition following initial chemical reduction of sodium sup(99m)Tc-pertechnetate by stannous chloride. A combined labelling efficiency range of 95-99% for sup(99m)Tc-labelled fibrinogen, immune gamma globulin and serum albumin is achieved. The actual amount of labelled protein content in the product is found to be 85-95% when assayed by ITLC and 74-85% by TCAA protein precipitation. In vitro experimental data indicate that sup(99m)Tc-fibrinogen contains an average of 85% clottable protein with an average clottability of 95%. This strongly suggests that the radioactive proteins retain much of their biological and physiological activities after the labelling process. (author)

  3. PASA - A Program for Automated Protein NMR Backbone Signal Assignment by Pattern-Filtering Approach

    International Nuclear Information System (INIS)

    Xu Yizhuang; Wang Xiaoxia; Yang Jun; Vaynberg, Julia; Qin Jun

    2006-01-01

    We present a new program, PASA (Program for Automated Sequential Assignment), for assigning protein backbone resonances based on multidimensional heteronuclear NMR data. Distinct from existing programs, PASA emphasizes a per-residue-based pattern-filtering approach during the initial stage of the automated 13 C α and/or 13 C β chemical shift matching. The pattern filter employs one or multiple constraints such as 13 C α /C β chemical shift ranges for different amino acid types and side-chain spin systems, which helps to rule out, in a stepwise fashion, improbable assignments as resulted from resonance degeneracy or missing signals. Such stepwise filtering approach substantially minimizes early false linkage problems that often propagate, amplify, and ultimately cause complication or combinatorial explosion of the automation process. Our program (http://www.lerner.ccf.org/moleccard/qin/) was tested on four representative small-large sized proteins with various degrees of resonance degeneracy and missing signals, and we show that PASA achieved the assignments efficiently and rapidly that are fully consistent with those obtained by laborious manual protocols. The results demonstrate that PASA may be a valuable tool for NMR-based structural analyses, genomics, and proteomics

  4. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C’ scalar couplings (3hbJNC’)

    NARCIS (Netherlands)

    Bonvin, A.M.J.J.; Houben, K.; Guenneugues, M.N.L.; Kaptein, R.; Boelens, R.

    2001-01-01

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein

  5. Reduced dimensionality (3,2)D NMR experiments and their automated analysis: implications to high-throughput structural studies on proteins.

    Science.gov (United States)

    Reddy, Jithender G; Kumar, Dinesh; Hosur, Ramakrishna V

    2015-02-01

    Protein NMR spectroscopy has expanded dramatically over the last decade into a powerful tool for the study of their structure, dynamics, and interactions. The primary requirement for all such investigations is sequence-specific resonance assignment. The demand now is to obtain this information as rapidly as possible and in all types of protein systems, stable/unstable, soluble/insoluble, small/big, structured/unstructured, and so on. In this context, we introduce here two reduced dimensionality experiments – (3,2)D-hNCOcanH and (3,2)D-hNcoCAnH – which enhance the previously described 2D NMR-based assignment methods quite significantly. Both the experiments can be recorded in just about 2-3 h each and hence would be of immense value for high-throughput structural proteomics and drug discovery research. The applicability of the method has been demonstrated using alpha-helical bovine apo calbindin-D9k P43M mutant (75 aa) protein. Automated assignment of this data using AUTOBA has been presented, which enhances the utility of these experiments. The backbone resonance assignments so derived are utilized to estimate secondary structures and the backbone fold using Web-based algorithms. Taken together, we believe that the method and the protocol proposed here can be used for routine high-throughput structural studies of proteins. Copyright © 2014 John Wiley & Sons, Ltd.

  6. Recovery of insulin sensitivity and optimal body composition after rapid weight loss in obese dogs fed a high-protein medium-carbohydrate diet.

    Science.gov (United States)

    André, A; Leriche, I; Chaix, G; Thorin, C; Burger, M; Nguyen, P

    2017-06-01

    This study investigated the effects of an experimental high-protein medium-carbohydrate diet (protein level, 46% metabolizable energy, ME). First, postprandial plasma glucose and insulin kinetics were determined in steady-state overweight/obese Beagle dogs (28%-41% excess body weight) for an experimental high-protein medium-carbohydrate diet (protein level, 46% ME) and a commercial high-carbohydrate medium-protein diet (protein level, 24%ME) in obese dogs. Secondly, all the dogs were included in a weight loss programme. They were fed the high-protein medium-carbohydrate diet, and the energy allocation was gradually reduced until they reached their optimal body weight. Insulin sensitivity and body composition were evaluated before and after weight loss using a euglycaemic-hyperinsulinaemic clamp and the deuterium oxide dilution technique respectively. For statistical analysis, linear mixed effect models were used with a significance level of 5%. Postprandial plasma glucose and insulin concentrations were substantially lower with the high-protein medium-carbohydrate diet than the high-carbohydrate medium-protein diet. These differences can be explained mainly by the difference in carbohydrate content between the two diets. Energy restriction (35% lower energy intake than in the obese state) resulted in a 2.23 ± 0.05% loss in body weight/week, and the dogs reached their optimal body weight in 12-16 weeks. Weight loss was associated with a significant increase in insulin sensitivity. The high-protein medium-carbohydrate diet allowed fat-free mass preservation despite a relatively high rate of weekly weight loss. The increase in insulin sensitivity indicated improved control of carbohydrate metabolism, possible due to weight loss and to the nature of the diet. Thus, a high-protein medium-carbohydrate diet is a good nutritional solution for managing the weight of overweight dogs. This diet may improve glycaemic control, which could be beneficial for preventing or

  7. Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling*

    Science.gov (United States)

    Liu, Rui; Kenney, Justin W.; Manousopoulou, Antigoni; Johnston, Harvey E.; Kamei, Makoto; Woelk, Christopher H.; Xie, Jianling; Schwarzer, Michael; Proud, Christopher G.

    2016-01-01

    Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory

  8. Isocratic Solid Phase Extraction-Liquid Chromatography (SPE-LC) Interfaced to High-Performance Tandem Mass Spectrometry for Rapid Protein Identification

    DEFF Research Database (Denmark)

    Hørning, Ole B; Kjeldsen, Frank; Theodorsen, Søren

    2008-01-01

    the isocratic solid phase extraction-liquid chromatography (SPE-LC) technology for rapid separation ( approximately 8 min) of simple peptide samples. We now extend these studies to demonstrate the potential of SPE-LC separation in combination with a hybrid linear ion trap-Orbitrap tandem mass spectrometer...

  9. Activation of AMP-activated protein kinase rapidly suppresses multiple pro-inflammatory pathways in adipocytes including IL-1 receptor-associated kinase-4 phosphorylation

    DEFF Research Database (Denmark)

    Mancini, Sarah J; White, Anna D; Bijland, Silvia

    2017-01-01

    Inflammation of adipose tissue in obesity is associated with increased IL-1β, IL-6 and TNF-α secretion and proposed to contribute to insulin resistance. AMP-activated protein kinase (AMPK) regulates nutrient metabolism and is reported to have anti-inflammatory actions in adipose tissue, yet the m...

  10. Evaluation of an Immunochromatographic Assay for Rapid Detection of Penicillin-Binding Protein 2a in Human and Animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi Clinical Isolates.

    Science.gov (United States)

    Arnold, A R; Burnham, C-A D; Ford, B A; Lawhon, S D; McAllister, S K; Lonsway, D; Albrecht, V; Jerris, R C; Rasheed, J K; Limbago, B; Burd, E M; Westblade, L F

    2016-03-01

    The performance of a rapid penicillin-binding protein 2a (PBP2a) detection assay, the Alere PBP2a culture colony test, was evaluated for identification of PBP2a-mediated beta-lactam resistance in human and animal clinical isolates of Staphylococcus intermedius group, Staphylococcus lugdunensis, and Staphylococcus schleiferi. The assay was sensitive and specific, with all PBP2a-negative and PBP2a-positive strains testing negative and positive, respectively. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Hydroxyapatite hierarchically nanostructured porous hollow microspheres: rapid, sustainable microwave-hydrothermal synthesis by using creatine phosphate as an organic phosphorus source and application in drug delivery and protein adsorption.

    Science.gov (United States)

    Qi, Chao; Zhu, Ying-Jie; Lu, Bing-Qiang; Zhao, Xin-Yu; Zhao, Jing; Chen, Feng; Wu, Jin

    2013-04-22

    Hierarchically nanostructured porous hollow microspheres of hydroxyapatite (HAP) are a promising biomaterial, owing to their excellent biocompatibility and porous hollow structure. Traditionally, synthetic hydroxyapatite is prepared by using an inorganic phosphorus source. Herein, we report a new strategy for the rapid, sustainable synthesis of HAP hierarchically nanostructured porous hollow microspheres by using creatine phosphate disodium salt as an organic phosphorus source in aqueous solution through a microwave-assisted hydrothermal method. The as-obtained products are characterized by powder X-ray diffraction (XRD), Fourier-transform IR (FTIR) spectroscopy, SEM, TEM, Brunauer-Emmett-Teller (BET) nitrogen sorptometry, dynamic light scattering (DLS), and thermogravimetric analysis (TGA). SEM and TEM micrographs show that HAP hierarchically nanostructured porous hollow microspheres consist of HAP nanosheets or nanorods as the building blocks and DLS measurements show that the diameters of HAP hollow microspheres are within the range 0.8-1.5 μm. The specific surface area and average pore size of the HAP porous hollow microspheres are 87.3 m(2) g(-1) and 20.6 nm, respectively. The important role of creatine phosphate disodium salt and the influence of the experimental conditions on the products were systematically investigated. This method is facile, rapid, surfactant-free and environmentally friendly. The as-prepared HAP porous hollow microspheres show a relatively high drug-loading capacity and protein-adsorption ability, as well as sustained drug and protein release, by using ibuprofen as a model drug and hemoglobin (Hb) as a model protein, respectively. These experiments indicate that the as-prepared HAP porous hollow microspheres are promising for applications in biomedical fields, such as drug delivery and protein adsorption. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical Aspergillus fumigatus in different culture conditions based on ribosomal proteins

    Directory of Open Access Journals (Sweden)

    Zhou L

    2017-12-01

    Full Text Available Longrong Zhou, Yongquan Chen, Yuanhong Xu Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Anhui, Hefei, People’s Republic of China Abstract: Fast and accurate discrimination of Aspergillus fumigatus is significant, since misidentification may lead to inappropriate clinical therapy. This study assessed VITEK mass spectrometry (MS V3.0 for A. fumigatus identification using extracted fungal ribosomal proteins. A total of 52 isolates preliminarily identified as A. fumigatus by traditional morphological methods were inoculated in three different culture media and cultured at two different temperatures. The specific spectral fingerprints of different culture time points (48, 72, 96, and 120 h were obtained. Of all strains, 88.5% (46/52 were discriminated as A. fumigatus, while the remaining 11.5% (6/52 produced results inconsistent with morphological analysis. Molecular sequencing, as a reference method for species identification, was used to validate the morphological analysis and matrix-assisted laser desorption/ionization time of flight MS. Chi-square tests (Χ2 test, P=0.05 demonstrated that the culture medium and incubation temperature had no effects on identification accuracy; however, identification accuracy of the strains in the 48-h group was lower than that in other groups. In addition, we found that ribosomal proteins extracted from A. fumigatus can be stored in different environments for at least 1 week, with their profiles remaining stable and strain identification results showing no change. This is beneficial for medical institutions with no mass spectrometer at hand. Overall, this study showed the powerful ability of VITEK MS V 3.0 in identifying A. fumigatus. Keywords: VITEK MS V 3.0, Aspergillus fumigatus, identification, ribosomal protein, spectral fingerprints, fungal, matrix assisted laser desorption ionization-time of flight mass spectrometry, MALDI-TOF MS

  13. Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical Aspergillus fumigatus in different culture conditions based on ribosomal proteins.

    Science.gov (United States)

    Zhou, Longrong; Chen, Yongquan; Xu, Yuanhong

    2017-01-01

    Fast and accurate discrimination of Aspergillus fumigatus is significant, since misidentification may lead to inappropriate clinical therapy. This study assessed VITEK mass spectrometry (MS) V3.0 for A. fumigatus identification using extracted fungal ribosomal proteins. A total of 52 isolates preliminarily identified as A. fumigatus by traditional morphological methods were inoculated in three different culture media and cultured at two different temperatures. The specific spectral fingerprints of different culture time points (48, 72, 96, and 120 h) were obtained. Of all strains, 88.5% (46/52) were discriminated as A. fumigatus , while the remaining 11.5% (6/52) produced results inconsistent with morphological analysis. Molecular sequencing, as a reference method for species identification, was used to validate the morphological analysis and matrix-assisted laser desorption/ionization time of flight MS. Chi-square tests ( χ 2 test, P =0.05) demonstrated that the culture medium and incubation temperature had no effects on identification accuracy; however, identification accuracy of the strains in the 48-h group was lower than that in other groups. In addition, we found that ribosomal proteins extracted from A. fumigatus can be stored in different environments for at least 1 week, with their profiles remaining stable and strain identification results showing no change. This is beneficial for medical institutions with no mass spectrometer at hand. Overall, this study showed the powerful ability of VITEK MS V 3.0 in identifying A. fumigatus .

  14. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C' scalar couplings (3hbJNC')

    Energy Technology Data Exchange (ETDEWEB)

    Bonvin, Alexandre M.J.J.; Houben, Klaartje; Guenneugues, Marc; Kaptein, Robert; Boelens, Rolf [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands)

    2001-11-15

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small {alpha}/{beta} protein chymotrypsin inhibitor 2. Dihedral angle restraints for the {phi} and {psi} angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond {sup 3hb}J{sub NC'} coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

  15. Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

    Directory of Open Access Journals (Sweden)

    Wan Zhixiang

    2005-07-01

    Full Text Available Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR-based assays and enzyme-linked immunosorbent assays (ELISA are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip, on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05 existed between the results determined by our assay and ELISA respectively. Conclusion

  16. A novel NMR-based assay to measure circulating concentrations of branched-chain amino acids: Elevation in subjects with type 2 diabetes mellitus and association with carotid intima media thickness.

    Science.gov (United States)

    Wolak-Dinsmore, Justyna; Gruppen, Eke G; Shalaurova, Irina; Matyus, Steven P; Grant, Russell P; Gegen, Ray; Bakker, Stephan J L; Otvos, James D; Connelly, Margery A; Dullaart, Robin P F

    2018-04-01

    Plasma branched-chain amino acid (BCAA) levels, measured on nuclear magnetic resonance (NMR) metabolomics research platforms or by mass spectrometry, have been shown to be associated with type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). We developed a new test for quantification of BCAA on a clinical NMR analyzer and used this test to determine the clinical correlates of BCAA in 2 independent cohorts. The performance of the NMR-based BCAA assay was evaluated. A method comparison study was performed with mass spectrometry (LC-MS/MS). Plasma BCAA were measured in the Insulin Resistance Atherosclerosis Study (IRAS, n = 1209; 376 T2DM subjects) and in a Groningen cohort (n = 123; 67 T2DM subjects). In addition, carotid intima media thickness (cIMT) was measured successfully in 119 subjects from the Groningen cohort. NMR-based BCAA assay results were linear over a range of concentrations. Coefficients of variation for inter- and intra-assay precision ranged from 1.8-6.0, 1.7-5.4, 4.4-9.1, and 8.8-21.3%, for total BCAA, valine, leucine, and isoleucine, respectively. BCAA quantified from the same samples using NMR and LC-MS/MS were highly correlated (R 2  = 0.97, 0.95 and 0.90 for valine, leucine and isoleucine). In both cohorts total and individual BCAA were elevated in T2DM (P = 0.01 to ≤0.001). Moreover, cIMT was associated with BCAA independent of age, sex, T2DM and metabolic syndrome (MetS) categorization or alternatively of individual MetS components. BCAA levels, measured by NMR in the clinical laboratory, are elevated in T2DM and may be associated with cIMT, a proxy of subclinical atherosclerosis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  17. “Omics” Prospective Monitoring of Bariatric Surgery: Roux-En-Y Gastric Bypass Outcomes Using Mixed-Meal Tolerance Test and Time-Resolved 1H NMR-Based Metabolomics

    Science.gov (United States)

    Lopes, Thiago I.B.; Geloneze, Bruno; Pareja, José C.; Calixto, Antônio R.; Ferreira, Márcia M.C.

    2016-01-01

    Abstract Roux-en-Y gastric bypass (RYGB) surgery goes beyond weight loss to induce early beneficial hormonal changes that favor glycemic control. In this prospective study, ten obese subjects diagnosed with type 2 diabetes underwent bariatric surgery. Mixed-meal tolerance test was performed before and 12 months after RYGB, and the outcomes were investigated by a time-resolved hydrogen nuclear magnetic resonance (1H NMR)-based metabolomics. To the best of our knowledge, no previous omics-driven study has used time-resolved 1H NMR-based metabolomics to investigate bariatric surgery outcomes. Our results presented here show a significant decrease in glucose levels after bariatric surgery (from 159.80 ± 61.43 to 100.00 ± 22.94 mg/dL), demonstrating type 2 diabetes remission (p < 0.05). The metabolic profile indicated lower levels of lactate, alanine, and branched chain amino acids for the operated subject at fasting state after the surgery. However, soon after food ingestion, the levels of these metabolites increased faster in operated than in nonoperated subjects. The lipoprotein profile achieved before and after RYGB at fasting was also significantly different, but converging 180 min after food ingestion. For example, the very low-density lipoprotein, low-density lipoprotein, N-acetyl-glycoproteins, and unsaturated lipid levels decreased after RYGB, while phosphatidylcholine and high-density lipoprotein increased. This study provides important insights on RYGB surgery and attendant type 2 diabetes outcomes using an “omics” systems science approach. Further research on metabolomic correlates of RYGB surgery in larger study samples is called for. PMID:27428253

  18. High speed capillary zone electrophoresis-mass spectrometry via an electrokinetically pumped sheath flow interface for rapid analysis of amino acids and a protein digest.

    Science.gov (United States)

    Schiavone, Nicole M; Sarver, Scott A; Sun, Liangliang; Wojcik, Roza; Dovichi, Norman J

    2015-06-01

    While capillary zone electrophoresis (CZE) has been used to produce very rapid and efficient separations, coupling these high-speed separations with mass spectrometry (MS) has been challenging. Now, with much faster and sensitive mass spectrometers, it is possible to take full advantage of the CZE speed and reconstruct the fast migrating peaks. Here are three high-speed CZE-MS analyses via an electrokinetically pumped sheath-flow interface. The first separation demonstrates CZE-ESI-MS of an amino acid mixture with a 2-min separation, >50,000 theoretical plates, low micromolar concentration detection limits, and subfemtomole mass detection limits (LTQ XL mass spectrometer). The second separation with our recently improved third-generation CE-MS interface illustrates a 20 amino acid separation in ∼7min with an average over 200,000 plate counts, and results in almost-baseline resolution of structural isomers, leucine and isoleucine. The third separation is of a BSA digest with a reproducible CZE separation and mass spectrometry detection in 2min. CZE-MS/MS analysis of the BSA digest identified 31 peptides, produced 52% sequence coverage, and generated a peak capacity of ∼40 across the 1-min separation window (Q-Exactive mass spectrometer). Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Rapid and Sensitive LC-MS/MS Method for the Determination of Metoprolol in Beagle Dog Plasma with a Simple Protein Precipitation Treatment and Its Pharmacokinetic Applications

    Directory of Open Access Journals (Sweden)

    Aihua Hong

    2012-03-01

    Full Text Available : A rapid LC-MS/MS method with good accuracy and sensitivity was developed and validated for the pharmacokinetics study of metoprolol (MP in beagle dogs. The plasma samples were simply precipitated by methanol and then analyzed by LC-MS/MS. An Ultimate XB-C18 column (150 × 2.1 mm ID, 5 μm was used for separation, with methanol-water containing 0.2% formic acid (65:35, v/v as the mobile phase at a flow rate of 0.2 mL/min. Monitoring ions of MP and internal standard (hydroxypioglitazone were m/z 268.1/115.6 and m/z 373.1/150.2, respectively. The linear range was 3.03–416.35 ng/mL with an average correlation coefficient of 0.9996, and the limit of quantification was 3.03 ng/mL. The intra- and inter-day precision was less than 15%. At low, middle and high concentrations, the recovery, the matrix effect and the accuracy was in the range of 76.06%–95.25%, 93.67%–104.19% and 95.20%–99.96% respectively. The method was applied for the pharmacokinetics study of MP tartrate tablets (50 mg. The AUC0-t, Tmax and Cmax were respectively 919.88 ± 195.67 μg/L·h, 0.96 ± 0.33 h, 349.12 ± 78.04 ng/mL.

  20. The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90.

    Science.gov (United States)

    Bartolini, Manuela; Wainer, Irving W; Bertucci, Carlo; Andrisano, Vincenza

    2013-01-25

    Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Latex agglutination using the periplasmic proteins antigen of Brucella melitensis is a successful, rapid, and specific serodiagnostic test for ovine brucellosis.

    Science.gov (United States)

    Ismael, Alaa Bassuny; Swelum, Ayman Abdel-Aziz; Mostafa, Salama A-H; Alhumiany, Abdel-Rahman A

    2016-09-01

    Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity. © The Author(s) 2016.

  2. Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein gammaTIP.

    Science.gov (United States)

    Balk, P A; de Boer, A D

    1999-09-01

    Many bulbous plants need a low-temperature treatment for flowering. Cold, for example, affects the elongation of the stalk, thereby influencing the quality of the cut flower. How the elongation of the stalk is promoted by cold and which physiological and biochemical mechanisms are involved have remained obscure. As invertase has been shown to be involved in the cold-induced elongation of the flower stalks of tulips (Lambrechts et al., 1994, Plant Physiol 104: 515-520), we further characterized this enzyme by cloning the cDNA and analysing its expression in various tissues of the tulip (Tulipa gesneriana L. cv. Apeldoorn) stalk. In addition, the role of sucrose synthase was investigated. Since turgor pressure is an important force driving cell elongation, the role of a water-channel protein (gammaTIP) was studied in relation to these two enzymes. The mRNA level of the invertase found was substantially up-regulated as a result of cold treatment. Analysis of the amino acid sequence of this invertase revealed the presence of a vacuolar targeting signal. Two different forms of sucrose synthase were found, the expression of one of them appeared to be restricted to the vascular tissue while the other form was present in the surrounding tissue. Both sucrose synthases were present in the stalk during the entire period of bulb storage and after planting, but their activities declined during stalk elongation. The expression of the gammaTIP gene was restricted mainly to the vascular tissue and its expression profile was identical to that of invertase. Simultaneous expression of invertase and gammaTIP possibly leads to an increase in osmotic potential and vacuolar water uptake, thus providing a driving force for stretching the stalk cells.

  3. Rapid screening method to study the reactivity of UV filter substances towards skin proteins by high-performance thin-layer chromatography.

    Science.gov (United States)

    Stiefel, C; Schwack, W

    2013-12-01

    Most UV filters used in sunscreens and other cosmetic products contain carbonyl groups, which generally are able to react with peptides or free amino acids of the human skin. To estimate their reactivity, we studied different prominent UV filter substances, octocrylene, ethylhexyl salicylate, 4-t-butyl-4'-methoxydibenzoylmethane, ethylhexyl methoxycinnamate, benzophenone-3, hydroxymethylbenzoyl sulphonic acid, octyldimethyl p-aminobenzoic acid, 3-benzylidene camphor, 4-methylbenzylidene camphor, diethylhexyl butamido triazone and ethylhexyl triazone. A simple screening method using an amino HPTLC plate as protein model was established. The influence of different reaction conditions like heating and irradiation was determined. The ketones BP-3, HMBS and BM-DBM revealed the highest binding rates after both irradiation and heating. After 1 h of irradiation, 82%, 28% and 96%, respectively, were bonded to the amino phase, while heating resulted in values of 52%, 36% and 16%. For BP-3 and HMBS, even storage in the dark at room temperature resulted in a low binding. Contrarily, for the two camphor derivatives 3-BC and 4-MBC, only irradiation led to a slightly turnover. UV filters with ester groups also showed a different behaviour depending on their main skeleton. While OCR especially reacted under heating with the amino phase, resulting in 36% of bound species after one hour, UV irradiation particularly encouraged a reaction of the other esters. After 1 h irradiation, 15% of EHMC, 38% of EHS and 48% of OD-PABA were bonded to the amino groups of the HPTLC plate, whereas the reactivity of the two triazones, EHT and DEBT, was comparatively low. Especially the UV filters BP-3, BM-DBM, HMBS, EHMC or OCR, which are commonly known to cause contact dermatitis, showed a high tendency to form adducts with the amino layer. Thus, the amino plate seems to be a proper tool to screen for skin sensitizers. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  4. Leucine-rich diet alters the 1H-NMR based metabolomic profile without changing the Walker-256 tumour mass in rats.

    Science.gov (United States)

    Viana, Laís Rosa; Canevarolo, Rafael; Luiz, Anna Caroline Perina; Soares, Raquel Frias; Lubaczeuski, Camila; Zeri, Ana Carolina de Mattos; Gomes-Marcondes, Maria Cristina Cintra

    2016-10-03

    Cachexia is one of the most important causes of cancer-related death. Supplementation with branched-chain amino acids, particularly leucine, has been used to minimise loss of muscle tissue, although few studies have examined the effect of this type of nutritional supplementation on the metabolism of the tumour-bearing host. Therefore, the present study evaluated whether a leucine-rich diet affects metabolomic derangements in serum and tumour tissues in tumour-bearing Walker-256 rats (providing an experimental model of cachexia). After 21 days feeding Wistar female rats a leucine-rich diet, distributed in L-leucine and LW-leucine Walker-256 tumour-bearing groups, we examined the metabolomic profile of serum and tumour tissue samples and compared them with samples from tumour-bearing rats fed a normal protein diet (C - control; W - tumour-bearing groups). We utilised 1 H-NMR as a means to study the serum and tumour metabolomic profile, tumour proliferation and tumour protein synthesis pathway. Among the 58 serum metabolites examined, we found that 12 were altered in the tumour-bearing group, reflecting an increase in activity of some metabolic pathways related to energy production, which diverted many nutrients toward tumour growth. Despite displaying increased tumour cell activity (i.e., higher Ki-67 and mTOR expression), there were no differences in tumour mass associated with changes in 23 metabolites (resulting from valine, leucine and isoleucine synthesis and degradation, and from the synthesis and degradation of ketone bodies) in the leucine-tumour group. This result suggests that the majority of nutrients were used for host maintenance. A leucine rich-diet, largely used to prevent skeletal muscle loss, did not affect Walker 256 tumour growth and led to metabolomic alterations that may partially explain the positive effects of leucine for the whole tumour-bearing host.

  5. Comparison of earthworm responses to petroleum hydrocarbon exposure in aged field contaminated soil using traditional ecotoxicity endpoints and 1H NMR-based metabolomics

    International Nuclear Information System (INIS)

    Whitfield Åslund, Melissa; Stephenson, Gladys L.; Simpson, André J.; Simpson, Myrna J.

    2013-01-01

    1 H NMR metabolomics and conventional ecotoxicity endpoints were used to examine the response of earthworms exposed to petroleum hydrocarbons (PHCs) in soil samples collected from a site that was contaminated with crude oil from a pipeline failure in the mid-1990s. The conventional ecotoxicity tests showed that the soils were not acutely toxic to earthworms (average survival ≥90%), but some soil samples impaired reproduction endpoints by >50% compared to the field control soil. Additionally, metabolomics revealed significant relationships between earthworm metabolic profiles (collected after 2 or 14 days of exposure) and soil properties including soil PHC concentration. Further comparisons by partial least squares regression revealed a significant relationship between the earthworm metabolomic data (collected after only 2 or 14 days) and the reproduction endpoints (measured after 63 days). Therefore, metabolomic responses measured after short exposure periods may be predictive of chronic, ecologically relevant toxicity endpoints for earthworms exposed to soil contaminants. -- Highlights: •Earthworm response to petroleum hydrocarbon exposure in soil is examined. •Metabolomics shows significant changes to metabolic profile after 2 days. •Significant relationships observed between metabolomic and reproduction endpoints. •Metabolomics may have value as a rapid screening tool for chronic toxicity. -- Earthworm metabolomic responses measured after 2 and 14 days are compared to traditional earthworm ecotoxicity endpoints (survival and reproduction) in petroleum hydrocarbon contaminated soil

  6. PONDEROSA-C/S: client-server based software package for automated protein 3D structure determination.

    Science.gov (United States)

    Lee, Woonghee; Stark, Jaime L; Markley, John L

    2014-11-01

    Peak-picking Of Noe Data Enabled by Restriction Of Shift Assignments-Client Server (PONDEROSA-C/S) builds on the original PONDEROSA software (Lee et al. in Bioinformatics 27:1727-1728. doi: 10.1093/bioinformatics/btr200, 2011) and includes improved features for structure calculation and refinement. PONDEROSA-C/S consists of three programs: Ponderosa Server, Ponderosa Client, and Ponderosa Analyzer. PONDEROSA-C/S takes as input the protein sequence, a list of assigned chemical shifts, and nuclear Overhauser data sets ((13)C- and/or (15)N-NOESY). The output is a set of assigned NOEs and 3D structural models for the protein. Ponderosa Analyzer supports the visualization, validation, and refinement of the results from Ponderosa Server. These tools enable semi-automated NMR-based structure determination of proteins in a rapid and robust fashion. We present examples showing the use of PONDEROSA-C/S in solving structures of four proteins: two that enable comparison with the original PONDEROSA package, and two from the Critical Assessment of automated Structure Determination by NMR (Rosato et al. in Nat Methods 6:625-626. doi: 10.1038/nmeth0909-625 , 2009) competition. The software package can be downloaded freely in binary format from http://pine.nmrfam.wisc.edu/download_packages.html. Registered users of the National Magnetic Resonance Facility at Madison can submit jobs to the PONDEROSA-C/S server at http://ponderosa.nmrfam.wisc.edu, where instructions, tutorials, and instructions can be found. Structures are normally returned within 1-2 days.

  7. Studies of single-walled carbon nanotubes-induced hepatotoxicity by NMR-based metabonomics of rat blood plasma and liver extracts

    Science.gov (United States)

    Lin, Bencheng; Zhang, Huashan; Lin, Zhiqing; Fang, Yanjun; Tian, Lei; Yang, Honglian; Yan, Jun; Liu, Huanliang; Zhang, Wei; Xi, Zhuge

    2013-05-01

    The toxicological effects of single-walled carbon nanotubes (SWCNTs) were investigated after intratracheal instillation in male Wistar rats over a 15-day period using metabonomic analysis of 1H (nuclear magnetic resonance) NMR spectra of blood plasma and liver tissue extracts. Concurrent liver histopathology examinations and plasma clinical chemistry analyses were also performed. Significant changes were observed in clinical chemistry features, including alkaline phosphatase, total protein, and total cholesterol, and in liver pathology, suggesting that SWCNTs clearly have hepatotoxicity in the rat. 1H NMR spectra and pattern recognition analyses from nanomaterial-treated rats showed remarkable differences in the excretion of lactate, trimethylamine oxide, bilineurin, phosphocholine, amylaceum, and glycogen. Indications of amino acid metabolism impairment were supported by increased lactate concentrations and decreased alanine concentrations in plasma. The rise in plasma and liver tissue extract concentrations of choline and phosphocholine, together with decreased lipids and lipoproteins, after SWCNTs treatment indicated a disruption of membrane fluidity caused by lipid peroxidation. Energy, amino acid, and fat metabolism appeared to be affected by SWCNTs exposure. Clinical chemistry and metabonomic approaches clearly indicated liver injury, which might have been associated with an indirect mechanism involving nanomaterial-induced oxidative stress.

  8. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Unknown

    2005-01-03

    Jan 3, 2005 ... covering all the systems, so far discovered.5,7,8,12. With the increasing ... Structural investigations on proteins by NMR are, currently ... rapid analysis of unfolded proteins. ...... and hence help in design of drugs against them.

  9. Icariin reverses corticosterone-induced depression-like behavior, decrease in hippocampal brain-derived neurotrophic factor (BDNF) and metabolic network disturbances revealed by NMR-based metabonomics in rats.

    Science.gov (United States)

    Gong, Meng-Juan; Han, Bin; Wang, Shu-mei; Liang, Sheng-wang; Zou, Zhong-jie

    2016-05-10

    Previously published reports have revealed the antidepressant-like effects of icariin in a chronic mild stress model of depression and in a social defeat stress model in mice. However, the therapeutic effect of icariin in an animal model of glucocorticoid-induced depression remains unclear. This study aimed to investigate antidepressant-like effect and the possible mechanisms of icariin in a rat model of corticosterone (CORT)-induced depression by using a combination of behavioral and biochemical assessments and NMR-based metabonomics. The depression model was established by subcutaneous injections of CORT for 21 consecutive days in rats, as evidenced by reduced sucrose intake and hippocampal brain-derived neurotrophic factor (BDNF) levels, together with an increase in immobility time in a forced swim test (FST). Icariin significantly increased sucrose intake and hippocampal BDNF level and decreased the immobility time in FST in CORT-induced depressive rats, suggesting its potent antidepressant activity. Moreover, metabonomic analysis identified eight, five and three potential biomarkers associated with depression in serum, urine and brain tissue extract, respectively. These biomarkers are primarily involved in energy metabolism, lipid metabolism, amino acid metabolism and gut microbe metabolism. Icariin reversed the pathological process of CORT-induced depression, partially via regulation of the disturbed metabolic pathways. These results provide important mechanistic insights into the protective effects of icariin against CORT-induced depression and metabolic dysfunction. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Rapid shallow breathing

    Science.gov (United States)

    Tachypnea; Breathing - rapid and shallow; Fast shallow breathing; Respiratory rate - rapid and shallow ... Shallow, rapid breathing has many possible medical causes, including: Asthma Blood clot in an artery in the ...

  11. Targeted isolation and identification of bioactive compounds lowering cholesterol in the crude extracts of crabapples using UPLC-DAD-MS-SPE/NMR based on pharmacology-guided PLS-DA.

    Science.gov (United States)

    Wen, Chao; Wang, Dongshan; Li, Xing; Huang, Tao; Huang, Cheng; Hu, Kaifeng

    2018-02-20

    The anti-hyperlipidemic effects of crude crabapple extracts derived from Malus 'Red jade', Malus hupehensis (Pamp.) Rehd. and Malus prunifolia (Willd.) Borkh. were evaluated on high-fat diet induced obese (HF DIO) mice. The results revealed that some of these extracts could lower serum cholesterol levels in HF DIO mice. The same extracts were also parallelly analyzed by LC-MS in both positive and negative ionization modes. Based on the pharmacological results, 22 LC-MS variables were identified to be correlated with the anti-hyperlipidemic effects using partial least square discriminant analysis (PLS-DA) and independent samples t-test. Further, under the guidance of the bioactivity-correlated LC-MS signals, 10 compounds were targetedly isolated and enriched using UPLC-DAD-MS-SPE and identified/elucidated by NMR together with MS/MS as citric acid(1), p-coumaric acid(2), hyperoside(3), myricetin(4), naringenin(5), quercetin(6), kaempferol(7), gentiopicroside(8), ursolic acid(9) and 8-epiloganic acid(10). Among these 10 compounds, 6 compounds, hyperoside(3), myricetin(4), naringenin(5), quercetin(6), kaempferol(7) and ursolic acid(9), were individually studied and reported to indeed have effects on lowering the serum lipid levels. These results demonstrated the efficiency of this strategy for drug discovery. In contrast to traditional routes to discover bioactive compounds in the plant extracts, targeted isolation and identification of bioactive compounds in the crude plant extracts using UPLC-DAD-MS-SPE/NMR based on pharmacology-guided PLS-DA of LC-MS data brings forward a new efficient dereplicated approach to natural products research for drug discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. On the mechanism of rapid postirradiation recovery of yeast

    International Nuclear Information System (INIS)

    Glazunov, A.V.; Kapul'tsevich, Yu.G.

    1983-01-01

    Rapid postirradiation recovery of diploid yeast Saccharomyces cerevisiae is equally effective both in water and in a liquid nutrition medium. In the haploid strains, rapid recovery occurs more readily in the log phase than in the stationary phase of growth. In the diploid strains, rapid recovery is more effective in the log phase than in the stationary phase. Rapid recovery of yeast does not require an additional protein synthesis. Damages induced by UV-light are not sub ected to rapid recovery

  13. Solvated protein-DNA docking using HADDOCK

    Energy Technology Data Exchange (ETDEWEB)

    Dijk, Marc van; Visscher, Koen M.; Kastritis, Panagiotis L.; Bonvin, Alexandre M. J. J., E-mail: a.m.j.j.bonvin@uu.nl [Utrecht University, Bijvoet Center for Biomolecular Research, Faculty of Science-Chemistry (Netherlands)

    2013-05-15

    Interfacial water molecules play an important role in many aspects of protein-DNA specificity and recognition. Yet they have been mostly neglected in the computational modeling of these complexes. We present here a solvated docking protocol that allows explicit inclusion of water molecules in the docking of protein-DNA complexes and demonstrate its feasibility on a benchmark of 30 high-resolution protein-DNA complexes containing crystallographically-determined water molecules at their interfaces. Our protocol is capable of reproducing the solvation pattern at the interface and recovers hydrogen-bonded water-mediated contacts in many of the benchmark cases. Solvated docking leads to an overall improvement in the quality of the generated protein-DNA models for cases with limited conformational change of the partners upon complex formation. The applicability of this approach is demonstrated on real cases by docking a representative set of 6 complexes using unbound protein coordinates, model-built DNA and knowledge-based restraints. As HADDOCK supports the inclusion of a variety of NMR restraints, solvated docking is also applicable for NMR-based structure calculations of protein-DNA complexes.

  14. Rapidly Progressive Quadriplegia and Encephalopathy.

    Science.gov (United States)

    Wynn, DonRaphael; McCorquodale, Donald; Peters, Angela; Juster-Switlyk, Kelsey; Smith, Gordon; Ansari, Safdar

    2016-11-01

    A woman aged 77 years was transferred to our neurocritical care unit for evaluation and treatment of rapidly progressive motor weakness and encephalopathy. Examination revealed an ability to follow simple commands only and abnormal movements, including myoclonus, tongue and orofacial dyskinesias, and opsoclonus. Imaging study findings were initially unremarkable, but when repeated, they demonstrated enhancement of the cauda equina nerve roots, trigeminal nerve, and pachymeninges. Cerebrospinal fluid examination revealed mildly elevated white blood cell count and protein levels. Serial electrodiagnostic testing demonstrated a rapidly progressive diffuse sensory motor axonopathy, and electroencephalogram findings progressed from generalized slowing to bilateral periodic lateralized epileptiform discharges. Critical details of her recent history prompted a diagnostic biopsy. Over time, the patient became completely unresponsive with no further abnormal movements and ultimately died. The differential diagnosis, pathological findings, and diagnosis are discussed with a brief review of a well-known yet rare diagnosis.

  15. Rapid deuterium exchange-in time for probing conformational change

    International Nuclear Information System (INIS)

    Dharmasiri, K.; Smith, D.L.

    1995-01-01

    Isotopic exchange of protein backbone amide hydrogens has been used extensively as a sensitive probe of protein structure. One of the salient features of hydrogen exchange is the vast range of exchange rates in one protein. Isotopic exchange methods have been used to study the structural features including protein folding and unfolding (1), functionally different forms of proteins (2), protein-protein complexation (3), and protein stability parameter. Many backbone amide protons that are surface accessible and are not involved in hydrogen bonding undergo rapid deuterium exchange. In order to study, fast exchanging amide protons, fast exchange-in times are necessary

  16. The protein protocols handbook

    National Research Council Canada - National Science Library

    Walker, John M

    2002-01-01

    .... The new chapters cover with many rapidly developing areas, particularly the application of mass spectrometry in protein characterization, as well as the now well-established 2-D PAGE technique in proteomics...

  17. Extra pulmonary tuberculosis: Rapid identification of Mycobacterium tuberculosis grown in Mycobacterium growth indicator tube 960 and Lowenstein-Jensen media, employing Standard diagnostics Bioline Mycobacterium tuberculosis protein 64 antigen detection kit

    Directory of Open Access Journals (Sweden)

    G Kandhakumari

    2015-01-01

    Full Text Available Background: Investigation of extra pulmonary tuberculosis (EPTB in and around Pondicherry is being carried out since August 2011 in our tertiary care super specialty hospital. Objectives: To compare the rapid Kit SD Bio-Line MPT 64 Ag with conventional and time consuming biochemical tests. Confirmation of Mycobacterium tuberculosis at a reasonable time frame is the main thrust. Materials and Methods: Thirty three Mycobacterium tuberculosis and four Non-Tuberculous Mycobacteria (NTM grown in MGIT960 system/Lowenstein-Jensen media (LJ were examined by the rapid MPT 64 antigen detection as well as a battery of conventional tests like niacin, nitrate reduction, paraminobenzoic acid susceptibility and cord formation. Results and Conclusion: . Both the rapid kit and conventional tests correctly identified 33 M.tuberculosis isolates. Keeping conventional identification as reference, sensitivity and specificity for rapid kit was 100%. Rapid kit which takes only 15 minutes is accurate, cost effective, and facilitates early treatment for these EPTB patients, whose clinical specimens are paucibacillary.

  18. Rapid Prototyping Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The ARDEC Rapid Prototyping (RP) Laboratory was established in December 1992 to provide low cost RP capabilities to the ARDEC engineering community. The Stratasys,...

  19. Rapid Tooling via Stereolithography

    OpenAIRE

    Montgomery, Eva

    2006-01-01

    Approximately three years ago, composite stereolithography (SL) resins were introduced to the marketplace, offering performance features beyond what traditional SL resins could offer. In particular, the high heat deflection temperatures and high stiffness of these highly filled resins have opened the door to several new rapid prototyping (RP) applications, including wind tunnel test modelling and, more recently, rapid tooling.

  20. Rapid improvement teams.

    Science.gov (United States)

    Alemi, F; Moore, S; Headrick, L; Neuhauser, D; Hekelman, F; Kizys, N

    1998-03-01

    Suggestions, most of which are supported by empirical studies, are provided on how total quality management (TQM) teams can be used to bring about faster organizationwide improvements. Ideas are offered on how to identify the right problem, have rapid meetings, plan rapidly, collect data rapidly, and make rapid whole-system changes. Suggestions for identifying the right problem include (1) postpone benchmarking when problems are obvious, (2) define the problem in terms of customer experience so as not to blame employees nor embed a solution in the problem statement, (3) communicate with the rest of the organization from the start, (4) state the problem from different perspectives, and (5) break large problems into smaller units. Suggestions for having rapid meetings include (1) choose a nonparticipating facilitator to expedite meetings, (2) meet with each team member before the team meeting, (3) postpone evaluation of ideas, and (4) rethink conclusions of a meeting before acting on them. Suggestions for rapid planning include reducing time spent on flowcharting by focusing on the future, not the present. Suggestions for rapid data collection include (1) sample patients for surveys, (2) rely on numerical estimates by process owners, and (3) plan for rapid data collection. Suggestions for rapid organizationwide implementation include (1) change membership on cross-functional teams, (2) get outside perspectives, (3) use unfolding storyboards, and (4) go beyond self-interest to motivate lasting change in the organization. Additional empirical investigations of time saved as a consequence of the strategies provided are needed. If organizations solve their problems rapidly, fewer unresolved problems may remain.

  1. Plant MAPK cascades: Just rapid signaling modules?

    KAUST Repository

    Boudsocq, Marie; Danquah, Agyemang; Zé licourt, Axel de; Hirt, Heribert; Colcombet, Jean

    2015-01-01

    rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests

  2. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    Science.gov (United States)

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  3. Rapid response systems.

    Science.gov (United States)

    Lyons, Patrick G; Edelson, Dana P; Churpek, Matthew M

    2018-07-01

    Rapid response systems are commonly employed by hospitals to identify and respond to deteriorating patients outside of the intensive care unit. Controversy exists about the benefits of rapid response systems. We aimed to review the current state of the rapid response literature, including evolving aspects of afferent (risk detection) and efferent (intervention) arms, outcome measurement, process improvement, and implementation. Articles written in English and published in PubMed. Rapid response systems are heterogeneous, with important differences among afferent and efferent arms. Clinically meaningful outcomes may include unexpected mortality, in-hospital cardiac arrest, length of stay, cost, and processes of care at end of life. Both positive and negative interventional studies have been published, although the two largest randomized trials involving rapid response systems - the Medical Early Response and Intervention Trial (MERIT) and the Effect of a Pediatric Early Warning System on All-Cause Mortality in Hospitalized Pediatric Patients (EPOCH) trial - did not find a mortality benefit with these systems, albeit with important limitations. Advances in monitoring technologies, risk assessment strategies, and behavioral ergonomics may offer opportunities for improvement. Rapid responses may improve some meaningful outcomes, although these findings remain controversial. These systems may also improve care for patients at the end of life. Rapid response systems are expected to continue evolving with novel developments in monitoring technologies, risk prediction informatics, and work in human factors. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins......, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA...

  5. An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

    Directory of Open Access Journals (Sweden)

    Ying Huang

    2013-11-01

    Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

  6. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs...

  7. Metabonomic Response to Milk Proteins after a Single Bout of Heavy Resistance Exercise Elucidated by 1H Nuclear Magnetic Resonance Spectroscopy

    Directory of Open Access Journals (Sweden)

    Hanne Christine Bertram

    2013-01-01

    Full Text Available In the present study, proton NMR-based metabonomics was applied on femoral arterial plasma samples collected from young male subjects (milk protein n = 12 in a crossover design; non-caloric control n = 8 at different time intervals (70, 220, 370 min after heavy resistance training and intake of either a whey or calcium caseinate protein drink in order to elucidate the impact of the protein source on post-exercise metabolism, which is important for muscle hypertrophy. Dynamic changes in the post-exercise plasma metabolite profile consisted of fluctuations in alanine, beta-hydroxybutyrate, branched amino acids, creatine, glucose, glutamine, glutamate, histidine, lipids and tyrosine. In comparison with the intake of a non-caloric drink, the same pattern of changes in low-molecular weight plasma metabolites was found for both whey and caseinate intake. However, the study indicated that whey and caseinate protein intake had a different impact on low-density and very-low-density lipoproteins present in the blood, which may be ascribed to different effects of the two protein sources on the mobilization of lipid resources during energy deficiency. In conclusion, no difference in the effects on low-molecular weight metabolites as measured by proton NMR-based metabonomics was found between the two protein sources.

  8. Rapid world modeling

    International Nuclear Information System (INIS)

    Little, Charles; Jensen, Ken

    2002-01-01

    Sandia National Laboratories has designed and developed systems capable of large-scale, three-dimensional mapping of unstructured environments in near real time. This mapping technique is called rapid world modeling and has proven invaluable when used by prototype systems consisting of sensory detection devices mounted on mobile platforms. These systems can be deployed into previously unmapped environments and transmit real-time 3-D visual images to operators located remotely. This paper covers a brief history of the rapid world modeling system, its implementation on mobile platforms, and the current state of the technology. Applications to the nuclear power industry are discussed. (author)

  9. JINR rapid communications

    International Nuclear Information System (INIS)

    1998-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate records on relativistic multiparticle processes in the central rapidity region at asymptotically high energies, a new experimental study of charged K→3π decays, pre-Cherenkov radiation as a phenomenon of 'light barrier', stable S=-2 H dibaryon found in Dubna, calculation of Green functions and gluon top in some unambiguous gauges, a method of a fast selection of inelastic nucleus-nucleus collisions for the CMS experiment and the manifestation of jet quenching in differential distributions of the total transverse energy in nucleus-nucleus collisions

  10. Rapid microbiology - raising awareness.

    Science.gov (United States)

    Bailie, Jonathan

    2016-01-01

    A 'high-level overview' of some of the emerging rapid microbiology technologies designed to help healthcare engineering and infection control teams working in hospitals and other healthcare facilities more rapidly identify potentially hazardous levels of waterborne microorganisms in their water systems, enabling them to take prompt remedial action, and a look at the some of the 'pros and cons' of such testing techniques, was given by Nalco technical director, Howard Barnes, the vice-chair of the Legionella Control Association (LCA), at a recent LCA open day. HEJ editor, Jonathan Bailie, reports.

  11. JINR rapid communications

    International Nuclear Information System (INIS)

    1998-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate records on invisible Z-boson width and restrictions on next-to-minimal supersymmetric standard model, cosmic test of honeycomb drift chambers, fission of 209 Bi, 232 Th, 235 U, 238 U and 237 Np in a spallation neutron field, rapid screening of spontaneous and radiation-induced structural changes at the vestigial gene of Drosophila melanogaster by polymerase chain reaction, gamma-ray multiplicities in sub-barrier fission of 226 Th and the decay constants of the scalar and pseudoscalar mesons in the quark models with quasilocal interaction

  12. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    Science.gov (United States)

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  13. Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

    Science.gov (United States)

    Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

    2011-10-28

    Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Navigate the Digital Rapids

    Science.gov (United States)

    Lindsay, Julie; Davis, Vicki

    2010-01-01

    How can teachers teach digital citizenship when the digital landscape is changing so rapidly? How can teachers teach proper online social interactions when the students are outside their classroom and thus outside their control? Will encouraging students to engage in global collaborative environments land teachers in hot water? These are the…

  15. Supplementation with a fish oil-enriched, high-protein medical food leads to rapid incorporation of EPA into white blood cells and modulates immune responses within one week in healthy men and women.

    Science.gov (United States)

    Faber, Joyce; Berkhout, Marloes; Vos, Arjan P; Sijben, John W C; Calder, Philip C; Garssen, Johan; van Helvoort, Ardy

    2011-05-01

    Immune modulatory effects of EPA and DHA are well described. However, these fatty acids must be effectively incorporated into cell membrane phospholipids to modify cell function. To address the absence of human data regarding short-term incorporation, the present study investigated the incorporation of EPA and DHA into white blood cells (WBC) at different time points during 1 wk of supplementation with a medical food, which is high in protein and leucine and enriched with fish oil and specific oligosaccharides. Additionally, the effects on ex vivo immune function were determined. In a single-arm, open label study, 12 healthy men and women consumed 2 × 200 mL of medical food providing 2.4 g EPA, 1.2 g DHA, 39.7 g protein (including 4.4 g L-leucine), and 5.6 g oligosaccharides daily. Blood samples were taken at d 0 (baseline), 1, 2, 4, and 7. Within 1 d of nutritional intervention, the percentage of EPA in phospholipids of WBC increased from 0.5% at baseline to 1.3% (P blood cultures was significantly increased within 1 wk. Nutritional supplementation with a fish oil-enriched medical food significantly increased the percentage of EPA in phospholipids of WBC within 1 wk. Simultaneously, ex vivo immune responsiveness to LPS increased significantly. These results hold promise for novel applications such as fast-acting nutritional interventions in cancer patients, which should be investigated in future studies.

  16. Rapid road repair vehicle

    Science.gov (United States)

    Mara, Leo M.

    1998-01-01

    Disclosed is a rapid road repair vehicle capable of moving over a surface to be repaired at near normal posted traffic speeds to scan for and find an the high rate of speed, imperfections in the pavement surface, prepare the surface imperfection for repair by air pressure and vacuum cleaning, applying a correct amount of the correct patching material to effect the repair, smooth the resulting repaired surface, and catalog the location and quality of the repairs for maintenance records of the road surface. The rapid road repair vehicle can repair surface imperfections at lower cost, improved quality, at a higher rate of speed than was was heretofor possible, with significantly reduced exposure to safety and health hazards associated with this kind of road repair activities in the past.

  17. Rapidly processable radiographic material

    International Nuclear Information System (INIS)

    Brabandere, L.A. de; Borginon, H.A.; Pattyn, H.A.; Pollet, R.J.

    1981-01-01

    A new rapidly processable radiographic silver halide material is described for use in mammography and non-destructive testing of industrial materials. The radiographic material is used for direct exposure to penetrating radiation without the use of fluorescent-intensifying screens. It consists of a transparent support with a layer of hydrophilic colloid silver halide emulsion on one or both sides. Examples of the preparation of three different silver halide emulsions are given including the use of different chemical sensitizers. These new radiographic materials have good resistance to the formation of pressure marks in rapid processing apparatus and they have improved sensitivity for direct exposure to penetrating radiation compared to conventional radiographic emulsions. (U.K.)

  18. Rapid manufacturing for microfluidics

    CSIR Research Space (South Africa)

    Land, K

    2012-10-01

    Full Text Available for microfluidics K. LAND, S. HUGO, M MBANJWA, L FOURIE CSIR Materials Science and Manufacturing P O Box 395, Pretoria 0001, SOUTH AFRICA Email: kland@csir.co.za INTRODUCTION Microfluidics refers to the manipulation of very small volumes of fluid.... Microfluidics is at the forefront of developing solutions for drug discovery, diagnostics (from glucose tests to malaria and TB testing) and environmental diagnostics (E-coli monitoring of drinking water). In order to quickly implement new designs, a rapid...

  19. Tiber Personal Rapid Transit

    Directory of Open Access Journals (Sweden)

    Diego Carlo D'agostino

    2011-02-01

    Full Text Available The project “Tiber Personal Rapid Transit” have been presented by the author at the Rome City Vision Competition1 2010, an ideas competition, which challenges architects, engineers, designers, students and creatives individuals to develop visionary urban proposals with the intention of stimulating and supporting the contemporary city, in this case Rome. The Tiber PRT proposal tries to answer the competition questions with the definition of a provocative idea: a Personal Rapid transit System on the Tiber river banks. The project is located in the central section of the Tiber river and aims at the renewal of the river banks with the insertion of a Personal Rapid Transit infrastructure. The project area include the riverbank of Tiber from Rome Transtevere RFI station to Piazza del Popolo, an area where main touristic and leisure attractions are located. The intervention area is actually no used by the city users and residents and constitute itself a strong barrier in the heart of the historic city.

  20. Rapid MR imaging

    International Nuclear Information System (INIS)

    Edelman, R.R.; Buxton, R.B.; Brady, T.J.

    1988-01-01

    Conventional magnetic resonance (MR) imaging methods typically require several minutes to produce an image, but the periods of respiration, cardiac motion and peristalsis are on the order of seconds or less. The need to reduce motion artifact, as well as the need to reduce imaging time for patient comfort and efficiency, have provided a strong impetus for the development of rapid imaging methods. For abdominal imaging, motion artifacts due to respiration can be significantly reduced by collecting the entire image during one breath hold. For other applications, such as following the kinetics of administered contrast agents, rapid imaging is essential to achieve adequate time resolution. A shorter imaging time entails a cost in image signal/noise (S/N), but improvements in recent years in magnet homogeneity, gradient and radiofrequency coil design have led to steady improvements in S/N and consequently in image quality. For many chemical applications the available S/N is greater than needed, and a trade-off of lower S/N for a shorter imaging time is acceptable. In this chapter, the authors consider the underlying principles of rapid imaging as well as clinical applications of these methods. The bulk of this review concentrates on short TR imaging, but methods that provide for a more modest decrease in imaging time as well as or those that dramatically shorten the imaging time to tens of milliseconds are also discussed

  1. Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet.

    Science.gov (United States)

    Brito, S M R C; Moura, M A F; Kawashita, N H; Festuccia, W T L; Garófalo, M A R; Kettelhut, I C; Migliorini, R H

    2005-06-01

    We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.

  2. Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis, cotyledons and endosperm.

    Science.gov (United States)

    Leung, D W; Reid, J S; Bewley, J D

    1979-01-01

    The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), α-galactosidase and β-mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo-β-mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.

  3. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  4. JINR rapid communications

    International Nuclear Information System (INIS)

    1998-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate records on decays of excited strange mesons in the extended NJL model, production of heavy evaporation residues in the reactions induced by an extracted 48 Ca beam on a 208 Pb target, scaling behaviour of tensor analyzing power (A yy ) in the inelastic scattering or relativistic deuterons,two-photon collisions at very low Q 2 from LEP2: forthcoming results, high magnetic field uniformity superconducting magnet for a movable polarized target, multichannel time-to-digital converter for drift detector and wavelet-analysis: application to Gaussian signals

  5. JINR rapid communications

    International Nuclear Information System (INIS)

    1995-01-01

    The present collection of rapid communications from JINR, Dubna, contains eight separate reports on the measurement of charge radii for Ti nuclei, spectroscopy of 13 Be, concentrations of hadrons and quark-gluon plasma in mixed phase, experimental results on one-spin pion asymmetry in the d↑ + A → π±(90 0 ) + X process, new results on cumulative pion and proton production in p-D collisions, investigation of charge exchange reactions, the study of the tensor analyzing power in cumulative particle production on a deuteron beam and an evidence for the excited states of the S = -2 stable light dibaryon. 32 figs., 6 tabs

  6. JINR rapid communications

    International Nuclear Information System (INIS)

    1996-01-01

    The present collection of rapid communications from JINR, Dubna, contains five separate reports on analytic QCD running coupling with finite IR behaviour and universal α bar s (0) value, quark condensate in the interacting pion- nucleon medium at finite temperature and baryon number density, γ-π 0 discrimination with a shower maximum detector using neural networks for the solenoidal tracker at RHIC, off-specular neutron reflection from magnetic media with nondiagonal reflectivity matrices and molecular cytogenetics of radiation-induced gene mutations in Drosophila melanogaster. 21 fig., 1 tab

  7. JINR rapid communications

    International Nuclear Information System (INIS)

    1999-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate records on additional conditions on eigenvectors in solving inverse problem for two-dimensional Schroedinger equation, on an absolute calibration of deuteron beam polarization at LHE, determination of the vector component of the polarization of the JINR synchrophasotron deuteron beam, wavelet-analysis: criterion of reliable signal selection, on asymptotics in inclusive production of antinuclei and nuclear fragments, use of neutron activation analysis at the IBR-2 reactor for atmospheric monitoring and impulse method for temperature measurement of silicon detectors

  8. JINR rapid communications

    International Nuclear Information System (INIS)

    1995-01-01

    The present collection of rapid communications from JINR, Dubna, contains six separate reports on Monte Carlo simulation of silicon detectors for the ALICE experiment at LHC, a study of single tagged multihadronic γγ* events at an average Q 2 of 90 GeV 2 , epithermal neutron activation analysis of moss, lichen and pine needles in atmospheric deposition monitoring, the theory of neutrino oscillation, coupled quadrupole and monopole vibrations of large amplitude and test of the Ellis-Jaffe sum rule using parametrization of the measured lepton-proton asymmetry. 21 figs., 18 tabs

  9. Rapidly variable relatvistic absorption

    Science.gov (United States)

    Parker, M.; Pinto, C.; Fabian, A.; Lohfink, A.; Buisson, D.; Alston, W.; Jiang, J.

    2017-10-01

    I will present results from the 1.5Ms XMM-Newton observing campaign on the most X-ray variable AGN, IRAS 13224-3809. We find a series of nine absorption lines with a velocity of 0.24c from an ultra-fast outflow. For the first time, we are able to see extremely rapid variability of the UFO features, and can link this to the X-ray variability from the inner accretion disk. We find a clear flux dependence of the outflow features, suggesting that the wind is ionized by increasing X-ray emission.

  10. Rapid Geophysical Surveyor

    International Nuclear Information System (INIS)

    Roybal, L.G.; Carpenter, G.S.; Josten, N.E.

    1993-01-01

    The Rapid Geophysical Surveyor (RGS) is a system designed to rapidly and economically collect closely-spaced geophysical data used for characterization of US Department of Energy waste sites. Geophysical surveys of waste sites are an important first step in the remediation and closure of these sites; especially older sites where historical records are inaccurate and survey benchmarks have changed because of refinements in coordinate controls and datum changes. Closely-spaced data are required to adequately differentiate pits, trenches, and soil vault rows whose edges may be only a few feet from each other. A prototype vehicle designed to collect magnetic field data was built at the Idaho National Engineering Laboratory (INEL) during the summer of 1992. The RGS was funded by the Buried Waste Integrated Demonstration program. This vehicle was demonstrated at the Subsurface Disposal Area (SDA) within the Radioactive Waste Management Complex at the INEL in September 1992. Magnetic data were collected over two areas in the SDA, with a total survey area of about 1.7 acres. Data were collected at a nominal density of 2 1/2 in. along survey lines spaced 1-ft apart. Over 350,000 data points were collected over a 6 day period corresponding to about 185 worker-days using conventional ground survey techniques

  11. NMR approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes.

    Science.gov (United States)

    Dias, David M; Ciulli, Alessio

    2014-01-01

    Nuclear magnetic resonance (NMR) spectroscopy is a pivotal method for structure-based and fragment-based lead discovery because it is one of the most robust techniques to provide information on protein structure, dynamics and interaction at an atomic level in solution. Nowadays, in most ligand screening cascades, NMR-based methods are applied to identify and structurally validate small molecule binding. These can be high-throughput and are often used synergistically with other biophysical assays. Here, we describe current state-of-the-art in the portfolio of available NMR-based experiments that are used to aid early-stage lead discovery. We then focus on multi-protein complexes as targets and how NMR spectroscopy allows studying of interactions within the high molecular weight assemblies that make up a vast fraction of the yet untargeted proteome. Finally, we give our perspective on how currently available methods could build an improved strategy for drug discovery against such challenging targets. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

    Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

  13. Rapid automated nuclear chemistry

    International Nuclear Information System (INIS)

    Meyer, R.A.

    1979-01-01

    Rapid Automated Nuclear Chemistry (RANC) can be thought of as the Z-separation of Neutron-rich Isotopes by Automated Methods. The range of RANC studies of fission and its products is large. In a sense, the studies can be categorized into various energy ranges from the highest where the fission process and particle emission are considered, to low energies where nuclear dynamics are being explored. This paper presents a table which gives examples of current research using RANC on fission and fission products. The remainder of this text is divided into three parts. The first contains a discussion of the chemical methods available for the fission product elements, the second describes the major techniques, and in the last section, examples of recent results are discussed as illustrations of the use of RANC

  14. JINR rapid communications

    International Nuclear Information System (INIS)

    1999-01-01

    The present collection of rapid communications from JINR, DUBNA, contains eight separate records on symmetry in modern physics (dedicated to the 100th anniversary of the birth of academician V.A.Fock), the double φ-meson production investigation on the Serpukhov accelerator, two-leptonic η-meson decays and SUSY without R parity, charge form factors and alpha-cluster internal structure of 12 C, increasing of muon-track reconstruction efficiency in ME1/1 Dubna prototype for the CMS/LHC, study of photon-structure function F 2 γ in the reaction e + e - → e + e - + hadrons at LEP2, jets reconstruction possibility in pAu and AuAu interactions at STAR RHIC and high-vacuum nondispersable gas absorber

  15. Rapid thermal pulse annealing

    International Nuclear Information System (INIS)

    Miller, M.G.; Koehn, B.W.; Chaplin, R.L.

    1976-01-01

    Characteristics of recovery processes have been investigated for cases of heating a sample to successively higher temperatures by means of isochronal annealing or by using a rapid pulse annealing. A recovery spectra shows the same features independent of which annealing procedure is used. In order to determine which technique provides the best resolution, a study was made of how two independent first-order processes are separated for different heating rates and time increments of the annealing pulses. It is shown that the pulse anneal method offers definite advantages over isochronal annealing when annealing for short time increments. Experimental data by means of the pulse anneal techniques are given for the various substages of stage I of aluminium. (author)

  16. JINR rapid communications

    International Nuclear Information System (INIS)

    1996-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate reports on the identification of events with a secondary vertex in the experiment EXCHARM, the zero degree calorimeter for CERN WA-98 experiment, a new approach to increase the resource of installation elements for super-high energy physics, a method of the in-flight production of exotic systems in the charge-exchange reactions, the neutron activation analysis for monitoring northern terrestrial ecosystems, a search for 28 O and study of the neutron-rich nuclei near the neutron closure N=20, a search for new neutron-rich nuclei with a 70A MeV 48 Ca beam. 33 figs., 4 tabs

  17. JINR Rapid Communications. Collection

    International Nuclear Information System (INIS)

    1994-01-01

    The present collection of rapid communications from JINR, Dubna, contains nine separate reports on quasi-classical description of one-nucleon transfer reactions with heavy ions, elastic and inelastic scattering in the high energy approximation, experimental study of fission and evaporation cross sections for 6 He + 209 Bi reaction, d ↑ + 12 C → p + X at Θ p = 0 o in the region of high internal momenta in the deuteron, the Nuclotron internal targets, actively screened superconducting magnets, using of polarized target in backward elastic dp scattering, application of transputers in the data acquisition system of the INESS-ALPHA spectrometer, narrow dibaryon resonances with isotopic spin I=2. 93 refs., 27 figs., 4 tabs

  18. JINR Rapid Communications. Collection

    International Nuclear Information System (INIS)

    1994-01-01

    The present collection of rapid communications from JINR, Dubna, contains eight separate reports on Lorentz transformations with superluminal velocities, photo chromic effect in HTSC films, the investigation of hypernuclei in the Nuclotron accelerator, a new hadron jets finding algorithm in the four-dimensional velocity space, investigations of neutral particle production by relativistic nuclei on the LHE 90-channel γ-spectrometer (results and perspectives), coherent meson production in the dp → 3 HeX reaction, the relativistic projectile nuclei fragmentation and A-dependence of nucleon Fermi-momenta, energy spectra of γ-quanta from d-propane interactions at momentum P d = 1.25 GeV/c per nucleon. 86 refs., 26 figs., 4 tabs

  19. JINR rapid communications

    International Nuclear Information System (INIS)

    1999-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate records on measurements of the total cross section difference Δσ L (np) at 1.59, 1.79, and 2.20 GeV, to the estimation of angular distributions of double charged spectator fragments in nucleus-nucleus interactions at superhigh energies, simulation dE/dx analysis results for silicon inner tracking system of ALICE set-up at LHC accelerator, high-multiplicity processes, triggering of high-multiplicity events using calorimetry, ORBIT-3.0 - a computer code for simulation and correction of the closed orbit and first turn in synchrotrons and determination of memory performance

  20. JINR rapid communications

    International Nuclear Information System (INIS)

    1999-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate records on yields of the rare-earth neutron-deficient isotopes in the reactions of Mo isotopes with 40 Ca ions, observations of slow components of solitonic-type wave structure excited by e-beam in massive copper sample, development and investigation of low-mass multilayer drift chambers (MDC-2) for inner part of the HADES spectrometer, temperature measurement of the uranium sample irradiated with secondary neutrons, edge effects in multiwire proportional chambers, the influence of the dielectric frame, an object-oriented framework for the hadronic Monte-Carlo event generators and uranium-238 as a source for electronuclear power production. 32 figs., 3 tabs

  1. JINR rapid communications

    International Nuclear Information System (INIS)

    1997-01-01

    The present collection of rapid communications from JINR, Dubna, contains nine separate reports on collective energy dissipation and fluctuations in elastoplastic systems, diagnostics system of the circulating beam of the NUCLOTRON based on microchannel plates, time-of-flight detector for WA98 CERN experiment, fractal structure formation on the surfaces of solids subjected to high intensity electron and ion treatment, production of nuclei in 32,34,36 S-induced reactions in the energy range 6-75 MeV/A, rare-earth elements in soil and pine needle from northern terrestrial ecosystems, 'thermal' multifragmentation in p + Au collisions at relativistic energies, search for effects of the OZI rule violation in φ and ω mesons production in polarized deuteron beam interaction with polarized proton target (project DPHE3) and fast detector for triggering on charged particle multiplicity for relativistic nucleus-nucleus collisions

  2. JINR rapid communications

    International Nuclear Information System (INIS)

    1997-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate reports on observation of transversal handedness in the diffractive production of pion triples, a possible experiment on the research of dibaryon states, Cherenkov beam counter system of the CERES/NA45 spectrometer for investigation with 160 GeV/n. lead ions, a profile-based gaseous detector with capacitive pad readout as the prototype of the shower maximum detector for the end-cap electromagnetic calorimeter for the STAR experiment, what DELPHI can get with an upgraded position for the very small angle tagger, estimation of the radiation environment and the shielding aspect for the point 2 area of the LHC and the orthopositronium decay puzzle

  3. Rapid chemical separations

    CERN Document Server

    Trautmann, N

    1976-01-01

    A survey is given on the progress of fast chemical separation procedures during the last few years. Fast, discontinuous separation techniques are illustrated by a procedure for niobium. The use of such techniques for the chemical characterization of the heaviest known elements is described. Other rapid separation methods from aqueous solutions are summarized. The application of the high speed liquid chromatography to the separation of chemically similar elements is outlined. The use of the gas jet recoil transport method for nuclear reaction products and its combination with a continuous solvent extraction technique and with a thermochromatographic separation is presented. Different separation methods in the gas phase are briefly discussed and the attachment of a thermochromatographic technique to an on-line mass separator is shown. (45 refs).

  4. JINR rapid communications

    International Nuclear Information System (INIS)

    1997-01-01

    The present collection of rapid communications from JINR, Dubna, contains seven separate reports on investigation of the tensor analyzing power A yy in the reaction A(d polarized, p)X at large transverse momenta of proton, double-differential ionization cross section calculations for fast collisions of ions and atoms, a study of the two-photon interactions tagged at an average 2 > of 90 GeV 2 , cluster and single-particle distributions in nucleus-nucleus interactions, the Coulomb interaction of charged pions in CC-and CTa-collisions at 4.2 A GeV/c, influence of nitrogen and oxygen gas admixtures on the response of the DELPHI HCAL and MUS detectors and an automation of physics research on base of open standards

  5. JINR rapid communications

    International Nuclear Information System (INIS)

    1997-01-01

    The present collection of rapid communications from JINR, Dubna, contains nine separate reports on effects arising from charged particles overcoming of the light velocity barrier, deformable templates for circle recognition, scintillation detectors for precise time measurements, atomic form factors and incoherent scattering functions of atoms and ions with the number of electrons N ≤ 10, experimental set-up ANOMALON for measurement of relativistic nuclear fragmentation cross sections, superconducting dipole magnet for ALICE dimuon arm spectrometer, analysis of transverse mass dependence of Bose-Einstein correlation radii using the DELPHI data, low-energy theorem in softly broken supersymmetry and study of the characteristics of particles in reactions π - , p, d, He, C + C with the total disintegration on carbon nucleus

  6. JINR rapid communications

    International Nuclear Information System (INIS)

    1998-01-01

    The present collection of rapid communications from JINR, Dubna, contains six separate records on test of a threshold aerogel Cherenkov counter on cosmic particles, first results of study of transversal dimension of region of cumulative particles production in d + C and d + Cu reactions for energy 2 GeV/nucleon, the evidence of σ[0 + (0 ++ 0)] meson at a mass of M π + π - = 750 ± 5 MeV/c 2 observed in π + π - combinations from the reaction np → npπ + π - at an incident momentum of P n (5.20 ± 0.16 GeV/c, inclusive spectra of protons and π - mesons emitted in 4 HeC and 12 CC interactions with total disintegration of nuclei, heavy quark-antiquark pair production by double pomeron exchange in pp and AA collisions on the CMS and global features of nucleus-nucleus collisions in ultrarelativistic domain

  7. Rapid Refresh (RAP) [13 km

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Rapid Refresh (RAP) numerical weather model took the place of the Rapid Update Cycle (RUC) on May 1, 2012. Run by the National Centers for Environmental...

  8. Rapid Refresh (RAP) [20 km

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Rapid Refresh (RAP) numerical weather model took the place of the Rapid Update Cycle (RUC) on May 1, 2012. Run by the National Centers for Environmental...

  9. Rapid population growth.

    Science.gov (United States)

    1972-01-01

    At the current rate of population growth, world population by 2000 is expected to reach 7 billion or more, with developing countries accounting for some 5.4 billion, and economically advanced nations accounting for 1.6 billion. 'Population explosion' is the result of falling mortality rates and continuing high birth rates. Many European countries, and Japan, have already completed what is termed as demographic transition, that is, birth rates have fallen to below 20 births per 1000 population, death rates to 10/1000 population, and annual growth rates are 1% or less; annual growth rates for less developed countries ranged from 2 to 3.5%. Less developed countries can be divided into 3 groups: 1) countries with both high birth and death rates; 2) countries with high birth rates and low death rates; and 3) countries with intermediate and declining birth rates and low death rates. Rapid population growth has serious economic consequences. It encourages inequities in income distribution; it limits rate of growth of gross national product by holding down level of savings and capital investments; it exerts pressure on agricultural production and land; and it creates unemployment problems. In addition, the quality of education for increasing number of chidren is adversely affected, as high proportions of children reduce the amount that can be spent for the education of each child out of the educational budget; the cost and adequacy of health and welfare services are affected in a similar way. Other serious consequences of rapid population growth are maternal death and illness, and physical and mental retardation of children of very poor families. It is very urgent that over a billion births be prevented in the next 30 years to reduce annual population growth rate from the current 2% to 1% per year.

  10. Rapid geophysical surveyor

    International Nuclear Information System (INIS)

    Roybal, L.G.; Carpenter, G.S.; Josten, N.E.

    1993-01-01

    The Rapid Geophysical Surveyor (RGS) is a system designed to rapidly and economically collect closely-spaced geophysical data used for characterization of Department of Energy (DOE) waste sites. Geophysical surveys of waste sites are an important first step in the remediation and closure of these sites; especially older sties where historical records are inaccurate and survey benchmarks have changed due to refinements in coordinate controls and datum changes. Closely-spaced data are required to adequately differentiate pits, trenches, and soil vault rows whose edges may be only a few feet from each other. A prototype vehicle designed to collect magnetic field data was built at the Idaho national Engineering Laboratory (INEL) during the summer of 1992. The RGS was one of several projects funded by the Buried Waste Integrated Demonstration (BWID) program. This vehicle was demonstrated at the Subsurface Disposal Area (SDA) within the Radioactive Waste Management Complex (RWMC) on the INEL in September of 1992. Magnetic data were collected over two areas in the SDA, with a total survey area of about 1.7 acres. Data were collected at a nominal density of 2 1/2 inches along survey lines spaced 1 foot apart. Over 350,000 data points were collected over a 6 day period corresponding to about 185 man-days using conventional ground survey techniques. This report documents the design and demonstration of the RGS concept including the presentation of magnetic data collected at the SDA. The surveys were able to show pit and trench boundaries and determine details of their spatial orientation never before achieved

  11. Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets

    International Nuclear Information System (INIS)

    Bellstedt, Peter; Seiboth, Thomas; Häfner, Sabine; Kutscha, Henriette; Ramachandran, Ramadurai; Görlach, Matthias

    2013-01-01

    NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ('scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided

  12. Dairy Proteins and Energy Balance

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist

    High protein diets affect energy balance beneficially through decreased hunger, enhanced satiety and increased energy expenditure. Dairy products are a major source of protein. Dairy proteins are comprised of two classes, casein (80%) and whey proteins (20%), which are both of high quality......, but casein is absorbed slowly and whey is absorbed rapidly. The present PhD study investigated the effects of total dairy proteins, whey, and casein, on energy balance and the mechanisms behind any differences in the effects of the specific proteins. The results do not support the hypothesis that dairy...... proteins, whey or casein are more beneficial than other protein sources in the regulation of energy balance, and suggest that dairy proteins, whey or casein seem to play only a minor role, if any, in the prevention and treatment of obesity....

  13. Rapid Evaporation of microbubbles

    Science.gov (United States)

    Gautam, Jitendra; Esmaeeli, Asghar

    2008-11-01

    When a liquid is heated to a temperature far above its boiling point, it evaporates abruptly. Boiling of liquid at high temperatures can be explosive and destructive, and poses a potential hazard for a host of industrial processes. Explosive boiling may occur if a cold and volatile liquid is brought into contact with a hot and non-volatile liquid, or if a liquid is superheated or depressurized rapidly. Such possibilities are realized, for example, in the depressurization of low boiling point liquefied natural gas (LNG) in the pipelines or storage tanks as a result of a leak. While boiling of highly heated liquids can be destructive at macroscale, the (nearly) instantaneous pace of the process and the release of large amount of kinetic energy make the phenomena extremely attractive at microscale where it is possible to utilize the released energy to derive micromechanical systems. For instance, there is currently a growing interest in micro-explosion of liquid for generation of micro bubbles for actuation purposes. The aim of the current study is to gain a fundamental understanding of the subject using direct numerical simulations. In particular, we seek to investigate the boundary between stable and unstable nucleus growth in terms of the degree of liquid superheat and to compare the dynamics of unstable and stable growth.

  14. Rapid flow imaging method

    International Nuclear Information System (INIS)

    Pelc, N.J.; Spritzer, C.E.; Lee, J.N.

    1988-01-01

    A rapid, phase-contrast, MR imaging method of imaging flow has been implemented. The method, called VIGRE (velocity imaging with gradient recalled echoes), consists of two interleaved, narrow flip angle, gradient-recalled acquisitions. One is flow compensated while the second has a specified flow encoding (both peak velocity and direction) that causes signals to contain additional phase in proportion to velocity in the specified direction. Complex image data from the first acquisition are used as a phase reference for the second, yielding immunity from phase accumulation due to causes other than motion. Images with pixel values equal to MΔΘ where M is the magnitude of the flow compensated image and ΔΘ is the phase difference at the pixel, are produced. The magnitude weighting provides additional vessel contrast, suppresses background noise, maintains the flow direction information, and still allows quantitative data to be retrieved. The method has been validated with phantoms and is undergoing initial clinical evaluation. Early results are extremely encouraging

  15. JINR rapid communications

    International Nuclear Information System (INIS)

    1995-01-01

    The present collection of rapid communications from JINR, Dubna, contains twelve separate reports on an estimation of the possibility of fusion reactions in water molecules, an analysis of pion spectra of the charge-exchange reaction Mg(t, 3 He), the results of simulation of e + e - pair production and detection in the ALICE experiment, the data on the edge effects in multiwire proportional chambers, standard and nonstandard applications of wavelet analysis, the design and study of light readout system for scintillator shower maximum detector for the endcap electromagnetic calorimeter for the STAR experiment at RHIC, a study of multiparticle azimuthal correlations in high energy interactions, coherent multifragmentation of relativistic nuclei, superposition of neutrino eigenstates and neutrino oscillation, simulation results and suggestions for possible design of gaseous shower maximum detector for the endcap electromagnetic calorimeter for the STAR experiment at RHIC, determination of the sizes of the pion emission region in np-interactions at P n =(5.2±0.16)GeV/c using the interference correlation method for identical particles, inelasticity of nucleus-nucleus collisions in the CMS experiment. 65 figs., 19 tabs

  16. Rapid Polymer Sequencer

    Science.gov (United States)

    Stolc, Viktor (Inventor); Brock, Matthew W (Inventor)

    2013-01-01

    Method and system for rapid and accurate determination of each of a sequence of unknown polymer components, such as nucleic acid components. A self-assembling monolayer of a selected substance is optionally provided on an interior surface of a pipette tip, and the interior surface is immersed in a selected liquid. A selected electrical field is impressed in a longitudinal direction, or in a transverse direction, in the tip region, a polymer sequence is passed through the tip region, and a change in an electrical current signal is measured as each polymer component passes through the tip region. Each of the measured changes in electrical current signals is compared with a database of reference electrical change signals, with each reference signal corresponding to an identified polymer component, to identify the unknown polymer component with a reference polymer component. The nanopore preferably has a pore inner diameter of no more than about 40 nm and is prepared by heating and pulling a very small section of a glass tubing.

  17. An Unusual Case of Rapidly Progressive Hyperbilirubinemia

    Directory of Open Access Journals (Sweden)

    Kimberly M. Thornton

    2013-01-01

    Full Text Available We present an unusual case of hyperbilirubinemia with rapid early progression leading to bilirubin encephalopathy in a term neonate. Despite early recognition and intervention, the total serum bilirubin reached a maximum level of 39 mg/dL at 32 hours of life. Prior to an emergent exchange transfusion, the patient’s diagnostic evaluation was significant for Coombs-negative microangiopathic hemolytic anemia and thrombocytopenia. Further testing revealed a deficiency of ADAMTS13 protein, or von Willebrand factor-cleaving protease, a finding diagnostic of congenital thrombotic thrombocytopenic purpura, or Upshaw-Schulman syndrome. This rare disease is often misdiagnosed, especially in the newborn period.

  18. Plant MAPK cascades: Just rapid signaling modules?

    KAUST Repository

    Boudsocq, Marie

    2015-08-27

    © 2015 Taylor & Francis Group, LLC. Abscisic acid (ABA) is a major phytohormone mediating important stress-related processes. We recently unveiled an ABA-activated MAPK signaling module constituted of MAP3K17/18-MKK3-MPK1/2/7/14. Unlike classical rapid MAPK activation, we showed that the activation of the new MAPK module is delayed and relies on the MAP3K protein synthesis. In this addendum, we discuss the role of this original and unexpected activation mechanism of MAPK cascades which suggests that MAPKs can regulate both early and longterm plant stress responses.

  19. A 1H NMR-based metabolomics approach to evaluate the geographical authenticity of herbal medicine and its application in building a model effectively assessing the mixing proportion of intentional admixtures: A case study of Panax ginseng: Metabolomics for the authenticity of herbal medicine.

    Science.gov (United States)

    Nguyen, Huy Truong; Lee, Dong-Kyu; Choi, Young-Geun; Min, Jung-Eun; Yoon, Sang Jun; Yu, Yun-Hyun; Lim, Johan; Lee, Jeongmi; Kwon, Sung Won; Park, Jeong Hill

    2016-05-30

    Ginseng, the root of Panax ginseng has long been the subject of adulteration, especially regarding its origins. Here, 60 ginseng samples from Korea and China initially displayed similar genetic makeup when investigated by DNA-based technique with 23 chloroplast intergenic space regions. Hence, (1)H NMR-based metabolomics with orthogonal projections on the latent structure-discrimination analysis (OPLS-DA) were applied and successfully distinguished between samples from two countries using seven primary metabolites as discrimination markers. Furthermore, to recreate adulteration in reality, 21 mixed samples of numerous Korea/China ratios were tested with the newly built OPLS-DA model. The results showed satisfactory separation according to the proportion of mixing. Finally, a procedure for assessing mixing proportion of intentionally blended samples that achieved good predictability (adjusted R(2)=0.8343) was constructed, thus verifying its promising application to quality control of herbal foods by pointing out the possible mixing ratio of falsified samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Fluorogen-activating proteins: beyond classical fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Shengnan Xu

    2018-05-01

    Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

  1. Rapid Modulation of Aromatase Activity in the Vertebrate Brain

    Directory of Open Access Journals (Sweden)

    Thierry D. Charlier

    2013-01-01

    Full Text Available Numerous steroid hormones, including 17β-estradiol (E2, activate rapid and transient cellular, physiological, and behavioral changes in addition to their well-described genomic effects. Aromatase is the key-limiting enzyme in the production of estrogens, and the rapid modulation of this enzymatic activity could produce rapid changes in local E2 concentrations. The mechanisms that might mediate such rapid enzymatic changes are not fully understood but are currently under intense scrutiny. Recent studies in our laboratory indicate that brain aromatase activity is rapidly inhibited by an increase in intracellular calcium concentration resulting from potassium-induced depolarization or from the activation of glutamatergic receptors. Phosphorylating conditions also reduce aromatase activity within minutes, and this inhibition is blocked by the addition of multiple protein kinase inhibitors. This rapid modulation of aromatase activity by phosphorylating conditions is a general mechanism observed in different cell types and tissues derived from a variety of species, including human aromatase expressed in various cell lines. Phosphorylation processes affect aromatase itself and do not involve changes in aromatase protein concentration. The control of aromatase activity by multiple kinases suggests that several amino acids must be concomitantly phosphorylated to modify enzymatic activity but site-directed mutagenesis of several amino acids alone or in combination has not to date revealed the identity of the targeted residue(s. Altogether, the phosphorylation processes affecting aromatase activity provide a new general mechanism by which the concentration of estrogens can be rapidly altered in the brain.

  2. Radioactive Lysine in Protein Metabolism Studies

    Science.gov (United States)

    Miller, L. L.; Bale, W. F.; Yuile, C. L.; Masters, R. E.; Tishkoff, G. H.; Whipple,, G. H.

    1950-01-09

    Studies of incorporation of DL-lysine in various body proteins of the dog; the time course of labeled blood proteins; and apparent rate of disappearance of labeled plasma proteins for comparison of behavior of the plasma albumin and globulin fractions; shows more rapid turn over of globulin fraction.

  3. Rapid prototyping: een veelbelovende methode

    NARCIS (Netherlands)

    Haverman, T.M.; Karagozoglu, K.H.; Prins, H.; Schulten, E.A.J.M.; Forouzanfar, T.

    2013-01-01

    Rapid prototyping is a method which makes it possible to produce a three-dimensional model based on two-dimensional imaging. Various rapid prototyping methods are available for modelling, such as stereolithography, selective laser sintering, direct laser metal sintering, two-photon polymerization,

  4. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  5. Comparison of Rapid Diagnostic Tests and Microscopy for Malaria ...

    African Journals Online (AJOL)

    Presumptive treatment of malaria results in significant overuse of antimalarials. This study compared the diagnostic accuracy of Histidine Rich Protein II and plasmodium lactate dehydrogenase (pLDH)-based Rapid Kits( RDTs)and using expert microscopy as the gold standard for the detection of falciparum and ...

  6. Extraction of alkaloids for NMR-based profiling

    DEFF Research Database (Denmark)

    Yilmaz, Ali; Nyberg, Nils; Jaroszewski, Jerzy W.

    2012-01-01

    A museum collection of Cinchona cortex samples (n = 117), from the period 1850–1950, were extracted with a mixture of chloroform-d1, methanol-d4, water-d2, and perchloric acid in the ratios 5:5:1:1. The extracts were directly analyzed using 1H NMR spectroscopy (600 MHz) and the spectra evaluated...

  7. NMR-based stable isotope resolved metabolomics in systems biochemistry

    International Nuclear Information System (INIS)

    Fan, Teresa W-M.; Lane, Andrew N.

    2011-01-01

    An important goal of metabolomics is to characterize the changes in metabolic networks in cells or various tissues of an organism in response to external perturbations or pathologies. The profiling of metabolites and their steady state concentrations does not directly provide information regarding the architecture and fluxes through metabolic networks. This requires tracer approaches. NMR is especially powerful as it can be used not only to identify and quantify metabolites in an unfractionated mixture such as biofluids or crude cell/tissue extracts, but also determine the positional isotopomer distributions of metabolites derived from a precursor enriched in stable isotopes such as 13 C and 15 N via metabolic transformations. In this article we demonstrate the application of a variety of 2-D NMR editing experiments to define the positional isotopomers of compounds present in polar and non-polar extracts of human lung cancer cells grown in either [U– 13 C]-glucose or [U– 13 C, 15 N]-glutamine as source tracers. The information provided by such experiments enabled unambiguous reconstruction of metabolic pathways, which is the foundation for further metabolic flux modeling.

  8. NMR-based metabolomic profiling of overweight adolescents

    DEFF Research Database (Denmark)

    Zheng, Hong; Yde, Christian C; Arnberg, Karina

    2014-01-01

    The plasma and urine metabolome of 192 overweight 12-15-year-old adolescents (BMI of 25.4 ± 2.3 kg/m(2)) were examined in order to elucidate gender, pubertal development measured as Tanner stage, physical activity measured as number of steps taken daily, and intra-/interindividual differences...... and the metabolome could be identified. The present study for the first time provides comprehensive information about associations between the metabolome and gender, pubertal development, and physical activity in overweight adolescents, which is an important subject group to approach in the prevention of obesity...... affecting the metabolome detected by proton NMR spectroscopy. Higher urinary excretion of citrate, creatinine, hippurate, and phenylacetylglutamine and higher plasma level of phosphatidylcholine and unsaturated lipid were found for girls compared with boys. The results suggest that gender differences...

  9. How Rapid is Rapid Prototyping? Analysis of ESPADON Programme Results

    Directory of Open Access Journals (Sweden)

    Ian D. Alston

    2003-05-01

    Full Text Available New methodologies, engineering processes, and support environments are beginning to emerge for embedded signal processing systems. The main objectives are to enable defence industry to field state-of-the-art products in less time and with lower costs, including retrofits and upgrades, based predominately on commercial off the shelf (COTS components and the model-year concept. One of the cornerstones of the new methodologies is the concept of rapid prototyping. This is the ability to rapidly and seamlessly move from functional design to the architectural design to the implementation, through automatic code generation tools, onto real-time COTS test beds. In this paper, we try to quantify the term “rapid” and provide results, the metrics, from two independent benchmarks, a radar and sonar beamforming application subset. The metrics show that the rapid prototyping process may be sixteen times faster than a conventional process.

  10. A new rapid method for isolating nucleoli.

    Science.gov (United States)

    Li, Zhou Fang; Lam, Yun Wah

    2015-01-01

    The nucleolus was one of the first subcellular organelles to be isolated from the cell. The advent of modern proteomic techniques has resulted in the identification of thousands of proteins in this organelle, and live cell imaging technology has allowed the study of the dynamics of these proteins. However, the limitations of current nucleolar isolation methods hinder the further exploration of this structure. In particular, these methods require the use of a large number of cells and tedious procedures. In this chapter we describe a new and improved nucleolar isolation method for cultured adherent cells. In this method cells are snap-frozen before direct sonication and centrifugation onto a sucrose cushion. The nucleoli can be obtained within a time as short as 20 min, and the high yield allows the use of less starting material. As a result, this method can capture rapid biochemical changes in nucleoli by freezing the cells at a precise time, hence faithfully reflecting the protein composition of nucleoli at the specified time point. This protocol will be useful for proteomic studies of dynamic events in the nucleolus and for better understanding of the biology of mammalian cells.

  11. Rapid deployment intrusion detection system

    International Nuclear Information System (INIS)

    Graham, R.H.

    1997-01-01

    A rapidly deployable security system is one that provides intrusion detection, assessment, communications, and annunciation capabilities; is easy to install and configure; can be rapidly deployed, and is reusable. A rapidly deployable intrusion detection system (RADIDS) has many potential applications within the DOE Complex: back-up protection for failed zones in a perimeter intrusion detection and assessment system, intrusion detection and assessment capabilities in temporary locations, protection of assets during Complex reconfiguration, and protection in hazardous locations, protection of assets during Complex reconfiguration, and protection in hazardous locations. Many DOE user-need documents have indicated an interest in a rapidly deployable intrusion detection system. The purpose of the RADIDS project is to design, develop, and implement such a system. 2 figs

  12. Rapid Continuous Multimaterial Extrusion Bioprinting

    NARCIS (Netherlands)

    Liu, Wanjun; Zhang, Yu Shrike; Heinrich, Marcel A.; De Ferrari, F; Jang, HL; Bakht, SM; Alvarez, MM; Yang, J; Li, YC; Trujillo-de Stantiago, G; Miri, AK; Zhu, K; Khoshakhlagh, P; Prakash, G; Cheng, H; Guan, X; Zhong, Z; Ju, J; Zhu, GH; Jin, X; Ryon Shin, Su; Dokmeci, M.R.; Khademhosseini, Ali

    The development of a multimaterial extrusion bioprinting platform is reported. This platform is capable of depositing multiple coded bioinks in a continuous manner with fast and smooth switching among different reservoirs for rapid fabrication of complex constructs, through digitally controlled

  13. Colour reconnections and rapidity gaps

    International Nuclear Information System (INIS)

    Loennblad, Leif

    1996-01-01

    I argue that the success of recently proposed models describing events with large rapidity gaps in DIS at HERA in terms of non-perturbative colour exchange is heavily reliant on suppression of perturbative gluon emission in the proton direction. There is little or no physical motivation for such suppression and I show that a model without this suppression cannot describe the rapidity gap events at HERA. (author)

  14. Dilepton distributions at backward rapidities

    International Nuclear Information System (INIS)

    Betemps, M. A.; Ducati, M. B. Gay; Oliveira, E. G. de

    2006-01-01

    The dilepton production at backward rapidities in pAu and pp collisions at RHIC and LHC energies is investigated in the dipole approach. The results are shown through the nuclear modification ratio R pA considering transverse momentum and rapidity spectra. The dilepton modification ratio presents interesting behavior at the backward rapidities when compared with the already known forward ones, since it is related with the large x kinematical region that is being probed. The rapidity dependence of the nuclear modification ratio in the dilepton production is strongly dependent on the Bjorken x behavior of the nuclear structure function ratio R F 2 =F 2 A /F 2 p . The R pA transverse momentum dependence at backward rapidities is modified due to the large x nuclear effects: at RHIC energies, for instance, the ratio R pA is reduced as p T increases, presenting an opposite behavior when compared with the forward one. It implies that the dilepton production at backward rapidities should carry information of the nuclear effects at large Bjorken x, as well as that it is useful to investigate the p T dependence of the observables in this kinematical regime

  15. Rapid

    Directory of Open Access Journals (Sweden)

    Nahla M. Wassim

    2013-01-01

    Full Text Available Members of Aedes caspius mosquitoes are incriminated to be a potential reservoir of “Rift Valley Fever Virus” (RVF during interepizootic periods in Egypt. Ae. caspius contains two distinct forms which are morphologically indistinguishable but differ in physiology and behavior; Ae. caspius form (a requires a blood meal for each egg batch(anautogeny, is unable to mate in confined spaces(eurygamous. The second form (b lays egg batch without blood meal (autogenous and can mate in confined spaces (stenogamous. In this work, we collected the autogenous and anautogenous forms of Ae. caspius from two different breeding habitats in the Qalyubia Governorate. Analysis of the Drosophila ace-Orthologous acetylecholinesterase gene revealed that a single polymorphic region characterized each species. Based on this region, specific primers were used to amplify the entire section of intron II, sections of Exon 2 and Exon 3 of ace-2 gene for differentiating the complex species of mosquitoes. The amplicons of anautogenous form sized 441 pb and increase 116 bp than autogenous form of Ae. caspius. High rates of point mutations were addressed; deletion/insertion events are 120 bases. The transversion mutations were 44 bases and were relatively close to the transtion mutations 43 base. The genetic distance was 0.01 between the two forms.

  16. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  17. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  18. Whey Protein

    Science.gov (United States)

    ... reliable information about the safety of taking whey protein if you are pregnant or breast feeding. Stay on the safe side and avoid use. Milk allergy: If you are allergic to cow's milk, avoid using whey protein.

  19. Differential Precipitation and Solubilization of Proteins.

    Science.gov (United States)

    Ryan, Barry J; Kinsella, Gemma K

    2017-01-01

    Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilized so as to reform functional, correctly folded proteins. Here, appropriate bioinformatics tools to aid in understanding a protein's propensity to aggregate and solubilize are explored as a backdrop for a typical protein extraction, precipitation, and selective resolubilization procedure, based on a recombinantly expressed protein.

  20. Hard diffraction and rapidity gaps

    International Nuclear Information System (INIS)

    Brandt, A.

    1995-09-01

    The field of hard diffraction, which studies events with a rapidity gap and a hard scattering, has expanded dramatically recently. A review of new results from CDF, D OE, H1 and ZEUS will be given. These results include diffractive jet production, deep-inelastic scattering in large rapidity gap events, rapidity gaps between high transverse energy jets, and a search for diffractive W-boson production. The combination of these results gives new insight into the exchanged object, believed to be the pomeron. The results axe consistent with factorization and with a hard pomeron that contains both quarks and gluons. There is also evidence for the exchange of a strongly interacting color singlet in high momentum transfer (36 2 ) events

  1. On the rapid melt quenching

    International Nuclear Information System (INIS)

    Usatyuk, I.I.; Novokhatskij, I.A.; Kaverin, Yu.F.

    1994-01-01

    Specific features of instrumentation of traditionally employed method of melt spinning (rapid quenching), its disadvantages being discussed, were analyzed. The necessity of the method upgrading as applied to the problems of studying fine structure of molten metals and glasses was substantiated. The principle flowsheet of experimental facility for extremely rapid quenching of the melts of metals is described, specificity of its original functional units being considered. The sequence and character of all the principal stages of the method developed were discussed. 18 refs.; 3 figs

  2. Furnace for rapid thermal processing

    NARCIS (Netherlands)

    Roozeboom, F.; Duine, P.A.; Sluis, P. van der

    2001-01-01

    A Method (1) for Rapid Thermal Processing of a wafer (7), wherein the wafer (7) is heated by lamps (9), and the heat radiation is reflected by an optical switching device (15,17) which is in the reflecting state during the heating stage. During the cooling stage of the wafer (7), the heat is

  3. Rapid thermal processing of semiconductors

    CERN Document Server

    Borisenko, Victor E

    1997-01-01

    Rapid thermal processing has contributed to the development of single wafer cluster processing tools and other innovations in integrated circuit manufacturing environments Borisenko and Hesketh review theoretical and experimental progress in the field, discussing a wide range of materials, processes, and conditions They thoroughly cover the work of international investigators in the field

  4. Rapid general microdetermination of fluorine

    NARCIS (Netherlands)

    Leuven, H.C.E. van; Rotscheid, G.J.; Buis, W.J.

    1979-01-01

    A rapid micromethod for the determination of fluorine in a wide variety of materials has been developed. The method is based on the liberation of the fluorine (as HF) from the sample by means of pyrohydrolysis with steam at 1120?? C, The amount of fluoride in the condensate is subsequently measured

  5. Rapid Prototyping Enters Mainstream Manufacturing.

    Science.gov (United States)

    Winek, Gary

    1996-01-01

    Explains rapid prototyping, a process that uses computer-assisted design files to create a three-dimensional object automatically, speeding the industrial design process. Five commercially available systems and two emerging types--the 3-D printing process and repetitive masking and depositing--are described. (SK)

  6. Portable Diagnostics and Rapid Germination

    Energy Technology Data Exchange (ETDEWEB)

    Dunn, Zachary Spencer [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2016-12-01

    In the Bioenergy and Defense Department of Sandia National Laboratories, characterization of the BaDx (Bacillus anthracis diagnostic cartridge) was performed and rapid germination chemistry was investigated. BaDx was tested with complex sample matrixes inoculated with Bacillus anthracis, and the trials proved that BaDx will detect Bacillus anthracis in a variety of the medium, such as dirt, serum, blood, milk, and horse fluids. The dimensions of the device were altered to accommodate an E. coli or Listeria lateral flow immunoassay, and using a laser printer, BaDx devices were manufactured to identify E. coli and Listeria. Initial testing with E. coli versions of BaDx indicate that the device will be viable as a portable diagnostic cartridge. The device would be more effective with faster bacteria germination; hence studies were performed the use of rapid germination chemistry. Trials with calcium dipicolinic acid displayed increased cell germination, as shown by control studies using a microplate reader. Upon lyophilization the rapid germination chemistry failed to change growth patterns, indicating that the calcium dipicolinic acid was not solubilized under the conditions tested. Although incompatible with the portable diagnostic device, the experiments proved that the rapid germination chemistry was effective in increasing cell germination.

  7. Text Mining for Protein Docking.

    Directory of Open Access Journals (Sweden)

    Varsha D Badal

    2015-12-01

    Full Text Available The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking. Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu. The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound

  8. Rapid-scan EPR imaging.

    Science.gov (United States)

    Eaton, Sandra S; Shi, Yilin; Woodcock, Lukas; Buchanan, Laura A; McPeak, Joseph; Quine, Richard W; Rinard, George A; Epel, Boris; Halpern, Howard J; Eaton, Gareth R

    2017-07-01

    In rapid-scan EPR the magnetic field or frequency is repeatedly scanned through the spectrum at rates that are much faster than in conventional continuous wave EPR. The signal is directly-detected with a mixer at the source frequency. Rapid-scan EPR is particularly advantageous when the scan rate through resonance is fast relative to electron spin relaxation rates. In such scans, there may be oscillations on the trailing edge of the spectrum. These oscillations can be removed by mathematical deconvolution to recover the slow-scan absorption spectrum. In cases of inhomogeneous broadening, the oscillations may interfere destructively to the extent that they are not visible. The deconvolution can be used even when it is not required, so spectra can be obtained in which some portions of the spectrum are in the rapid-scan regime and some are not. The technology developed for rapid-scan EPR can be applied generally so long as spectra are obtained in the linear response region. The detection of the full spectrum in each scan, the ability to use higher microwave power without saturation, and the noise filtering inherent in coherent averaging results in substantial improvement in signal-to-noise relative to conventional continuous wave spectroscopy, which is particularly advantageous for low-frequency EPR imaging. This overview describes the principles of rapid-scan EPR and the hardware used to generate the spectra. Examples are provided of its application to imaging of nitroxide radicals, diradicals, and spin-trapped radicals at a Larmor frequency of ca. 250MHz. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Non-Protein Coding RNAs

    CERN Document Server

    Walter, Nils G; Batey, Robert T

    2009-01-01

    This book assembles chapters from experts in the Biophysics of RNA to provide a broadly accessible snapshot of the current status of this rapidly expanding field. The 2006 Nobel Prize in Physiology or Medicine was awarded to the discoverers of RNA interference, highlighting just one example of a large number of non-protein coding RNAs. Because non-protein coding RNAs outnumber protein coding genes in mammals and other higher eukaryotes, it is now thought that the complexity of organisms is correlated with the fraction of their genome that encodes non-protein coding RNAs. Essential biological processes as diverse as cell differentiation, suppression of infecting viruses and parasitic transposons, higher-level organization of eukaryotic chromosomes, and gene expression itself are found to largely be directed by non-protein coding RNAs. The biophysical study of these RNAs employs X-ray crystallography, NMR, ensemble and single molecule fluorescence spectroscopy, optical tweezers, cryo-electron microscopy, and ot...

  10. Rapid screening of monoclonal antibodies: new 'microstick' radioimmunoassay

    International Nuclear Information System (INIS)

    Scheinberg, D.A.; Strand, M.; Wilsnack, R.

    1983-01-01

    A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antigen specific antibody, and immunoglobulin isotypes could be measured under sterile conditions directly in the original fusion mixture wells and much greater rapidity than with the microtiter plate assay. (Auth.)

  11. Rapid shift in substrate utilization driven by hypothalamic Agrp neurons

    OpenAIRE

    Cavalcanti-De-Albuquerque, Joao; Dietrich, Marcelo; Bober, Jeremy; Zimmer, Marcelo

    2016-01-01

    Agrp neurons drive feeding. To what extend these neurons participate in the regulation of other homeostatic processes is not well understood. We investigated the role of Agrp neurons in substrate utilization in mice. Activation of Agrp neurons was sufficient to rapidly increase RER and carbohydrate utilization, while decreasing fat utilization. These metabolic changes were linearly correlated with carbohydrates ingested, but not protein or fat ingestion. However, even in the absence of ingest...

  12. Rapid and sustained cost management

    International Nuclear Information System (INIS)

    Hanson, D.

    2009-01-01

    Accenture helps clients develop comprehensive, process-driven strategies for rapid and sustained cost management that leverage deep insights and analytics. This approach enables companies to gain operating cost advantages by rationalizing, simplifying and automating current operating capabilities. It drives structural cost advantages by optimizing business mix, capital structure, organizational structure and geographic presence. This paper discussed how successful companies achieve high performance during times of economic turmoil. It also discussed the value of the winner's strategy in terms of rapid and sustained cost management (RSCM). It discussed how Accenture operates and its leveraged capabilities, improved efficiency, margins and cash flow while maintaining customer service levels. Building structural advantage and the Accenture difference were also discussed. It was concluded that RSCM is one vital way that Accenture can help companies achieve success. 4 figs

  13. Rapid mask prototyping for microfluidics.

    Science.gov (United States)

    Maisonneuve, B G C; Honegger, T; Cordeiro, J; Lecarme, O; Thiry, T; Fuard, D; Berton, K; Picard, E; Zelsmann, M; Peyrade, D

    2016-03-01

    With the rise of microfluidics for the past decade, there has come an ever more pressing need for a low-cost and rapid prototyping technology, especially for research and education purposes. In this article, we report a rapid prototyping process of chromed masks for various microfluidic applications. The process takes place out of a clean room, uses a commercially available video-projector, and can be completed in less than half an hour. We quantify the ranges of fields of view and of resolutions accessible through this video-projection system and report the fabrication of critical microfluidic components (junctions, straight channels, and curved channels). To exemplify the process, three common devices are produced using this method: a droplet generation device, a gradient generation device, and a neuro-engineering oriented device. The neuro-engineering oriented device is a compartmentalized microfluidic chip, and therefore, required the production and the precise alignment of two different masks.

  14. Rapidity correlations test stochastic hydrodynamics

    International Nuclear Information System (INIS)

    Zin, C; Gavin, S; Moschelli, G

    2017-01-01

    We show that measurements of the rapidity dependence of transverse momentum correlations can be used to determine the characteristic time τ π that dictates the rate of isotropization of the stress energy tensor, as well as the shear viscosity ν = η/sT . We formulate methods for computing these correlations using second order dissipative hydrodynamics with noise. Current data are consistent with τ π /ν ∼ 10 but targeted measurements can improve this precision. (paper)

  15. Rapid duodenal and jejunal intubation

    International Nuclear Information System (INIS)

    Nolan, D.J.

    1979-01-01

    A size 12 French radiopaque catheter, 135 cm long, suitable for rapid duodenal and jejunal intubation, is described. Its size and flexibility enable it to be passed with ease through the nose, stomach and duodenum. A guide wire is used to act as a stiffener as the catheter is passed through the stomach. The catheter is suitable for infusing barium directly into the small intestine and for performing hypotonic duodenography. The technique for duodenal and jejunal intubation is discussed. (author)

  16. Rapid synthesis of beta zeolites

    Science.gov (United States)

    Fan, Wei; Chang, Chun -Chih; Dornath, Paul; Wang, Zhuopeng

    2015-08-18

    The invention provides methods for rapidly synthesizing heteroatom containing zeolites including Sn-Beta, Si-Beta, Ti-Beta, Zr-Beta and Fe-Beta. The methods for synthesizing heteroatom zeolites include using well-crystalline zeolite crystals as seeds and using a fluoride-free, caustic medium in a seeded dry-gel conversion method. The Beta zeolite catalysts made by the methods of the invention catalyze both isomerization and dehydration reactions.

  17. Rapid reconnection of flux lines

    International Nuclear Information System (INIS)

    Samain, A.

    1982-01-01

    The rapid reconnection of flux lines in an incompressible fluid through a singular layer of the current density is discussed. It is shown that the liberated magnetic energy must partially appear in the form of plasma kinetic energy. A laminar structure of the flow is possible, but Alfven velocity must be achieved in eddies of growing size at the ends of the layer. The gross structure of the flow and the magnetic configuration may be obtained from variational principles. (author)

  18. Single wafer rapid thermal multiprocessing

    International Nuclear Information System (INIS)

    Saraswat, K.C.; Moslehi, M.M.; Grossman, D.D.; Wood, S.; Wright, P.; Booth, L.

    1989-01-01

    Future success in microelectronics will demand rapid innovation, rapid product introduction and ability to react to a change in technological and business climate quickly. These technological advances in integrated electronics will require development of flexible manufacturing technology for VLSI systems. However, the current approach of establishing factories for mass manufacturing of chips at a cost of more than 200 million dollars is detrimental to flexible manufacturing. The authors propose concepts of a micro factory which may be characterized by more economical small scale production, higher flexibility to accommodate many products on several processes, and faster turnaround and learning. In-situ multiprocessing equipment where several process steps can be done in sequence may be a key ingredient in this approach. For this environment to be flexible, the equipment must have ability to change processing environment, requiring extensive in-situ measurements and real time control. This paper describes the development of a novel single wafer rapid thermal multiprocessing (RTM) reactor for next generation flexible VLSI manufacturing. This reactor will combine lamp heating, remote microwave plasma and photo processing in a single cold-wall chamber, with applications for multilayer in-situ growth and deposition of dielectrics, semiconductors and metals

  19. Rapid Sampling from Sealed Containers

    International Nuclear Information System (INIS)

    Johnston, R.G.; Garcia, A.R.E.; Martinez, R.K.; Baca, E.T.

    1999-01-01

    The authors have developed several different types of tools for sampling from sealed containers. These tools allow the user to rapidly drill into a closed container, extract a sample of its contents (gas, liquid, or free-flowing powder), and permanently reseal the point of entry. This is accomplished without exposing the user or the environment to the container contents, even while drilling. The entire process is completed in less than 15 seconds for a 55 gallon drum. Almost any kind of container can be sampled (regardless of the materials) with wall thicknesses up to 1.3 cm and internal pressures up to 8 atm. Samples can be taken from the top, sides, or bottom of a container. The sampling tools are inexpensive, small, and easy to use. They work with any battery-powered hand drill. This allows considerable safety, speed, flexibility, and maneuverability. The tools also permit the user to rapidly attach plumbing, a pressure relief valve, alarms, or other instrumentation to a container. Possible applications include drum venting, liquid transfer, container flushing, waste characterization, monitoring, sampling for archival or quality control purposes, emergency sampling by rapid response teams, counter-terrorism, non-proliferation and treaty verification, and use by law enforcement personnel during drug or environmental raids

  20. Rapid glutamate receptor 2 trafficking during retinal degeneration

    Directory of Open Access Journals (Sweden)

    Lin Yanhua

    2012-02-01

    Full Text Available Abstract Background Retinal degenerations, such as age-related macular degeneration (AMD and retinitis pigmentosa (RP, are characterized by photoreceptor loss and anomalous remodeling of the surviving retina that corrupts visual processing and poses a barrier to late-stage therapeutic interventions in particular. However, the molecular events associated with retinal remodeling remain largely unknown. Given our prior evidence of ionotropic glutamate receptor (iGluR reprogramming in retinal degenerations, we hypothesized that the edited glutamate receptor 2 (GluR2 subunit and its trafficking may be modulated in retinal degenerations. Results Adult albino Balb/C mice were exposed to intense light for 24 h to induce light-induced retinal degeneration (LIRD. We found that prior to the onset of photoreceptor loss, protein levels of GluR2 and related trafficking proteins, including glutamate receptor-interacting protein 1 (GRIP1 and postsynaptic density protein 95 (PSD-95, were rapidly increased. LIRD triggered neuritogenesis in photoreceptor survival regions, where GluR2 and its trafficking proteins were expressed in the anomalous dendrites. Immunoprecipitation analysis showed interaction between KIF3A and GRIP1 as well as PSD-95, suggesting that KIF3A may mediate transport of GluR2 and its trafficking proteins to the novel dendrites. However, in areas of photoreceptor loss, GluR2 along with its trafficking proteins nearly vanished in retracted retinal neurites. Conclusions All together, LIRD rapidly triggers GluR2 plasticity, which is a potential mechanism behind functionally phenotypic revisions of retinal neurons and neuritogenesis during retinal degenerations.

  1. Protein Adsorption in Three Dimensions

    Science.gov (United States)

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  2. Linearization of the Bradford Protein Assay

    OpenAIRE

    Ernst, Orna; Zor, Tsaffrir

    2010-01-01

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

  3. Insect Cells as Hosts for Recombinat Proteins

    OpenAIRE

    Murwani, Retno

    1997-01-01

    Since the development of recombinant baculovirus expression system, insect cell culture has rapidly gain popularity as the method of choice for production of a variety of biologically active proteins. Up to date tens of recombinant protein have been produced by this method commercially or non-commercially and have been widely used for research. This review describes the basic concept of baculovirus expression vector and the use of insect cells as host for recombinant proteins. Examples of the...

  4. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  5. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  6. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  7. Rapid Adaptation in Digital Transformation

    DEFF Research Database (Denmark)

    Hansen, Anne Mette; Kræmmergaard, Pernille; Mathiassen, Lars

    2011-01-01

    landscape. In this article, we share insights gained from two public sector organizations in which IS and business leaders used the Participatory Process Model (PPM) designed by the authors to share their assumptions about IS leadership, challenge existing IT strategies and collaboration patterns and adapt...... the organization’s digitization approach. We demonstrate in detail how the leaders within these two organizations were engaged and offer recommendations for how other organizations can use the PPM to rapidly adapt their approaches to digital transformation through more effective IS leadership roles....

  8. Rapid thermal processing by stamping

    Science.gov (United States)

    Stradins, Pauls; Wang, Qi

    2013-03-05

    A rapid thermal processing device and methods are provided for thermal processing of samples such as semiconductor wafers. The device has components including a stamp (35) having a stamping surface and a heater or cooler (40) to bring it to a selected processing temperature, a sample holder (20) for holding a sample (10) in position for intimate contact with the stamping surface; and positioning components (25) for moving the stamping surface and the stamp (35) in and away from intimate, substantially non-pressured contact. Methods for using and making such devices are also provided. These devices and methods allow inexpensive, efficient, easily controllable thermal processing.

  9. Rapidly Adaptable Instrumentation Tester (RAIT)

    International Nuclear Information System (INIS)

    Vargo, Timothy D.

    1999-01-01

    Emerging technologies in the field of ''Test ampersand Measurement'' have recently enabled the development of the Rapidly Adaptable Instrumentation Tester (RAIT). Based on software developed with LabVIEW, the RAIT design enables quick reconfiguration to test and calibrate a wide variety of telemetry systems. The consequences of inadequate testing could be devastating if a telemetry system were to fail during an expensive flight mission. Supporting both open-bench testing as well as automated test sequences, the RAIT has significantly lowered total time required to test and calibrate a system. This has resulted in an overall lower per unit testing cost than has been achievable in the past

  10. Manipulating the glycosylation pathway in bacterial and lower eukaryotes for production of therapeutic proteins

    DEFF Research Database (Denmark)

    Anyaogu, Diana Chinyere; Mortensen, Uffe Hasbro

    2015-01-01

    The medical use of pharmaceutical proteins is rapidly increasing and cheap, fast and efficient production is therefore attractive. Microbial production hosts are promising candidates for development and production of pharmaceutical proteins. However, as most therapeutic proteins are secreted...... to produce proteins with humanlike glycan structures setting the stage for production of pharmaceutical proteins in bacteria, yeasts and algae....

  11. Chemical cross-linking and mass spectrometry for protein structural modeling

    NARCIS (Netherlands)

    Back, Jaap Willem; de Jong, Luitzen; Muijsers, Anton O.; de Koster, Chris G.

    2003-01-01

    The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described,

  12. Wyoming Basin Rapid Ecoregional Assessment

    Science.gov (United States)

    Carr, Natasha B.; Melcher, Cynthia P.

    2015-08-28

    The Wyoming Basin Rapid Ecoregional Assessment was conducted in partnership with the Bureau of Land Management (BLM). The overall goals of the BLM Rapid Ecoregional Assessments (REAs) are to identify important ecosystems and wildlife habitats at broad spatial scales; identify where these resources are at risk from Change Agents, including development, wildfire, invasive species, disease and climate change; quantify cumulative effects of anthropogenic stressors; and assess current levels of risk to ecological resources across a range of spatial scales and jurisdictional boundaries by assessing all lands within an ecoregion. There are several components of the REAs. Management Questions, developed by the BLM and stakeholders for the ecoregion, identify the regionally significant information needed for addressing land-management responsibilities. Conservation Elements represent regionally significant species and ecological communities that are of management concern. Change Agents that currently affect or are likely to affect the condition of species and communities in the future are identified and assessed. REAs also identify areas that have high conservation potential that are referred to as “large intact areas.” At the ecoregion level, the ecological value of large intact areas is based on the assumption that because these areas have not been greatly altered by human activities (such as development), they are more likely to contain a variety of plant and animal communities and to be resilient and resistant to changes resulting from natural disturbances such as fire, insect outbreaks, and disease.

  13. Direct, rapid RNA sequence analysis

    International Nuclear Information System (INIS)

    Peattie, D.A.

    1987-01-01

    The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

  14. Rapid learning: a breakthrough agenda.

    Science.gov (United States)

    Etheredge, Lynn M

    2014-07-01

    A "rapid-learning health system" was proposed in a 2007 thematic issue of Health Affairs. The system was envisioned as one that uses evidence-based medicine to quickly determine the best possible treatments for patients. It does so by drawing on electronic health records and the power of big data to access large volumes of information from a variety of sources at high speed. The foundation for a rapid-learning health system was laid during 2007-13 by workshops, policy papers, large public investments in databases and research programs, and developing learning systems. Challenges now include implementing a new clinical research system with several hundred million patients, modernizing clinical trials and registries, devising and funding research on national priorities, and analyzing genetic and other factors that influence diseases and responses to treatment. Next steps also should aim to improve comparative effectiveness research; build on investments in health information technology to standardize handling of genetic information and support information exchange through apps and software modules; and develop new tools, data, and information for clinical decision support. Further advances will require commitment, leadership, and public-private and global collaboration. Project HOPE—The People-to-People Health Foundation, Inc.

  15. Structural Mass Spectrometry of Proteins Using Hydroxyl Radical Based Protein Footprinting

    OpenAIRE

    Wang, Liwen; Chance, Mark R.

    2011-01-01

    Structural MS is a rapidly growing field with many applications in basic research and pharmaceutical drug development. In this feature article the overall technology is described and several examples of how hydroxyl radical based footprinting MS can be used to map interfaces, evaluate protein structure, and identify ligand dependent conformational changes in proteins are described.

  16. Rapid chemical analysis of allanite

    International Nuclear Information System (INIS)

    Nishiyama, Goro; Hayashi, Hiroshi

    1981-01-01

    Rapid chemical analysis of allanite was studied by atomic absorption spectrophotometry. Powdered sample was fused with mixture of sodium carbonate anhydrous and borax (4 : 1 weight) in platinum crucible and sample solution was prepared. SiO 2 , Fe 2 O 3 , Al 2 O 3 , MnO and rare earth metals were determined by atomic absorption spectrophotometry, CaO, MgO and Ce 2 O 3 by titration, ThO 2 by colorimetry, and La 2 O 3 by flame photometry respectively. For sample solution treated with hydrofluoric acid and sulfuric acid. Na 2 O and K 2 O were determined by atomic absorption spectrophotometry, TiO 2 and P 2 O 5 by colorimetry. Chemical analyses for four samples were carried out and gave consistent results. (author)

  17. Rapid world modelling for robotics

    International Nuclear Information System (INIS)

    Littile, C.Q.; Wilson, C.W.

    1996-01-01

    The ability to use an interactive world model, whether it is for robotics simulation or most other virtual graphical environments, relies on the users ability to create an accurate world model. Typically this is a tedious process, requiring many hours to create 3-D CAD models of the surfaces within a workspace. The goal of this ongoing project is to develop usable methods to rapidly build world models of real world workspaces. This brings structure to an unstructured environment and allows graphical based robotics control to be accomplished in a reasonable time frame when traditional CAD modelling is not enough. To accomplish this, 3D range sensors are deployed to capture surface data within the workspace. This data is then transformed into surface maps, or models. A 3D world model of the workspace is built quickly and accurately, without ever having to put people in the environment

  18. On rapid rotation in stellarators

    International Nuclear Information System (INIS)

    Helander, Per

    2008-01-01

    The conditions under which rapid plasma rotation may occur in a three-dimensional magnetic field, such as that of a stellarator, are investigated. Rotation velocities comparable to the ion thermal speed are found to be attainable only in magnetic fields which are approximately isometric. In an isometric magnetic field the dependence of the magnetic field strength B on the arc length l along the field is the same for all field lines on each flux surface ψ. Only in fields where the departure from exact isometry, B=B(ψ,l), is of the order of the ion gyroradius divided by the macroscopic length scale are rotation speeds comparable to the ion thermal speed possible. Moreover, it is shown that the rotation must be in the direction of the vector ∇ψx∇B. (author)

  19. Microstructure of rapidly solidified materials

    Science.gov (United States)

    Jones, H.

    1984-07-01

    The basic features of rapidly solidified microstructures are described and differences arising from alternative processing strategies are discussed. The possibility of achieving substantial undercooling prior to solidification in processes such as quench atomization and chill block melt spinning can give rise to striking microstructural transitions even when external heat extraction is nominally Newtonian. The increased opportunity in laser and electron beam surface melting for epitaxial growth on the parent solid at an accelerating rate, however, does not exclude the formation of nonequilibrium phases since the required undercooling can be locally attained at the solidification front which is itself advancing at a sufficiently high velocity. The effects of fluid flow indicated particularly in melt spinning and surface melting are additional to the transformational and heat flow considerations that form the present basis for interpretation of such microstructural effects.

  20. Rapid iconic erasure without masking.

    Science.gov (United States)

    Tijus, Charles Albert; Reeves, Adam

    2004-01-01

    We report on the erasure of the iconic memory of an array of 12 black letters flashed on a continuously- present white field. Erasure is accomplished by replacing the 16 ms letter array (frame 1) with a blank white frame for 16 ms (frame 2). The letter array returns in frame 3, with from one to six letters missing. Report of the missing letters is accurate without the blank white frame but is impoverished with it, as if interposing the blank erases the icon. Erasure occurs without any obvious luminance masking, 'mud splashes', pattern masking (backward, forward, or metacontrast), lateral masking, or masking by object substitution. Erasure is greatly decreased if the blank is presented one frame earlier or later. We speculate that erasure is due to a rapid reset of the icon produced by an informational mis-match.

  1. Rapid onset aggressive vertebral haemangioma.

    Science.gov (United States)

    Cheung, Nicholas K; Doorenbosch, Xenia; Christie, John G

    2011-03-01

    Vertebral haemangiomas are generally benign asymptomatic vascular tumours seen commonly in the adult population. Presentations in paediatric populations are extremely rare, which can result in rapid onset of neurological symptoms. We present a highly unusual case of an aggressive paediatric vertebral haemangioma causing significant cord compression. A 13-year-old boy presented with only 2 weeks duration of progressive gait disturbance, truncal ataxia and loss of bladder control. Magnetic resonance imaging (MRI) of the spine revealed a large vascular epidural mass extending between T6 and T8 vertebral bodies. Associated displacement and compression of the spinal cord was present. A highly vascular bony lesion was found during surgery. Histopathology identified this tumour to be a vertebral haemangioma. We present an extremely unusual acute presentation of a paediatric vertebral haemangioma. This study highlights the need for early diagnosis, MRI for investigation and urgent surgical management. © Springer-Verlag 2011

  2. Customer-experienced rapid prototyping

    Science.gov (United States)

    Zhang, Lijuan; Zhang, Fu; Li, Anbo

    2008-12-01

    In order to describe accurately and comprehend quickly the perfect GIS requirements, this article will integrate the ideas of QFD (Quality Function Deployment) and UML (Unified Modeling Language), and analyze the deficiency of prototype development model, and will propose the idea of the Customer-Experienced Rapid Prototyping (CE-RP) and describe in detail the process and framework of the CE-RP, from the angle of the characteristics of Modern-GIS. The CE-RP is mainly composed of Customer Tool-Sets (CTS), Developer Tool-Sets (DTS) and Barrier-Free Semantic Interpreter (BF-SI) and performed by two roles of customer and developer. The main purpose of the CE-RP is to produce the unified and authorized requirements data models between customer and software developer.

  3. Spin-Label CW Microwave Power Saturation and Rapid Passage with Triangular Non-Adiabatic Rapid Sweep (NARS) and Adiabatic Rapid Passage (ARP) EPR Spectroscopy

    Science.gov (United States)

    Kittell, Aaron W.; Hyde, James S.

    2015-01-01

    Non-adiabatic rapid passage (NARS) electron paramagnetic resonance (EPR) spectroscopy was introduced by Kittell, A.W., Camenisch, T.G., Ratke, J.J. Sidabras, J.W., Hyde, J.S., 2011 as a general purpose technique to collect the pure absorption response. The technique has been used to improve sensitivity relative to sinusoidal magnetic field modulation, increase the range of inter-spin distances that can be measured under near physiological conditions, and enhance spectral resolution in copper (II) spectra. In the present work, the method is extended to CW microwave power saturation of spin-labeled T4 Lysozyme (T4L). As in the cited papers, rapid triangular sweep of the polarizing magnetic field was superimposed on slow sweep across the spectrum. Adiabatic rapid passage (ARP) effects were encountered in samples undergoing very slow rotational diffusion as the triangular magnetic field sweep rate was increased. The paper reports results of variation of experimental parameters at the interface of adiabatic and non-adiabatic rapid sweep conditions. Comparison of the forward (up) and reverse (down) triangular sweeps is shown to be a good indicator of the presence of rapid passage effects. Spectral turning points can be distinguished from spectral regions between turning points in two ways: differential microwave power saturation and differential passage effects. Oxygen accessibility data are shown under NARS conditions that appear similar to conventional field modulation data. However, the sensitivity is much higher, permitting, in principle, experiments at substantially lower protein concentrations. Spectral displays were obtained that appear sensitive to rotational diffusion in the range of rotational correlation times of 10−3 to 10−7 s in a manner that is analogous to saturation transfer spectroscopy. PMID:25917132

  4. Cell-free protein synthesis: applications in proteomics and biotechnology.

    Science.gov (United States)

    He, Mingyue

    2008-01-01

    Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

  5. Omalizumab facilitates rapid oral desensitization for peanut allergy.

    Science.gov (United States)

    MacGinnitie, Andrew J; Rachid, Rima; Gragg, Hana; Little, Sara V; Lakin, Paul; Cianferoni, Antonella; Heimall, Jennifer; Makhija, Melanie; Robison, Rachel; Chinthrajah, R Sharon; Lee, John; Lebovidge, Jennifer; Dominguez, Tina; Rooney, Courtney; Lewis, Megan Ott; Koss, Jennifer; Burke-Roberts, Elizabeth; Chin, Kimberly; Logvinenko, Tanya; Pongracic, Jacqueline A; Umetsu, Dale T; Spergel, Jonathan; Nadeau, Kari C; Schneider, Lynda C

    2017-03-01

    Peanut oral immunotherapy is a promising approach to peanut allergy, but reactions are frequent, and some patients cannot be desensitized. The anti-IgE medication omalizumab (Xolair; Genentech, South San Francisco, Calif) might allow more rapid peanut updosing and decrease reactions. We sought to evaluate whether omalizumab facilitated rapid peanut desensitization in highly allergic patients. Thirty-seven subjects were randomized to omalizumab (n = 29) or placebo (n = 8). After 12 weeks of treatment, subjects underwent a rapid 1-day desensitization of up to 250 mg of peanut protein, followed by weekly increases up to 2000 mg. Omalizumab was then discontinued, and subjects continued on 2000 mg of peanut protein. Subjects underwent an open challenge to 4000 mg of peanut protein 12 weeks after stopping study drug. If tolerated, subjects continued on 4000 mg of peanut protein daily. The median peanut dose tolerated on the initial desensitization day was 250 mg for omalizumab-treated subjects versus 22.5 mg for placebo-treated subject. Subsequently, 23 (79%) of 29 subjects randomized to omalizumab tolerated 2000 mg of peanut protein 6 weeks after stopping omalizumab versus 1 (12%) of 8 receiving placebo (P omalizumab versus 1 subject receiving placebo passed the 4000-mg food challenge. Overall reaction rates were not significantly lower in omalizumab-treated versus placebo-treated subjects (odds ratio, 0.57; P = .15), although omalizumab-treated subjects were exposed to much higher peanut doses. Omalizumab allows subjects with peanut allergy to be rapidly desensitized over as little as 8 weeks of peanut oral immunotherapy. In the majority of subjects, this desensitization is sustained after omalizumab is discontinued. Additional studies will help clarify which patients would benefit most from this approach. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Rapid Prototyping in Instructional Design: Creating Competencies

    Science.gov (United States)

    Fulton, Carolyn D.

    2010-01-01

    Instructional designers working in rapid prototyping environments currently do not have a list of competencies that help to identify the knowledge, skills, and attitudes (KSAs) required in these workplaces. This qualitative case study used multiple cases in an attempt to identify rapid prototyping competencies required in a rapid prototyping…

  7. Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

    OpenAIRE

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDI...

  8. Delineating slowly and rapidly evolving fractions of the Drosophila genome.

    Science.gov (United States)

    Keith, Jonathan M; Adams, Peter; Stephen, Stuart; Mattick, John S

    2008-05-01

    Evolutionary conservation is an important indicator of function and a major component of bioinformatic methods to identify non-protein-coding genes. We present a new Bayesian method for segmenting pairwise alignments of eukaryotic genomes while simultaneously classifying segments into slowly and rapidly evolving fractions. We also describe an information criterion similar to the Akaike Information Criterion (AIC) for determining the number of classes. Working with pairwise alignments enables detection of differences in conservation patterns among closely related species. We analyzed three whole-genome and three partial-genome pairwise alignments among eight Drosophila species. Three distinct classes of conservation level were detected. Sequences comprising the most slowly evolving component were consistent across a range of species pairs, and constituted approximately 62-66% of the D. melanogaster genome. Almost all (>90%) of the aligned protein-coding sequence is in this fraction, suggesting much of it (comprising the majority of the Drosophila genome, including approximately 56% of non-protein-coding sequences) is functional. The size and content of the most rapidly evolving component was species dependent, and varied from 1.6% to 4.8%. This fraction is also enriched for protein-coding sequence (while containing significant amounts of non-protein-coding sequence), suggesting it is under positive selection. We also classified segments according to conservation and GC content simultaneously. This analysis identified numerous sub-classes of those identified on the basis of conservation alone, but was nevertheless consistent with that classification. Software, data, and results available at www.maths.qut.edu.au/-keithj/. Genomic segments comprising the conservation classes available in BED format.

  9. Protection Conferred by recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System

    National Research Council Canada - National Science Library

    Santi, Luca; Giritch, Anatoli; Roy, Chad J; Marillonnet, Sylvestre; Klimyuk, Victor; Gleba, Yuri; Webb, Robert; Arntzen, Charles J; Mason, Hugh S

    2006-01-01

    ... have highlighted the need for a safe, efficacious, and rapidly producible vaccine. The use of F1 and V antigens and the derived protein fusion F1-V has shown great potential as a protective vaccine in animal studies...

  10. Molecular landscape of the interaction between the urease accessory proteins UreE and UreG.

    Science.gov (United States)

    Merloni, Anna; Dobrovolska, Olena; Zambelli, Barbara; Agostini, Federico; Bazzani, Micaela; Musiani, Francesco; Ciurli, Stefano

    2014-09-01

    Urease, the most efficient enzyme so far discovered, depends on the presence of nickel ions in the catalytic site for its activity. The transformation of inactive apo-urease into active holo-urease requires the insertion of two Ni(II) ions in the substrate binding site, a process that involves the interaction of four accessory proteins named UreD, UreF, UreG and UreE. This study, carried out using calorimetric and NMR-based structural analysis, is focused on the interaction between UreE and UreG from Sporosarcina pasteurii, a highly ureolytic bacterium. Isothermal calorimetric protein-protein titrations revealed the occurrence of a binding event between SpUreE and SpUreG, entailing two independent steps with positive cooperativity (Kd1=42±9μM; Kd2=1.7±0.3μM). This was interpreted as indicating the formation of the (UreE)2(UreG)2 hetero-oligomer upon binding of two UreG monomers onto the pre-formed UreE dimer. The molecular details of this interaction were elucidated using high-resolution NMR spectroscopy. The occurrence of SpUreE chemical shift perturbations upon addition of SpUreG was investigated and analyzed to establish the protein-protein interaction site. The latter appears to involve the Ni(II) binding site as well as mobile portions on the C-terminal and the N-terminal domains. Docking calculations based on the information obtained from NMR provided a structural basis for the protein-protein contact site. The high sequence and structural similarity within these protein classes suggests a generality of the interaction mode among homologous proteins. The implications of these results on the molecular details of the urease activation process are considered and analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Rapid charging of nickel-cadmium accumulators

    Energy Technology Data Exchange (ETDEWEB)

    Bruck, F

    1972-01-01

    Four types of charging of gas-tight Ni-Cd accumulators (a) normal; (b) accelerated; (c) rapid; and (d) ultra-rapid are described. For rapid charging, a built-in temperature sensor cuts off charging current at a prescribed point. In ultra-rapid charging, 50% charge can be attained in 3.5 min. and 25% charge within 50 sec. In the second phase of ultra-rapid charging, a surplus of oxygen is released at the positive electrode and a safety valve is provided for pressure reduction. Characteristic curves are given for various rates of charging and some data on discharge rates is also given.

  12. Metabolic behavior of cell surface biotinylated proteins

    International Nuclear Information System (INIS)

    Hare, J.F.; Lee, E.

    1989-01-01

    The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-epsilon-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N'-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from undervatized cells or from derivatized cells treated with free avidin at 4 degrees C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved greater than 100 BNHS-derivatized proteins and greater than 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18 degrees C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins

  13. Rapid typing of Coxiella burnetii.

    Directory of Open Access Journals (Sweden)

    Heidie M Hornstra

    Full Text Available Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels among multispacer sequence typing (MST loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies.

  14. Rapid contextual conditioning in autoshaping.

    Science.gov (United States)

    Balsam, P D; Schwartz, A L

    1981-10-01

    Two experiments are reported which investigate the speed of contextual conditioning in autoshaping. In both experiments, a procedure was employed in which ring doves were magazine trained in one context prior to the manipulation of background values in a second context. In Experiment 1, subjects were exposed to 4, 8, 64, 128, or 256 US-only presentations prior to autoshaping. Acquisition speed and maintained response measures were monotonically related to the number of pretraining trials. Subjects in Group 4 acquired the key-peck response fastest, and retardation was maximal within 64 pretraining trials. In Experiment 2, subjects given 20 pretraining trials were significantly more retarded than subjects given 2 pretraining trials, but only when pretraining and testing were conducted in the same context. Overall, the results of these experiments show that in autoshaping, contextual conditioning is very rapid; this demonstrates the plausibility of theoretical accounts of Pavlovian conditioning which assert that the development of the conditioned response depends on the associative values of both the CS and background stimuli.

  15. Rapid Gradient-Echo Imaging

    Science.gov (United States)

    Hargreaves, Brian

    2012-01-01

    Gradient echo sequences are widely used in magnetic resonance imaging (MRI) for numerous applications ranging from angiography to perfusion to functional MRI. Compared with spin-echo techniques, the very short repetition times of gradient-echo methods enable very rapid 2D and 3D imaging, but also lead to complicated “steady states.” Signal and contrast behavior can be described graphically and mathematically, and depends strongly on the type of spoiling: fully balanced (no spoiling), gradient spoiling, or RF-spoiling. These spoiling options trade off between high signal and pure T1 contrast while the flip angle also affects image contrast in all cases, both of which can be demonstrated theoretically and in image examples. As with spin-echo sequences, magnetization preparation can be added to gradient-echo sequences to alter image contrast. Gradient echo sequences are widely used for numerous applications such as 3D perfusion imaging, functional MRI, cardiac imaging and MR angiography. PMID:23097185

  16. Rapidly developing market regions : Brazil

    International Nuclear Information System (INIS)

    Britto, A.

    1997-01-01

    Brazil and the State of Rio Grande do Sul are experiencing a period of rapid industrial development. Global investment has been forecast to reach $240 billion over the next five to seven years. This level of development is likely to result in a sharp increase in the consumption of plastic products made from olefins and from aromatic products. Accordingly, Copesul, the centre of raw materials for the State complex, is expected to increase its production of ethane from 685 tonnes to 1.13 million tonnes after 1999. The government has established a program of incentives to stimulate investment in third generation industries. Also, the State petrochemical industry has been rendered more competitive as a result of the purchase of the latest generation equipment. The principal challenges that exist for the petrochemical industry in Brazil and for that matter, around the world, are to reduce production costs and to preserve the natural environment. Another challenge, also world-wide, is to address the issue of plastic residues and to eliminate such residues through plastic recycling programs

  17. Rapidly Progressive Corticobasal Degeneration Syndrome

    Directory of Open Access Journals (Sweden)

    Ana Herrero Valverde

    2011-08-01

    Full Text Available Introduction: Corticobasal syndrome (CBS has a heterogeneous clinical presentation with no specific pathologic substratum. Its accurate diagnosis is a challenge for neurologists; in order to establish CBS definitively, postmortem confirmation is required. Some clinical and radiological features can help to distinguish it from other neurodegenerative conditions, such as Creutzfeldt-Jakob disease (CJD. Clinical Case: A 74-year-old woman presented with language impairment, difficulty in walking and poor attentiveness that had begun 10 days before. Other symptoms, such as asymmetrical extra-pyramidal dysfunction, limb dystonia and ‘alien limb’ phenomena, were established over the next 2 months, with rapid progression. Death occurred 3 months after symptom onset. Laboratory results were normal. Initially, imaging only showed restricted diffusion with bilateral parieto-occipital gyri involvement on DWI-MRI, with unspecific EEG changes. An autopsy was performed. Brain neuropathology confirmed sporadic CJD (sCJD. Conclusions: CBS is a heterogeneous clinical syndrome whose differential diagnosis is extensive. CJD can occasionally present with clinical characteristics resembling CBS. MRI detection of abnormalities in some sequences (FLAIR, DWI, as previously reported, has high diagnostic utility for sCJD diagnosis – especially in early stages – when other tests can still appear normal. Abnormalities on DWI sequencing may not correlate with neuropathological findings, suggesting a functional basis to explain the changes found.

  18. Rapid purification of recombinant histones.

    Science.gov (United States)

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  19. Printing Proteins as Microarrays for High-Throughput Function Determination

    Science.gov (United States)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  20. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  1. Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

    Science.gov (United States)

    Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

    2006-09-06

    Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

  2. Fragment-based discovery of potent inhibitors of the anti-apoptotic MCL-1 protein.

    Science.gov (United States)

    Petros, Andrew M; Swann, Steven L; Song, Danying; Swinger, Kerren; Park, Chang; Zhang, Haichao; Wendt, Michael D; Kunzer, Aaron R; Souers, Andrew J; Sun, Chaohong

    2014-03-15

    Apoptosis is regulated by the BCL-2 family of proteins, which is comprised of both pro-death and pro-survival members. Evasion of apoptosis is a hallmark of malignant cells. One way in which cancer cells achieve this evasion is thru overexpression of the pro-survival members of the BCL-2 family. Overexpression of MCL-1, a pro-survival protein, has been shown to be a resistance factor for Navitoclax, a potent inhibitor of BCL-2 and BCL-XL. Here we describe the use of fragment screening methods and structural biology to drive the discovery of novel MCL-1 inhibitors from two distinct structural classes. Specifically, cores derived from a biphenyl sulfonamide and salicylic acid were uncovered in an NMR-based fragment screen and elaborated using high throughput analog synthesis. This culminated in the discovery of selective and potent inhibitors of MCL-1 that may serve as promising leads for medicinal chemistry optimization efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability.

    Science.gov (United States)

    Surya, Wahyu; Torres, Jaume

    2015-04-02

    Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.

  4. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  5. Protein Crystal Growth

    Science.gov (United States)

    2003-01-01

    In order to rapidly and efficiently grow crystals, tools were needed to automatically identify and analyze the growing process of protein crystals. To meet this need, Diversified Scientific, Inc. (DSI), with the support of a Small Business Innovation Research (SBIR) contract from NASA s Marshall Space Flight Center, developed CrystalScore(trademark), the first automated image acquisition, analysis, and archiving system designed specifically for the macromolecular crystal growing community. It offers automated hardware control, image and data archiving, image processing, a searchable database, and surface plotting of experimental data. CrystalScore is currently being used by numerous pharmaceutical companies and academic and nonprofit research centers. DSI, located in Birmingham, Alabama, was awarded the patent Method for acquiring, storing, and analyzing crystal images on March 4, 2003. Another DSI product made possible by Marshall SBIR funding is VaporPro(trademark), a unique, comprehensive system that allows for the automated control of vapor diffusion for crystallization experiments.

  6. RAPID TRANSFER ALIGNMENT USING FEDERATED KALMAN FILTER

    Institute of Scientific and Technical Information of China (English)

    GUDong-qing; QINYong-yuan; PENGRong; LIXin

    2005-01-01

    The dimension number of the centralized Kalman filter (CKF) for the rapid transfer alignment (TA) is as high as 21 if the aircraft wing flexure motion is considered in the rapid TA. The 21-dimensional CKF brings the calculation burden on the computer and the difficulty to meet a high filtering updating rate desired by rapid TA. The federated Kalman filter (FKF) for the rapid TA is proposed to solve the dilemma. The structure and the algorithm of the FKF, which can perform parallel computation and has less calculation burden, are designed.The wing flexure motion is modeled, and then the 12-order velocity matching local filter and the 15-order attitud ematching local filter are devised. Simulation results show that the proposed EKE for the rapid TA almost has the same performance as the CKF. Thus the calculation burden of the proposed FKF for the rapid TA is markedly decreased.

  7. Theory of hard diffraction and rapidity gaps

    International Nuclear Information System (INIS)

    Del Duca, V.

    1995-06-01

    In this talk we review the models describing the hard diffractive production of jets or more generally high-mass states in presence of rapidity gaps in hadron-hadron and lepton-hadron collisions. By rapidity gaps we mean regions on the lego plot in (pseudo)-rapidity and azimuthal angle where no hadrons are produced, between the jet(s) and an elastically scattered hadron (single hard diffraction) or between two jets (double hard diffraction). (orig.)

  8. Theory of hard diffraction and rapidity gaps

    International Nuclear Information System (INIS)

    Del Duca, V.

    1996-01-01

    In this talk we review the models describing the hard diffractive production of jets or more generally high-mass states in presence of rapidity gaps in hadron-hadron and lepton-hadron collisions. By rapidity gaps we mean regions on the lego plot in (pseudo)-rapidity and azimuthal angle where no hadrons are produced, between the jet(s) and an elastically scattered hadron (single hard diffraction) or between two jets (double hard diffraction). copyright 1996 American Institute of Physics

  9. Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome.

    Directory of Open Access Journals (Sweden)

    Jason T Popesku

    2010-08-01

    Full Text Available Dopamine (DA is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA.Female goldfish were injected (i.p. with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555 and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively.The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system.

  10. Parallel, Rapid Diffuse Optical Tomography of Breast

    National Research Council Canada - National Science Library

    Yodh, Arjun

    2001-01-01

    During the last year we have experimentally and computationally investigated rapid acquisition and analysis of informationally dense diffuse optical data sets in the parallel plate compressed breast geometry...

  11. Parallel, Rapid Diffuse Optical Tomography of Breast

    National Research Council Canada - National Science Library

    Yodh, Arjun

    2002-01-01

    During the last year we have experimentally and computationally investigated rapid acquisition and analysis of informationally dense diffuse optical data sets in the parallel plate compressed breast geometry...

  12. Numerology on pion and proton rapidity

    International Nuclear Information System (INIS)

    Gugelot, P.C.

    1987-01-01

    The pseudo-rapidity of pion jets which were measured for 50 GeV and 150 GeV incident pions and protons on carbon, copper and lead targets is analysed. The shape of the rapidity distribution for a ''fireball'' which emits particles isotropically in its center of mass is a cosh -2 y distribution. It is possible to unfold all measured distributions into three groups which correspond to a low rapidity originating from the target fragmentation, a middle group which is a function of the center of mass of the projectile and target rapidity and a fast group which is due to the projectile. 11 refs., 8 figs. (author)

  13. Rapid Automated Mission Planning System, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed innovation is an automated UAS mission planning system that will rapidly identify emergency (contingency) landing sites, manage contingency routing, and...

  14. Rapid Robot Design Validation, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Energid Technologies will create a comprehensive software infrastructure for rapid validation of robot designs. The software will support push-button validation...

  15. Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life.

    Science.gov (United States)

    Sojo, Victor; Dessimoz, Christophe; Pomiankowski, Andrew; Lane, Nick

    2016-11-01

    Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    International Nuclear Information System (INIS)

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-01-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined