Determining the solution conformational entropy of oligosaccharides by SEC with on-line viscometry detection.

PubMed

Morris, Mallory J; Striegel, André M

2014-06-15

Introduced here is a method for determining the solution conformational entropy of oligosaccharides (-ΔS) that relies on the on-line coupling of size-exclusion chromatography (SEC), an entropically-controlled separation technique, and differential viscometry (VISC). Results from this SEC/VISC method were compared, for the same injections of the same sample dissolutions and under identical solvent/temperature conditions, to results from a benchmark SEC/differential refractometry (SEC/DRI) method which has been applied successfully over the last decade to determining -ΔS of a variety of mono-, di-, and oligosaccharides. The accuracy (as compared to SEC/DRI) and precision of SEC/VISC were found to be excellent, as was the sensitivity of the viscometer in the oligomeric region. The experiments presented here contrast three sets of (1→4)-β-d-oligosaccharides, namely manno-, cello-, and N-acetylchitooligosaccharides of degree of polymerization (DP) 2 through 6. For each series, the dependence of -ΔS on DP was found to be monotonic while, between series, differences at each DP could be ascribed to either the additional degrees of freedom imparted by large, multi-atomic substituent groups, or to the presence or absence of additional intramolecular hydrogen bonds, depending on the axial versus equatorial arrangement of particular hydroxyl groups. An hypothesis is advanced to explain the unexpectedly high sensitivity of viscometric detection for low-molar-mass analytes. The method presented can be extended to the analysis of oligosaccharides other than those studied here. Published by Elsevier Ltd.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PubMed Central

Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.

PubMed

Park, Seong-Jun; Ahn, Hee-Sung; Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.

Stochastic theory of size exclusion chromatography by the characteristic function approach.

PubMed

Dondi, Francesco; Cavazzini, Alberto; Remelli, Maurizio; Felinger, Attila; Martin, Michel

2002-01-18

A general stochastic theory of size exclusion chromatography (SEC) able to account for size dependence on both pore ingress and egress processes, moving zone dispersion and pore size distribution, was developed. The relationship between stochastic-chromatographic and batch equilibrium conditions are discussed and the fundamental role of the 'ergodic' hypothesis in establishing a link between them is emphasized. SEC models are solved by means of the characteristic function method and chromatographic parameters like plate height, peak skewness and excess are derived. The peak shapes are obtained by numerical inversion of the characteristic function under the most general conditions of the exploited models. Separate size effects on pore ingress and pore egress processes are investigated and their effects on both retention selectivity and efficiency are clearly shown. The peak splitting phenomenon and peak tailing due to incomplete sample sorption near to the exclusion limit is discussed. An SEC model for columns with two types of pores is discussed and several effects on retention selectivity and efficiency coming from pore size differences and their relative abundance are singled out. The relevance of moving zone dispersion on separation is investigated. The present approach proves to be general and able to account for more complex SEC conditions such as continuous pore size distributions and mixed retention mechanism.

Size exclusion chromatography for analyses of fibroin in silk: optimization of sampling and separation conditions

NASA Astrophysics Data System (ADS)

Pawcenis, Dominika; Koperska, Monika A.; Milczarek, Jakub M.; Łojewski, Tomasz; Łojewska, Joanna

2014-02-01

A direct goal of this paper was to improve the methods of sample preparation and separation for analyses of fibroin polypeptide with the use of size exclusion chromatography (SEC). The motivation for the study arises from our interest in natural polymers included in historic textile and paper artifacts, and is a logical response to the urgent need for developing rationale-based methods for materials conservation. The first step is to develop a reliable analytical tool which would give insight into fibroin structure and its changes caused by both natural and artificial ageing. To investigate the influence of preparation conditions, two sets of artificially aged samples were prepared (with and without NaCl in sample solution) and measured by the means of SEC with multi angle laser light scattering detector. It was shown that dialysis of fibroin dissolved in LiBr solution allows removal of the salt which destroys stacks chromatographic columns and prevents reproducible analyses. Salt rich (NaCl) water solutions of fibroin improved the quality of chromatograms.

Characterization of Extracellular Vesicles by Size-Exclusion High-Performance Liquid Chromatography (HPLC).

PubMed

Huang, Tao; He, Jiang

2017-01-01

Extracellular vesicles (EVs) have recently attracted substantial attention due to the potential diagnostic and therapeutic relevance. Although a variety of techniques have been used to isolate and analyze EVs, it is still far away from satisfaction. Size-exclusion chromatography (SEC), which separates subjects by size, has been widely applied in protein purification and analysis. The purpose of this chapter is to show the applications of size-exclusion high-performance liquid chromatography (HPLC) as methods for EV characterization of impurities or contaminants of small size, and thus for quality assay for the purity of the samples of EVs.

Quantitative LC-MS of polymers: determining accurate molecular weight distributions by combined size exclusion chromatography and electrospray mass spectrometry with maximum entropy data processing.

PubMed

Gruendling, Till; Guilhaus, Michael; Barner-Kowollik, Christopher

2008-09-15

We report on the successful application of size exclusion chromatography (SEC) combined with electrospray ionization mass spectrometry (ESI-MS) and refractive index (RI) detection for the determination of accurate molecular weight distributions of synthetic polymers, corrected for chromatographic band broadening. The presented method makes use of the ability of ESI-MS to accurately depict the peak profiles and retention volumes of individual oligomers eluting from the SEC column, whereas quantitative information on the absolute concentration of oligomers is obtained from the RI-detector only. A sophisticated computational algorithm based on the maximum entropy principle is used to process the data gained by both detectors, yielding an accurate molecular weight distribution, corrected for chromatographic band broadening. Poly(methyl methacrylate) standards with molecular weights up to 10 kDa serve as model compounds. Molecular weight distributions (MWDs) obtained by the maximum entropy procedure are compared to MWDs, which were calculated by a conventional calibration of the SEC-retention time axis with peak retention data obtained from the mass spectrometer. Comparison showed that for the employed chromatographic system, distributions below 7 kDa were only weakly influenced by chromatographic band broadening. However, the maximum entropy algorithm could successfully correct the MWD of a 10 kDa standard for band broadening effects. Molecular weight averages were between 5 and 14% lower than the manufacturer stated data obtained by classical means of calibration. The presented method demonstrates a consistent approach for analyzing data obtained by coupling mass spectrometric detectors and concentration sensitive detectors to polymer liquid chromatography.

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

PubMed Central

Murphy, Patrick J. M.

2014-01-01

Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes. PMID:25254496

Quantitative characterization of gold nanoparticles by size-exclusion and hydrodynamic chromatography, coupled to inductively coupled plasma mass spectrometry and quasi-elastic light scattering.

PubMed

Pitkänen, Leena; Montoro Bustos, Antonio R; Murphy, Karen E; Winchester, Michael R; Striegel, André M

2017-08-18

The physicochemical characterization of nanoparticles (NPs) is of paramount importance for tailoring and optimizing the properties of these materials as well as for evaluating the environmental fate and impact of the NPs. Characterizing the size and chemical identity of disperse NP sample populations can be accomplished by coupling size-based separation methods to physical and chemical detection methods. Informed decisions regarding the NPs can only be made, however, if the separations themselves are quantitative, i.e., if all or most of the analyte elutes from the column within the course of the experiment. We undertake here the size-exclusion chromatographic characterization of Au NPs spanning a six-fold range in mean size. The main problem which has plagued the size-exclusion chromatography (SEC) analysis of Au NPs, namely lack of quantitation accountability due to generally poor NP recovery from the columns, is overcome by carefully matching eluent formulation with the appropriate stationary phase chemistry, and by the use of on-line inductively coupled plasma mass spectrometry (ICP-MS) detection. Here, for the first time, we demonstrate the quantitative analysis of Au NPs by SEC/ICP-MS, including the analysis of a ternary NP blend. The SEC separations are contrasted to HDC/ICP-MS (HDC: hydrodynamic chromatography) separations employing the same stationary phase chemistry. Additionally, analysis of Au NPs by HDC with on-line quasi-elastic light scattering (QELS) allowed for continuous determination of NP size across the chromatographic profiles, circumventing issues related to the shedding of fines from the SEC columns. The use of chemically homogeneous reference materials with well-defined size range allowed for better assessment of the accuracy and precision of the analyses, and for a more direct interpretation of results, than would be possible employing less rigorously characterized analytes. Published by Elsevier B.V.

CHARACTERIZATION OF HUMIC ACID SIZE FRACTIONS BY SEC AND MALS (R822832)

EPA Science Inventory

Latahco silt-loam humic acid was separated on a preparatory scale by size exclusion chromatography (SEC) on a gravity-fed Sepharose column. Four fractions from this separation were collected and further analyzed, along with whole humic acid, by high-performance SEC coupled with a...

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

PubMed

Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

2014-01-01

Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.

Size exclusion chromatography and viscometry in paper degradation studies. New Mark-Houwink coefficients for cellulose in cupri-ethylenediamine.

PubMed

Łojewski, Tomasz; Zieba, Katarzyna; Lojewska, Joanna

2010-10-15

The paper deals with the application of size exclusion chromatography (SEC) for the studies of paper degradation phenomena. The goal is to solve some of the technical problems connected with the calibration of multi-detector SEC system and to find the correlation between SEC and viscometric results of degree of polymerization of cellulose. The results gathered for the paper samples degraded by acidic air pollutant (NO(2)) are used as an example of SEC-MALLS application. From the correlation between intrinsic viscosities and absolute value of molecular masses obtained with SEC/MALLS (Multi Angle Laser Light Scattering) technique, Mark-Houwink coefficients for cellulose in cupri-ethylenediamine solution were determined. Thus obtained coefficients were used for the determination of viscometric degree of polymerization (molecular mass) of the aged samples. An excellent correlation was found between the chromatographic values of molecular masses obtained with SEC-UV/VIS detection and the viscometric ones utilizing the improved values of Mark-Houwink coefficients. Copyright © 2010 Elsevier B.V. All rights reserved.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

Pyrolysis-gas chromatography-mass spectrometry for studying N-vinyl-2-pyrrolidone-co-vinyl acetate copolymers and their dissolution behaviour.

PubMed

Chojnacka, Aleksandra; Ghaffar, Abdul; Feilden, Andrew; Treacher, Kevin; Janssen, Hans-Gerd; Schoenmakers, Peter

2011-11-14

Knowledge on the solubility behaviour and dissolution rate of speciality and commodity polymers is very important for the use of such materials in high-tech applications. We have developed methods for the quantification and characterization of dissolved copolymers of N-vinyl-2-pyrrolidone (VP) and vinyl acetate (VA) during dissolution in water. The methods are based on pyrolysis (Py) performed in a programmed-temperature vaporization injector with subsequent identification and quantification of the components in the pyrolysate using capillary gas chromatography-mass spectrometry (GC-MS). By injecting large volumes and applying cryo-focussing at the top of the column, low detection limits could be achieved. The monomer ratio was found to have the greatest effect on the dissolution rate of the PVP-co-VA copolymers. The material with the highest amount of VA (50%) dissolves significantly slower than the other grades. Size-exclusion chromatography (SEC) and Py-GC-MS were used to measure molecular weights and average chemical compositions, respectively. Combined off-line SEC//Py-GC-MS was used to determine the copolymer composition (VP/VA ratio), as a function of the molecular weight for the pure polymers. In the dissolution experiments, a constant VP/VA ratio across the dissolution curve was observed for all copolymers analysed. This suggests a random distribution of the two monomers over the molecules. Copyright © 2011 Elsevier B.V. All rights reserved.

A deadenylase assay by size-exclusion chromatography.

PubMed

He, Guang-Jun; Yan, Yong-Bin

2012-01-01

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.

Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.

PubMed

He, Feng; Phan, Duke H; Hogan, Sabine; Bailey, Robert; Becker, Gerald W; Narhi, Linda O; Razinkov, Vladimir I

2010-06-01

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles. (c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Absolute molecular weight determination of hypromellose acetate succinate by size exclusion chromatography: use of a multi angle laser light scattering detector and a mixed solvent.

PubMed

Chen, Raymond; Ilasi, Nicholas; Sekulic, Sonja S

2011-12-05

Molecular weight distribution is an important quality attribute for hypromellose acetate succinate (HPMCAS), a pharmaceutical excipient used in spray-dried dispersions. Our previous study showed that neither relative nor universal calibration method of size exclusion chromatography (SEC) works for HPMCAS polymers. We here report our effort to develop a SEC method using a mass sensitive multi angle laser light scattering detector (MALLS) to determine molecular weight distributions of HPMCAS polymers. A solvent screen study reveals that a mixed solvent (60:40%, v/v 50mM NaH(2)PO(4) with 0.1M NaNO(3) buffer: acetonitrile, pH* 8.0) is the best for HPMCAS-LF and MF sub-classes. Use of a mixed solvent creates a challenging condition for the method that uses refractive index detector. Therefore, we thoroughly evaluated the method performance and robustness. The mean weight average molecular weight of a polyethylene oxide standard has a 95% confidence interval of (28,443-28,793) g/mol vs. 28,700g/mol from the Certificate of Analysis. The relative standard deviations of average molecular weights for all polymers are 3-6%. These results and the Design of Experiments study demonstrate that the method is accurate and robust. Copyright © 2011 Elsevier B.V. All rights reserved.

Ultrahigh pressure fast size exclusion chromatography for top-down proteomics.

PubMed

Chen, Xin; Ge, Ying

2013-09-01

Top-down MS-based proteomics has gained a solid growth over the past few years but still faces significant challenges in the LC separation of intact proteins. In top-down proteomics, it is essential to separate the high mass proteins from the low mass species due to the exponential decay in S/N as a function of increasing molecular mass. SEC is a favored LC method for size-based separation of proteins but suffers from notoriously low resolution and detrimental dilution. Herein, we reported the use of ultrahigh pressure (UHP) SEC for rapid and high-resolution separation of intact proteins for top-down proteomics. Fast separation of intact proteins (6-669 kDa) was achieved in < 7 min with high resolution and high efficiency. More importantly, we have shown that this UHP-SEC provides high-resolution separation of intact proteins using a MS-friendly volatile solvent system, allowing the direct top-down MS analysis of SEC-eluted proteins without an additional desalting step. Taken together, we have demonstrated that UHP-SEC is an attractive LC strategy for the size separation of proteins with great potential for top-down proteomics. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Online stable isotope analysis of dissolved organic carbon size classes using size exclusion chromatography coupled to an isotope ratio mass spectrometer.

PubMed

Malik, Ashish; Scheibe, Andrea; LokaBharathi, P A; Gleixner, Gerd

2012-09-18

Stable isotopic content of dissolved organic carbon (δ(13)C-DOC) provides valuable information on its origin and fate. In an attempt to get additional insights into DOC cycling, we developed a method for δ(13)C measurement of DOC size classes by coupling high-performance liquid chromatography (HPLC)-size exclusion chromatography (SEC) to online isotope ratio mass spectrometry (IRMS). This represents a significant methodological contribution to DOC research. The interface was evaluated using various organic compounds, thoroughly tested with soil-water from a C3-C4 vegetation change experiment, and also applied to riverine and marine DOC. δ(13)C analysis of standard compounds resulted in excellent analytical precision (≤0.3‰). Chromatography resolved soil DOC into 3 fractions: high molecular weight (HMW; 0.4-10 kDa), low molecular weight (LMW; 50-400 Da), and retained (R) fraction. Sample reproducibility for measurement of δ(13)C-DOC size classes was ±0.25‰ for HMW fraction, ± 0.54‰ for LMW fraction, and ±1.3‰ for R fraction. The greater variance in δ(13)C values of the latter fractions was due to their lower concentrations. The limit of quantification (SD ≤0.6‰) for each size fraction measured as a peak is 200 ng C (2 mg C/L). δ(13)C-DOC values obtained in SEC mode correlated significantly with those obtained without column in the μEA mode (p < 0.001, intercept 0.17‰), which rules out SEC-associated isotopic effects or DOC loss. In the vegetation change experiment, fractions revealed a clear trend in plant contribution to DOC; those in deeper soils and smaller size fractions had less plant material. It was also demonstrated that the technique can be successfully applied to marine and riverine DOC without further sample pretreatment.

Caveats when Analyzing Ultra-high Molar Mass Polymers by SEC

USDA-ARS?s Scientific Manuscript database

The analysis of ultra-high molar mass (M > 1 million g/mol) polymers via size-exclusion chromatography (SEC) presents a number of non-trivial challenges. Dissolution and full solvation may take days, as is the case for cellulose dissolution in non-complexing non degrading solvents; very low concent...

Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples

PubMed Central

Lozano-Ramos, Inés; Bancu, Ioana; Oliveira-Tercero, Anna; Armengol, María Pilar; Menezes-Neto, Armando; Del Portillo, Hernando A.; Lauzurica-Valdemoros, Ricardo; Borràs, Francesc E.

2015-01-01

Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs. When analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm–Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species. To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches. PMID:26025625

Comparison of Diafiltration and Size-Exclusion Chromatography to Recover Hemicelluloses From Process Water From Thermomechanical Pulping of Spruce

NASA Astrophysics Data System (ADS)

Andersson, Alexandra; Persson, Tobias; Zacchi, Guido; Stålbrand, Henrik; Jönsson, Ann-Sofi

Hemicelluloses constitute one of the most abundant renewable resources on earth. To increase their utilization, the isolation of hemicelluloses from industrial biomass side-streams would be beneficial. A method was investigated to isolate hemicelluloses from process water from a thermomechanical pulp mill. The method consists of three steps: removal of solids by microfiltration, preconcentration of the hemicelluloses by ultrafiltration, and purification by either size-exclusion chromatography (SEC) or diafiltration. The purpose of the final purification step is to separate hemicelluloses from small oligosaccharides, monosaccharides, and salts. The ratio between galactose, glucose, and mannose in oligo- and polysaccharides after preconcentration was 0.8∶1∶2.8, which is similar to that found in galactoglucomannan. Continuous diafiltration was performed using a composite fluoro polymer membrane with cutoff of 1000 Da. After diafiltration with four diavolumes the purity of the hemicelluloses was 77% (gram oligo- and polysaccharides/ gram total dissolved solids) and the recovery was 87%. Purification by SEC was performed with 5, 20, and 40% sample loadings, respectively and a flow rate of 12 or 25 mL/min (9 or 19 cm/h). The purity of hemicelluloses after SEC was approx 82%, and the recovery was above 99%. The optimal sample load and flow rate were 20% and 25 mL/min, respectively. The process water from thermomechanical pulping of spruce is inexpensive. Thus, the recovery of hemicelluloses is not of main importance. If the purity of 77%, obtained with diafiltration, is sufficient for the utilization of the hemicelluloses, diafiltration probably offers a less expensive alternative in this application.

Composition and Molecular Weight Distribution of Carob Germ Proteins Fractions

USDA-ARS?s Scientific Manuscript database

Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and electrophoretic analysis. Using a mo...

Synthesis and chromatographic characterization of dextran-coated zirconia high-performance liquid chromatographic stationary phases.

PubMed

Dunlap, C J; Carr, P W

1996-10-11

Porous zirconia particles made by the oil emulsion (OE) method and the polymerization-induced colloid aggregation (PICA) method have been coated with a small, carboxymethylated (approximately 5%) dextran polymer and crosslinked in place. The parameters of the coating process (dextran concentration, adsorption time and crosslinker concentration) have all been examined and an optimum value for each determined. The coated and uncoated materials were characterized by nitrogen sorptometry and size-exclusion chromatography (SEC) using solutes (polystyrenes and dextrans) of well-defined molecular masses. Nitrogen sorptometry results show that the PICA material has a much lower pore volume and smaller pore diameter than do the OE materials. Despite this, the elution volumes of the SEC probes change very little upon polymer coating the PICA material while the OE material shows a very large change upon coating.

A new non-degradative method to purify glycogen.

PubMed

Tan, Xinle; Sullivan, Mitchell A; Gao, Fei; Li, Shihan; Schulz, Benjamin L; Gilbert, Robert G

2016-08-20

Liver glycogen, a complex branched glucose polymer containing a small amount of protein, is important for maintaining glucose homeostasis (blood-sugar control) in humans. It has recently been found that glycogen molecular structure is impaired in diabetes. Isolating the carbohydrate polymer and any intrinsically-attached protein(s) is an essential prerequisite for studying this structural impairment. This requires an effective, non-degradative and efficient purification method to exclude the many other proteins present in liver. Proteins and glycogen have different ranges of molecular sizes. Despite the plethora of proteins that might still be present in significant abundance after other isolation techniques, SEC (size exclusion chromatography, also known as GPC), which separates by molecular size, should separate those extraneous to glycogen from glycogen with any intrinsically associated protein(s). A novel purification method is developed for this, based on preparative SEC following sucrose gradient centrifugation. Proteomics is used to show that the new method compares favourably with current methods in the literature. Copyright © 2016 Elsevier Ltd. All rights reserved.

Examination of soil contaminated by coal-liquids by size exclusion chromatography in 1-methyl-2-pyrrolidinone solution to evaluate interference from humic and fulvic acids and extracts from peat.

PubMed

Morgan, T J; Herod, A A; Brain, S A; Chambers, F M; Kandiyoti, R

2005-11-18

Soil from a redundant coke oven site has been examined by extraction of soluble materials using 1-methyl-2-pyrrolidinone (NMP) followed by size exclusion chromatography (SEC) of the extracted material. The extracted material was found to closely resemble a high temperature coal tar pitch. Standard humic and fulvic acids were also examined since these materials are very soluble in NMP and would be extracted with pitch if present in the soil. Humic substances derived from peat samples and NMP-extracts of peats were also examined. The results show that the humic and fulvic substances were not extracted directly by NMP from peats. They were extracted using caustic soda solution and were different from the peat extracts in NMP. These results indicate that humic and fulvic acids were soluble in NMP in the protonated polyelectrolyte form but not in the original native polyelectrolyte form. The extraction of soil using NMP followed by SEC appears to be a promising method for identifying contamination by coal-based industries.

Size exclusion chromatography with superficially porous particles.

PubMed

Schure, Mark R; Moran, Robert E

2017-01-13

A comparison is made using size-exclusion chromatography (SEC) of synthetic polymers between fully porous particles (FPPs) and superficially porous particles (SPPs) with similar particle diameters, pore sizes and equal flow rates. Polystyrene molecular weight standards with a mobile phase of tetrahydrofuran are utilized for all measurements conducted with standard HPLC equipment. Although it is traditionally thought that larger pore volume is thermodynamically advantageous in SEC for better separations, SPPs have kinetic advantages and these will be shown to compensate for the loss in pore volume compared to FPPs. The comparison metrics include the elution range (smaller with SPPs), the plate count (larger for SPPs), the rate production of theoretical plates (larger for SPPs) and the specific resolution (larger with FPPs). Advantages to using SPPs for SEC are discussed such that similar separations can be conducted faster using SPPs. SEC using SPPs offers similar peak capacities to that using FPPs but with faster operation. This also suggests that SEC conducted in the second dimension of a two-dimensional liquid chromatograph may benefit with reduced run time and with equivalently reduced peak width making SPPs advantageous for sampling the first dimension by the second dimension separator. Additional advantages are discussed for biomolecules along with a discussion of optimization criteria for size-based separations. Copyright © 2016 Elsevier B.V. All rights reserved.

Filling the gap: Calibration of the low molar-mass range of cellulose in size exclusion chromatography with cello-oligomers.

PubMed

Oberlerchner, J T; Vejdovszky, P; Zweckmair, T; Kindler, A; Koch, S; Rosenau, T; Potthast, A

2016-11-04

Degraded celluloses are becoming increasingly important as part of product streams coming from various biorefinery scenarios. Analysis of the molar mass distribution of such fractions is a challenge, since neither established methods for mono- or disaccharides nor common methods for polysaccharide characterization cover the intermediate oligomer range appropriately. Size exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS), the standard approach for celluloses, suffers from decreased scattering intensities in the lower-molar mass range. The limitation in the low-molecular range can, in principle, be overcome by calibration, but calibration standards for such "short" celluloses are either not readily available or structurally remote and thus questionable. In this paper, we present the calibration of a SEC system- for the first time - with monodisperse cellooligomer standards up to about 3400gmol -1 . These cellooligomers are "short-chain celluloses" and can be seen as the "true" standard compounds, by contrast to commonly used standards that are chemically different from cellulose, such as pullulan, dextran, polystyrene, or poly(methyl methacrylate). The calibration is compared against those commercial standards and correction factors are calculated. Calibrations with non-cellulose standards can now be adjusted to yield better fitting results, and data already available can be corrected retrospectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of branched ultrahigh molar mass polymers by asymmetrical flow field-flow fractionation and size exclusion chromatography.

PubMed

Otte, T; Pasch, H; Macko, T; Brüll, R; Stadler, F J; Kaschta, J; Becker, F; Buback, M

2011-07-08

The molar mass distribution (MMD) of synthetic polymers is frequently analyzed by size exclusion chromatography (SEC) coupled to multi angle light scattering (MALS) detection. For ultrahigh molar mass (UHM) or branched polymers this method is not sufficient, because shear degradation and abnormal elution effects falsify the calculated molar mass distribution and information on branching. High temperatures above 130 °C have to be applied for dissolution and separation of semi-crystalline materials like polyolefins which requires special hardware setups. Asymmetrical flow field-flow fractionation (AF4) offers the possibility to overcome some of the main problems of SEC due to the absence of an obstructing porous stationary phase. The SEC-separation mainly depends on the pore size distribution of the used column set. The analyte molecules can enter the pores of the stationary phase in dependence on their hydrodynamic volume. The archived separation is a result of the retention time of the analyte species inside SEC-column which depends on the accessibility of the pores, the residence time inside the pores and the diffusion ability of the analyte molecules. The elution order in SEC is typically from low to high hydrodynamic volume. On the contrary AF4 separates according to the diffusion coefficient of the analyte molecules as long as the chosen conditions support the normal FFF-separation mechanism. The separation takes place in an empty channel and is caused by a cross-flow field perpendicular to the solvent flow. The analyte molecules will arrange in different channel heights depending on the diffusion coefficients. The parabolic-shaped flow profile inside the channel leads to different elution velocities. The species with low hydrodynamic volume will elute first while the species with high hydrodynamic volume elute later. The AF4 can be performed at ambient or high temperature (AT-/HT-AF4). We have analyzed one low molar mass polyethylene sample and a number of narrow distributed polystyrene standards as reference materials with known structure by AT/HT-SEC and AT/HT-AF4. Low density polyethylenes as well as polypropylene and polybutadiene, containing high degrees of branching and high molar masses, have been analyzed with both methods. As in SEC the relationship between the radius of gyration (R(g)) or the molar mass and the elution volume is curved up towards high elution volumes, a correct calculation of the MMD and the molar mass average or branching ratio is not possible using the data from the SEC measurements. In contrast to SEC, AF4 allows the precise determination of the MMD, the molar mass averages as well as the degree of branching because the molar mass vs. elution volume curve and the conformation plot is not falsified in this technique. In addition, higher molar masses can be detected using HT-AF4 due to the absence of significant shear degradation in the channel. As a result the average molar masses obtained from AF4 are higher compared to SEC. The analysis time in AF4 is comparable to that of SEC but the adjustable cross-flow program allows the user to influence the separation efficiency which is not possible in SEC without a costly change of the whole column combination. Copyright © 2011 Elsevier B.V. All rights reserved.

Guidance to Achieve Accurate Aggregate Quantitation in Biopharmaceuticals by SV-AUC.

PubMed

Arthur, Kelly K; Kendrick, Brent S; Gabrielson, John P

2015-01-01

The levels and types of aggregates present in protein biopharmaceuticals must be assessed during all stages of product development, manufacturing, and storage of the finished product. Routine monitoring of aggregate levels in biopharmaceuticals is typically achieved by size exclusion chromatography (SEC) due to its high precision, speed, robustness, and simplicity to operate. However, SEC is error prone and requires careful method development to ensure accuracy of reported aggregate levels. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is an orthogonal technique that can be used to measure protein aggregation without many of the potential inaccuracies of SEC. In this chapter, we discuss applications of SV-AUC during biopharmaceutical development and how characteristics of the technique make it better suited for some applications than others. We then discuss the elements of a comprehensive analytical control strategy for SV-AUC. Successful implementation of these analytical control elements ensures that SV-AUC provides continued value over the long time frames necessary to bring biopharmaceuticals to market. © 2015 Elsevier Inc. All rights reserved.

Evaluating the impact of different exogenous factors on silk textiles deterioration with use of size exclusion chromatography

NASA Astrophysics Data System (ADS)

Pawcenis, Dominika; Smoleń, Mariusz; Aksamit-Koperska, Monika A.; Łojewski, Tomasz; Łojewska, Joanna

2016-06-01

Size exclusion chromatography (SEC), especially coupled with multiple angle laser light scattering detector (MALLS) is a powerful tool in diagnostics of deterioration of historic and art objects to evaluate their condition. In this paper, SEC-UV-MALLS-DRI technique was applied to study degradation of silk fibroin samples ( Bombyx mori) artificially aged under various conditions: in the presence of oxygen, in different amount of water vapour and in volatile organic products (VOCs), all at temperature of 90 °C. Conditions were chosen in such a way that it mimicked real conditions of textiles' storing during exhibitions and in show cases. The influence of temperature, moisture and VOCs content on the state of silk textiles was examined with the use of size exclusion chromatography. Pseudo-zero-order Ekenstam equation was applied to study degradation rates of fibroin with use of the approximated values of DP of fibroin.

Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation

PubMed Central

2017-01-01

Polymeric nanoparticles have become indispensable in modern society with a wide array of applications ranging from waterborne coatings to drug-carrier-delivery systems. While a large range of techniques exist to determine a multitude of properties of these particles, relating physicochemical properties of the particle to the chemical structure of the intrinsic polymers is still challenging. A novel, highly orthogonal separation system based on comprehensive two-dimensional liquid chromatography (LC × LC) has been developed. The system combines hydrodynamic chromatography (HDC) in the first-dimension to separate the particles based on their size, with ultrahigh-performance size-exclusion chromatography (SEC) in the second dimension to separate the constituting polymer molecules according to their hydrodynamic radius for each of 80 to 100 separated fractions. A chip-based mixer is incorporated to transform the sample by dissolving the separated nanoparticles from the first-dimension online in tetrahydrofuran. The polymer bands are then focused using stationary-phase-assisted modulation to enhance sensitivity, and the water from the first-dimension eluent is largely eliminated to allow interaction-free SEC. Using the developed system, the combined two-dimensional distribution of the particle-size and the molecular-size of a mixture of various polystyrene (PS) and polyacrylate (PACR) nanoparticles has been obtained within 60 min. PMID:28745485

Conversion of calibration curves for accurate estimation of molecular weight averages and distributions of polyether polyols by conventional size exclusion chromatography.

PubMed

Xu, Xiuqing; Yang, Xiuhan; Martin, Steven J; Mes, Edwin; Chen, Junlan; Meunier, David M

2018-08-17

Accurate measurement of molecular weight averages (M¯ n, M¯ w, M¯ z ) and molecular weight distributions (MWD) of polyether polyols by conventional SEC (size exclusion chromatography) is not as straightforward as it would appear. Conventional calibration with polystyrene (PS) standards can only provide PS apparent molecular weights which do not provide accurate estimates of polyol molecular weights. Using polyethylene oxide/polyethylene glycol (PEO/PEG) for molecular weight calibration could improve the accuracy, but the retention behavior of PEO/PEG is not stable in THF-based (tetrahydrofuran) SEC systems. In this work, two approaches for calibration curve conversion with narrow PS and polyol molecular weight standards were developed. Equations to convert PS-apparent molecular weight to polyol-apparent molecular weight were developed using both a rigorous mathematical analysis and graphical plot regression method. The conversion equations obtained by the two approaches were in good agreement. Factors influencing the conversion equation were investigated. It was concluded that the separation conditions such as column batch and operating temperature did not have significant impact on the conversion coefficients and a universal conversion equation could be obtained. With this conversion equation, more accurate estimates of molecular weight averages and MWDs for polyether polyols can be achieved from conventional PS-THF SEC calibration. Moreover, no additional experimentation is required to convert historical PS equivalent data to reasonably accurate molecular weight results. Copyright © 2018. Published by Elsevier B.V.

Size-exclusion chromatography (HPLC-SEC) technique optimization by simplex method to estimate molecular weight distribution of agave fructans.

PubMed

Moreno-Vilet, Lorena; Bostyn, Stéphane; Flores-Montaño, Jose-Luis; Camacho-Ruiz, Rosa-María

2017-12-15

Agave fructans are increasingly important in food industry and nutrition sciences as a potential ingredient of functional food, thus practical analysis tools to characterize them are needed. In view of the importance of the molecular weight on the functional properties of agave fructans, this study has the purpose to optimize a method to determine their molecular weight distribution by HPLC-SEC for industrial application. The optimization was carried out using a simplex method. The optimum conditions obtained were at column temperature of 61.7°C using tri-distilled water without salt, adjusted pH of 5.4 and a flow rate of 0.36mL/min. The exclusion range is from 1 to 49 of polymerization degree (180-7966Da). This proposed method represents an accurate and fast alternative to standard methods involving multiple-detection or hydrolysis of fructans. The industrial applications of this technique might be for quality control, study of fractionation processes and determination of purity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization by Tin-Specific Size Exclusion Chromatography of the Free Radical Copolymerization of Tributyltin Methacrylate and Methyl Methacrylate,

DTIC Science & Technology

1980-12-11

Characterization by Tin-Specific Size Exclusion Chromatography of the Free Radical Copolymerization of Tributyltin Methacrylate and -~~~ ~~ ety Me aryate1...81 ~ 9 1 7 29 2 ABSTRACT Copolymers of tributyltin methacrylate (TBTM) and methyl methacrylate (MMA) comprise an important class of biocidal slow...exclusion chromatography (SEC); tin-specific graphite furnace atomic absorp- tion (GFAA); tributyltin methacrylate; ultraviolet absorbance; weight

Evaluation of a non-Arrhenius model for therapeutic monoclonal antibody aggregation.

PubMed

Kayser, Veysel; Chennamsetty, Naresh; Voynov, Vladimir; Helk, Bernhard; Forrer, Kurt; Trout, Bernhardt L

2011-07-01

Understanding antibody aggregation is of great significance for the pharmaceutical industry. We studied the aggregation of five different therapeutic monoclonal antibodies (mAbs) with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC), fluorescence spectroscopy, electron microscopy, and light scattering methods at various temperatures with the aim of gaining insight into the aggregation process and developing models of it. In particular, we find that the kinetics can be described by a second-order model and are non-Arrhenius. Thus, we develop a non-Arrhenius model to connect accelerated aggregation experiments at high temperature to long-term storage experiments at low temperature. We evaluate our model by predicting mAb aggregation and comparing it with long-term behavior. Our results suggest that the number of monomers and mAb conformations within aggregates vary with the size and age of the aggregates, and that only certain sizes of aggregates are populated in the solution. We also propose a kinetic model based on conformational changes of proteins and monomer peak loss kinetics from SEC-HPLC. This model could be employed for a detail analysis of mAb aggregation kinetics. Copyright © 2011 Wiley-Liss, Inc. and the American Pharmacists Association

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

PubMed

Börner, Tim; Grey, Carl; Adlercreutz, Patrick

2016-08-01

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unraveling the mystery of natural rubber biosythesis part I: investigation of the composition and growth of in vitro natural rubber using high resolution size exclusion chromatography

USDA-ARS?s Scientific Manuscript database

Monitoring the growth of in vitro natural rubberwas accomplished by high resolution size exclusion chromatography, SEC.Washed rubber particles isolated from H. brasiliensis latex, containing the rubber transferase enzyme, were used to catalyze the polymerization of synthetic isopentenyl pyrophosphat...

Direct quantification of molar masses of copolymers by online liquid chromatography under critical conditions-nuclear magnetic resonance and size exclusion chromatography-nuclear magnetic resonance.

PubMed

Hehn, Mathias; Wagner, Thomas; Hiller, Wolf

2014-01-07

Online LCCC-NMR and SEC-NMR are compared regarding the determination of molar masses of block copolymers. Two different direct referencing methods are particularly demonstrated in LCCC-NMR for a detailed characterization of diblock copolymers and their co-monomers. First, an intramolecular reference group was used for the direct determination of block lengths and molar masses. For the first time, it was shown that LCCC-NMR can be used for an accurate determination of Mw and Mn of copolymers. These data were in perfect agreement with SEC-NMR measurements using the same intramolecular referencing method. In contrast, the determination of molar masses with common relative methods based on calibrations with homopolymers delivered inaccurate results for all investigated diblock copolymers due to different hydrodynamic volumes of the diblock copolymer compared to their homopolymers. The intramolecular referencing method provided detailed insights in the co-monomer behavior during the chromatographic separation of LCCC. Especially, accurate chain lengths and chemical compositions of the "invisible" and "visible" blocks were quantified during the elution under critical conditions and provided new aspects to the concept of critical conditions. Second, an external reference NMR signal was used to directly determine concentrations and molar masses of the block copolymers from the chromatographic elution profile. Consequently, the intensity axes of the resulting chromatograms were converted to molar amounts and masses, allowing for determination of the amount of polymer chains with respect to elution volume, the evaluation of the limiting magnitude of concentration for LCCC-NMR, and determination of the molar masses of copolymers.

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

PubMed

Acosta, Mariano; Torres, Sabier; Mariño-Repizo, Leonardo; Martinez, Luis D; Gil, Raúl A

2018-06-02

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering.

PubMed

Pollock, Jacob F; Ashton, Randolph S; Rode, Nikhil A; Schaffer, David V; Healy, Kevin E

2012-09-19

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.

Using an FPLC to promote active learning of the principles of protein structure and purification.

PubMed

Robinson, Rebekah L; Neely, Amy E; Mojadedi, Wais; Threatt, Katie N; Davis, Nicole Y; Weiland, Mitch H

2017-01-02

The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and non-native). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

Nano-fabricated size exclusion chromatograph

NASA Technical Reports Server (NTRS)

Svehla, D.; Feldman, S.; Feldman, J.; Grunthaner, F.; Shakkottai, P.; Castillo, L. del; White, V.

2002-01-01

This paper describes the development of a nano-fabricated size exclusion chromatograph (nSEC) based on the principle that molecules traveling through amicrocolumn containing nano-fabricated features will have characteristic elution times that directly correlate to molecular weight. Compared to conventional size exclusion chromatography, the nSEC offers greater control over the size exclusion process; mass fabrication; integration of the separation column with associated valves, pumps, and detectors; and dramatic reductions in instrument mass and power requirements.

Determination of the MRI contrast agent Gd-DTPA by SEC-ICP-MS.

PubMed

Loreti, Valeria; Bettmer, Jörg

2004-08-01

The simultaneous determination of Gd(3+) and Gd-DTPA (DTPA: diethylenetriamino-pentaacetic acid), often used as contrast agent, is described. The proposed approach combines size-exclusion chromatography (SEC) and inductively coupled plasma-mass spectrometry (ICP-MS) for element-selective detection in order to determine also high-molecular Gd-complexes if present. This method was applied to the analysis of urine samples of a patient to whom Gd-DTPA was intravenously administered. The results showed that no conversion or adsorption of Gd-DTPA could be observed in any sample, even free Gd(3+) could not be detected. Urine excretion behaviour was monitored and it was proved that Gd-DTPA was almost completely (>99%) excreted by urination within one day. Traces of Gd-DTPA could be measured in hair samples, but extraction with tetramethylammonium hydroxide (TMAH) resulted in degradation of Gd-DTPA.

Separation of large DNA molecules by applying pulsed electric field to size exclusion chromatography-based microchip

NASA Astrophysics Data System (ADS)

Azuma, Naoki; Itoh, Shintaro; Fukuzawa, Kenji; Zhang, Hedong

2018-02-01

Through electrophoresis driven by a pulsed electric field, we succeeded in separating large DNA molecules with an electrophoretic microchip based on size exclusion chromatography (SEC), which was proposed in our previous study. The conditions of the pulsed electric field required to achieve the separation were determined by numerical analyses using our originally proposed separation model. From the numerical results, we succeeded in separating large DNA molecules (λ DNA and T4 DNA) within 1600 s, which was approximately half of that achieved under a direct electric field in our previous study. Our SEC-based electrophoresis microchip will be one of the effective tools to meet the growing demand of faster and more convenient separation of large DNA molecules, especially in the field of epidemiological research of infectious diseases.

Single-step isolation of extracellular vesicles by size-exclusion chromatography

PubMed Central

Böing, Anita N.; van der Pol, Edwin; Grootemaat, Anita E.; Coumans, Frank A. W.; Sturk, Auguste; Nieuwland, Rienk

2014-01-01

Background Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim To develop a single-step protocol to isolate vesicles from human body fluids. Methods Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n=3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results Fractions 9–12 contained the highest concentrations of particles larger than 70 nm and platelet-derived vesicles (46%±6 and 61%±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8%±1 and 0.65%±0.3, respectively). HDL was present mainly in fractions 18–20 (32%±2 of total), and protein in fractions 19–21 (36%±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43%±23 in fractions 9–12, with an 8-fold and 70-fold enrichment compared to HDL and protein. Conclusions SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles. PMID:25279113

Oligomeric cationic polymethacrylates: a comparison of methods for determining molecular weight.

PubMed

Locock, Katherine E S; Meagher, Laurence; Haeussler, Matthias

2014-02-18

This study compares three common laboratory methods, size-exclusion chromatography (SEC), (1)H nuclear magnetic resonance (NMR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), to determine the molecular weight of oligomeric cationic copolymers. The potential bias for each method was examined across a series of polymers that varied in molecular weight and cationic character (both choice of cation (amine versus guanidine) and relative proportion present). SEC was found to be the least accurate, overestimating Mn by an average of 140%, owing to the lack of appropriate cationic standards available, and the complexity involved in estimating the hydrodynamic volume of copolymers. MALDI-TOF approximated Mn well for the highly monodisperse (Đ < 1.1), low molecular weight (degree of polymerization (DP) <50) species but appeared unsuitable for the largest polymers in the series due to the mass bias associated with the technique. (1)H NMR was found to most accurately estimate Mn in this study, differing to theoretical values by only 5.2%. (1)H NMR end-group analysis is therefore an inexpensive and facile, primary quantitative method to estimate the molecular weight of oliogomeric cationic polymethacrylates if suitably distinct end-groups signals are present in the spectrum.

Simulation of size-exclusion chromatography distribution coefficients of comb-shaped molecules in spherical pores comparison of simulation and experiment.

PubMed

Radke, Wolfgang

2004-03-05

Simulations of the distribution coefficients of linear polymers and regular combs with various spacings between the arms have been performed. The distribution coefficients were plotted as a function of the number of segments in order to compare the size exclusion chromatography (SEC)-elution behavior of combs relative to linear molecules. By comparing the simulated SEC-calibration curves it is possible to predict the elution behavior of comb-shaped polymers relative to linear ones. In order to compare the results obtained by computer simulations with experimental data, a variety of comb-shaped polymers varying in side chain length, spacing between the side chains and molecular weights of the backbone were analyzed by SEC with light-scattering detection. It was found that the computer simulations could predict the molecular weights of linear molecules having the same retention volume with an accuracy of about 10%, i.e. the error in the molecular weight obtained by calculating the molecular weight of the comb-polymer based on a calibration curve constructed using linear standards and the results of the computer simulations are of the same magnitude as the experimental error of absolute molecular weight determination.

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

PubMed Central

Dernick, Gregor; Obermüller, Stefan; Mangold, Cyrill; Magg, Christine; Matile, Hugues; Gutmann, Oliver; von der Mark, Elisabeth; Handschin, Corinne; Maugeais, Cyrille; Niesor, Eric J.

2011-01-01

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique. PMID:21971713

Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography.

PubMed

Shanmuga Doss, Sreeja; Bhatt, Nirav Pravinbhai; Jayaraman, Guhan

2017-08-15

There is an unreasonably high variation in the literature reports on molecular weight of hyaluronic acid (HA) estimated using conventional size exclusion chromatography (SEC). This variation is most likely due to errors in estimation. Working with commercially available HA molecular weight standards, this work examines the extent of error in molecular weight estimation due to two factors: use of non-HA based calibration and concentration of sample injected into the SEC column. We develop a multivariate regression correlation to correct for concentration effect. Our analysis showed that, SEC calibration based on non-HA standards like polyethylene oxide and pullulan led to approximately 2 and 10 times overestimation, respectively, when compared to HA-based calibration. Further, we found that injected sample concentration has an effect on molecular weight estimation. Even at 1g/l injected sample concentration, HA molecular weight standards of 0.7 and 1.64MDa showed appreciable underestimation of 11-24%. The multivariate correlation developed was found to reduce error in estimations at 1g/l to <4%. The correlation was also successfully applied to accurately estimate the molecular weight of HA produced by a recombinant Lactococcus lactis fermentation. Copyright © 2017 Elsevier B.V. All rights reserved.

Additional band broadening of peptides in the first size-exclusion chromatographic dimension of an automated stop-flow two-dimensional high performance liquid chromatography.

PubMed

Xu, Jucai; Sun-Waterhouse, Dongxiao; Qiu, Chaoying; Zhao, Mouming; Sun, Baoguo; Lin, Lianzhu; Su, Guowan

2017-10-27

The need to improve the peak capacity of liquid chromatography motivates the development of two-dimensional analysis systems. This paper presented a fully automated stop-flow two-dimensional liquid chromatography system with size exclusion chromatography followed by reversed phase liquid chromatography (SEC×RPLC) to efficiently separate peptides. The effects of different stop-flow operational parameters (stop-flow time, peak parking position, number of stop-flow periods and column temperature) on band broadening in the first dimension (1 st D) SEC column were quantitatively evaluated by using commercial small proteins and peptides. Results showed that the effects of peak parking position and the number of stop-flow periods on band broadening were relatively small. Unlike stop-flow analysis of large molecules with a long running time, additional band broadening was evidently observed for small molecule analytes due to the relatively high effective diffusion coefficient (D eff ). Therefore, shorter analysis time and lower 1 st D column temperature were suggested for analyzing small molecules. The stop-flow two-dimensional liquid chromatography (2D-LC) system was further tested on peanut peptides and an evidently improved resolution was observed for both stop-flow heart-cutting and comprehensive 2D-LC analysis (in spite of additional band broadening in SEC). The stop-flow SEC×RPLC, especially heart-cutting analysis with shorter analysis time and higher 1 st D resolution for selected fractions, offers a promising approach for efficient analysis of complex samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Anhydrous trifluoroacetic acid pretreatment converts insoluble polyglutamine peptides to soluble monomers.

PubMed

Burra, Gunasekhar; Thakur, Ashwani Kumar

2015-12-01

The data provided in this article are related to the research article entitled "Unaided trifluoroacetic acid pretreatment solubilizes polyglutamine (polyGln) peptides and retains their biophysical properties of aggregation" by Burra and Thakur (in press) [1]. This research article reports data from size exclusion chromatography (SEC), reversed phase-high performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) assays. This data show that trifluoroacetic acid (TFA) has the ability to convert insoluble polyGln peptides to soluble monomers. The data also clarify the possibility of trifluoroacetylation modification caused due to TFA. We hope the data presented here will enhance the understanding of polyGln disaggregation and solubilization. For more insightful and useful discussions, see the research article published in Analytical Biochemistry: Methods in the Biological Sciences (Burra and Thakur, in press [1]).

Determination of molecular weight distributions in native and pretreated wood.

PubMed

Leskinen, Timo; Kelley, Stephen S; Argyropoulos, Dimitris S

2015-03-30

The analysis of native wood components by size-exclusion chromatography (SEC) is challenging. Isolation, derivatization and solubilization of wood polymers is required prior to the analysis. The present approach allowed the determination of molecular weight distributions of the carbohydrates and of lignin in native and processed woods, without preparative component isolation steps. For the first time a component selective SEC analysis of sawdust preparations was made possible by the combination of two selective derivatization methods, namely; ionic liquid assisted benzoylation of the carbohydrate fraction and acetobromination of the lignin in acetic acid media. These were optimized for wood samples. The developed method was thus used to examine changes in softwood samples after degradative mechanical and/or chemical treatments, such as ball milling, steam explosion, green liquor pulping, and chemical oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). The methodology can also be applied to examine changes in molecular weight and lignin-carbohydrate linkages that occur during wood-based biorefinery operations, such as pretreatments, and enzymatic saccharification. Copyright © 2014 Elsevier Ltd. All rights reserved.

Liquid Chromatography at Critical Conditions: Balancing size exclusion and adsorption in nanopores

NASA Astrophysics Data System (ADS)

Abdulahad, Asem; Amos, Jeffrey; Ryu, Chang

2009-03-01

Liquid chromatography at critical condition (LCCC) is a measure to identify thermodynamic conditions, in which polymers elute independently of molar mass during high performance liquid chromatography. Under these critical conditions the entropic exclusions that dominate size exclusion chromatography (SEC) and the enthalpic adsorption that governs adsorption-based interaction chromatography (IC) are said to negate one another resulting in simultaneous elution of the polymer of different molecular weights. Using multiple C18-bonded silica columns with different average nanopore sizes (from 5 nm to 30 nm), we will study the LCCC conditions of PS in methylene chloride/acetonitrile solvent mixture at different temperature. In addition, we will show that the separation of polystyrene can be fine tuned using a refined temperature gradient interaction chromatography (TGIC) that employs multiple columns of varying pore size in sequence.

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format.

PubMed

Alvarez, Melissa; Tremintin, Guillaume; Wang, Jennifer; Eng, Marian; Kao, Yung-Hsiang; Jeong, Justin; Ling, Victor T; Borisov, Oleg V

2011-12-01

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography-mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

Determination of 4-nonylphenol in water samples using 4-(2,6-dimethylhept-3-yl)phenol as new internal standard.

PubMed

Fischer, Axel R; Lan, Nham Thi Phuong; Wiedemann, Cornelia; Heide, Petra; Werner, Peter; Schmidt, Arndt W; Theumer, Gabriele; Knölker, Hans-Joachim

2010-04-23

A new method for determining the endocrine disrupting substance 4-nonylphenol (technical grade=mixture of isomers, 4-NP) from water samples has been developed by using 4-(2,6-dimethylhept-3-yl)phenol (4-sec-NP) as model compound. This branched monoalkylphenol is shown to serve as internal standard (IS) for the determination of technical 4-nonylphenol. To the best of our knowledge, 4-(2,6-dimethylhept-3-yl)phenol (racemic mixture) is a newly synthesized 4-nonylphenol isomer and has not been described elsewhere. Recoveries have been determined by analyzing spiked water samples from distilled water, river water and wastewater. Following acetylation, the compounds were enriched via solid phase extraction (SPE). Analyses of the compounds were performed by capillary column gas chromatography/mass spectrometry (GC/MS), operating in selected ion-monitoring (SIM) mode. The recovery of technical 4-NP using either the newly prepared 4-sec-NP or 4-n-nonylphenol (4-n-NP) as IS have been compared. 4-sec-NP showed slightly better results. However, in the first series of experiments using wastewater, the yields for the derivatization of the two standard compounds were remarkably different. The yield for derivatization of 4-n-NP was approximately 20%, probably due to the difficult matrix of the wastewater. In contrast, the yield for the derivatization of 4-sec-NP was considerably higher (approximately 63%). This problem can be solved by increasing the concentration of the reagent used for derivatization. For better control of the clean-up process, we recommend application of 4-sec-NP as internal standard, at least in water samples with complex matrices (e.g., high content of hydroxylated compounds). Copyright 2010 Elsevier B.V. All rights reserved.

Complete Molecular Weight Profiling of Low-Molecular Weight Heparins Using Size Exclusion Chromatography-Ion Suppressor-High-Resolution Mass Spectrometry.

PubMed

Zaia, Joseph; Khatri, Kshitij; Klein, Joshua; Shao, Chun; Sheng, Yuewei; Viner, Rosa

2016-11-01

Low-molecular weight heparins (LMWH) prepared by partial depolymerization of unfractionated heparin are used globally to treat coagulation disorders on an outpatient basis. Patent protection for several LMWH has expired and abbreviated new drug applications have been approved by the Food and Drug Administration. As a result, reverse engineering of LMWH for biosimilar LMWH has become an active global endeavor. Traditionally, the molecular weight distributions of LMWH preparations have been determined using size exclusion chromatography (SEC) with optical detection. Recent advances in liquid chromatography-mass spectrometry methods have enabled exact mass measurements of heparin saccharides roughly up to degree-of-polymerization 20, leaving the high molecular weight half of the LMWH preparation unassigned. We demonstrate a new LC-MS system capable of determining the exact masses of complete LMWH preparations, up to dp30. This system employed an ion suppressor cell to desalt the chromatographic effluent online prior to the electrospray mass spectrometry source. We expect this new capability will impact the ability to define LMWH mixtures favorably.

Influence of connection tubing in modern size exclusion chromatography and its impact on the characterization of mAbs.

PubMed

Fekete, Szabolcs; Guillarme, Davy

2018-02-05

The goal of the study was to evaluate the impact of connection tubing in modern size exclusion chromatography (SEC), since it may strongly impact the apparent column efficiency, as the compounds are not retained in SEC. For this purpose, a reference SEC column of 150×4.6mm, 1.8μm was considered, and various proteins were tested as model compounds. Different tube geometries (lengths and internal diameters) and materials (stainless steel and PEEK) were evaluated in a systematic way. Large proteins always showed larger tube dispersion vs. small molecules, especially when the residence time in the tube was long (at low flow rate). This confirms the need to drastically reduce the tube volume (using the shortest and narrowest connector tubing) to attain the full benefits of UHPSEC columns. In addition, PEEK tubing were found to be more appropriate than stainless steel tubing, since adsorption of proteins was less pronounced, and higher plate count can be obtained. Finally, after a careful system optimization, up to 40% increase of apparent column efficiency can be achieved compared to a regular UHPLC system, when using a 150×4.6mm UHPSEC columns packed with sub-3μm particles. Copyright © 2017 Elsevier B.V. All rights reserved.

Composition and molecular weight distribution of carob germ protein fractions.

PubMed

Smith, Brennan M; Bean, Scott R; Schober, Tilman J; Tilley, Michael; Herald, Thomas J; Aramouni, Fadi

2010-07-14

Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC) coupled with multiangle laser light scattering (SEC-MALS), and electrophoretic analysis. Using a modified Osborne extraction procedure, carob germ flour proteins were found to contain approximately 32% albumin and globulin and approximately 68% glutelin with no prolamins detected. The albumin and globulin fraction was found to contain low amounts of disulfide-bonded polymers with relatively low M(w) ranging up to 5 x 10(6) Da. The glutelin fraction, however, was found to contain large amounts of high molecular weight disulfide-bonded polymers with M(w) up to 8 x 10(7) Da. When extracted under nonreducing conditions and divided into soluble and insoluble proteins as typically done for wheat gluten, carob germ proteins were found to be almost entirely ( approximately 95%) in the soluble fraction with only ( approximately 5%) in the insoluble fraction. As in wheat, SEC-MALS analysis showed that the insoluble proteins had a greater M(w) than the soluble proteins and ranged up to 8 x 10(7) Da. The lower M(w) distribution of the polymeric proteins of carob germ flour may account for differences in functionality between wheat and carob germ flour.

Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red.

PubMed

Sutter, Marc; Oliveira, Sabrina; Sanders, Niek N; Lucas, Bart; van Hoek, Arie; Hink, Mark A; Visser, Antonie J W G; De Smedt, Stefaan C; Hennink, Wim E; Jiskoot, Wim

2007-03-01

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with beta-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.

PubMed

Blans, Kristine; Hansen, Maria S; Sørensen, Laila V; Hvam, Michael L; Howard, Kenneth A; Möller, Arne; Wiking, Lars; Larsen, Lotte B; Rasmussen, Jan T

2017-01-01

Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

PubMed Central

Blans, Kristine; Hansen, Maria S.; Sørensen, Laila V.; Hvam, Michael L.; Howard, Kenneth A.; Möller, Arne; Wiking, Lars; Larsen, Lotte B.; Rasmussen, Jan T.

2017-01-01

ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk. PMID:28386391

First report of phytochelatins in a mushroom: induction of phytochelatins by metal exposure in Boletus edulis.

PubMed

Collin-Hansen, Christian; Pedersen, Sindre A; Andersen, Rolf A; Steinnes, Eiliv

2007-01-01

Some species of macromycetes (mushrooms) consistently are found to contain high concentrations of toxic metals such as cadmium (Cd) and mercury (Hg), and consumption of wild-growing mushrooms is acknowledged as a significant source for Cd and Hg in humans. Yet little is known about the speciation of Cd and Hg in mushroom tissues. Here we present the first evidence of peptides of the phytochelatin family being responsible for binding a large fraction of Cd in caps of the macromycete Boletus edulis exposed to excess metals. Concentrations of Cd, Zn, Cu and Hg, as well as cytosolic Cd-binding capacity (CCBC), glutathione (GSH) and free proline (Pro) were quantified in fruiting bodies of B. edulis differentially exposed to a wide range of metals. Metal distribution among cytosolic compounds were investigated by size exclusion chromatography (SEC), followed by metal determinations with atomic absorption chromatography (AAS) and HR-ICP-MS. Cd-binding compounds in SEC elutates were investigated further by high performance liquid chromatography-mass spectrometry (HPLC-MS). CCBC was >90 times higher in the exposed group relative to the reference group (Mann-Whitney's P < 0.001), whereas concentrations of free Pro were almost identical for the two groups. For the whole study selection, CCBC correlated positively with metal exposure (Spearman's P < 0.001 for all four metals), suggesting dose-dependent induction of Cd-binding compounds by exposure to these metals, possibly as a defense mechanism. The presence of phytochelatins (PCs), a family of cystein-rich oligopeptides, was confirmed in Cd-containing SEC fractions by HPLC-MS. The appearance of more complex PCs was coupled to declining concentrations of GSH. To our knowledge this is the first report demonstrating the presence of PCs in a macromycete.

A study on characteristic of different sample pretreatment methods to evaluate the entrapment efficiency of liposomes.

PubMed

Ran, Congcong; Chen, Dan; Xu, Meng; Du, Chaohui; Li, Qinglian; Jiang, Ye

2016-08-15

To examine how methods affect the evaluation of entrapment efficiency (EE) of liposomes, four different sample pretreatment methods were adopted in the experiment. The four sample pretreatment methods were size-exclusion chromatography (SEC), solid-phase extraction (SPE), centrifugation ultrafiltration (CF-UF) and hollow fiber centrifugal ultrafiltration (HF-CF-UF). Amphotericin B (AmB), which could self-associate to form aggregates in water is adopted as the model drugs in this paper. In the present work, it was found that the characterization results of four methods were quite different. The EE of liposome by SEC was about 93%, only 5-13% using C18 or HLB columns, and approximately 100% by CF-UF. The EE of HF-CF-UF reached up to nearly 99.0%. Further, this paper revealed the reasons making the difference of EE among four methods. Conventional SEC may distort the authentic of EE of liposomes with mainly employing some small liposomes or excessive water as eluent. For SPE, cholesterol on liposome surface could interact with the stationary phase making it hard to elute with water, and increase the risk of liposome leakage. While for CF-UF, concentration polarization was a main limitation hindering unentrapped drug to pass through membrane, making unentrapped drug undetectable in liposome. HF-CF-UF could truly reflect EE of liposomes with the concentration of unentrapped AmB lower than 25.0μg/mL. However, when the concentration was higher than 25.0μg/mL, AmB aggregates could be entrapped by hollow fiber. From the above analysis, this paper came to the conclusion that each method had its own feature in characterization. This study provided a reasonable guideline for choosing methods to character the EE of liposome. Copyright © 2016 Elsevier B.V. All rights reserved.

Liquid chromatography of polymers under limiting conditions of desorption II. Tandem injection and quantitative molar mass determination.

PubMed

Snauko, Marián; Berek, Dusan

2005-11-11

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.

Quantification and varietal variation of low molecular weight glutenin subunits (LMW-GS) using size exclusion chromatography.

PubMed

Dangi, Priya; Khatkar, B S

2018-03-01

Crude glutenin of four commercial wheat varieties viz. C 306, HI 977, HW 2004 and PBW 550 of diverse origin and breadmaking quality were fractionated by size-exclusion chromatography into three fractions of decreasing molecular weights. The relative quantity of peak II, containing LMW-GS specifically, varied considerably among the varieties as reflected from their discrete SEC profiles. The area % of peak II, containing protein of interest, was maximal for C 306 (22.08%) followed by PBW 550 (15.86%). The least proportion of LMW-GS were recovered from variety HW 2004 (9.68%). As the concentration of the sample extract injected to the column increased, the resolution of the peak declined in association with the slight shifting of retention time to the higher values. The best results were obtained for variety C 306 at 100 mg protein concentration with 3 M urea buffer. Consequently, the optimized conditions for purification of LMW-GS in appreciable amounts using SEC were established.

Sequential extractions of selenium soils from Stewart Lake: total selenium and speciation measurements with ICP-MS detection.

PubMed

Ponce de León, Claudia A; DeNicola, Katie; Montes Bayón, Maria; Caruso, Joseph A

2003-06-01

Different techniques have been employed in order to evaluate the most efficient procedure for the extraction of selenium from soil as required for speciation. Selenium contaminated sediments from Stewart Lake Wetland, California were used. A strong acid mineralization of the samples gives quantitative total selenium, which is then used to estimate recoveries for the milder extraction methods. The different extraction methodologies involve the sequential use of water, buffer (phosphate, pH 7) and either acid solution (e.g. HNO3 or HCl) or basic solutions (e.g. ammonium acetate, NaOH or TMAH). Pyrophosphate extraction was also evaluated and showed that selenium was not associated with humic acids. The extractants were subsequently analyzed by size exclusion chromatography (SEC) with UV (254 and 400 nm) and on-line ICP-MS detection; anion exchange chromatography, and ion-pair reversed phase chromatography with ICP-MS detection. For sequential extractions the extraction efficiencies showed that the basic extractions were more efficient than the acidic. The difference between the acidic and the basic extraction efficiency is carried to the sulfite extraction, suggesting that whatever is not extracted by the acid is subsequently extracted by the sulfite. The species identified with the different chromatographies were selenate, selenite, elemental selenium and some organic selenium.

Hyphenating size‐exclusion chromatography with electrospray mass spectrometry; using on‐line liquid‐liquid extraction to study the lipid composition of lipoprotein particles

PubMed Central

Osei, Michael; Griffin, Julian L.

2015-01-01

Rationale Lipoproteins belong to the most commonly measured clinical biochemical parameters. Lipidomics is an orthogonal approach and aims to profile the individual lipid molecules that jointly form the lipoprotein particles. However, in the first step of the extraction of lipid molecules from serum, an organic solvent is used leading to dissociation of the lipoproteins. Thus far it has been impossible to combine lipidomics and lipoprotein analysis in one analytical system. Methods Human plasma was diluted in phosphate‐buffered saline (PBS) and injected onto a Superose 6 PC 3.2 column with PBS as a mobile phase to separate lipoproteins. The eluent was led to a Syrris FLLEX module, which also received CHCl3/MeOH (3:1). The two phases were mixed and subsequently separated using a Teflon membrane in an especially designed pressurized flow chamber. The organic phase was led to a standard electrospray source of an Orbitrap mass spectrometer. Results Size‐exclusion chromatography (SEC) has been commonly applied to separate lipoproteins and is considered a practical alternative to ultracentrifugation. Through the on‐line liquid‐liquid extraction method it becomes possible to obtained detailed mass spectra of lipids across different lipoprotein fractions. The extracted ion chromatograms of specific lipid signals showed their distribution against the size of lipoprotein particles. Conclusions The application of on‐line liquid‐liquid extraction allows for the continuous electrospray‐based mass spectral analysis of SEC eluent, providing the detailed lipid composition of lipoprotein particles separated by size. This approach provides new possibilities for the study of the biochemistry of lipoproteins. © 2015 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd. PMID:26443395

Metallomics investigations on potential binding partners of methylmercury in tuna fish muscle tissue using complementary mass spectrometric techniques.

PubMed

Kutscher, Daniel J; Sanz-Medel, Alfredo; Bettmer, Jörg

2012-08-01

In this study, the binding behaviour of methylmercury (MeHg(+)) towards proteins is investigated. Free sulfhydryl groups in cysteine residues are known to be the most likely binding partners, due to the high affinity of mercury to sulphur. However, detailed knowledge about discrete binding sites in living organisms has been so far scarce. A metallomics approach using different methods like size-exclusion chromatography (SEC) and liquid chromatography (LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) as well as complementary mass spectrometric techniques (electrospray ionisation-tandem mass spectrometry, ESI-MS/MS) are combined to sequence and identify possible target proteins or peptides after enzymatic digestion. Potential targets for MeHg(+) in tuna fish muscle tissue are investigated using the certified reference material CRM464 as a model tissue. Different extraction procedures appropriate for the extraction of proteins are evaluated for their efficiency using isotope dilution analysis for the determination of total Hg in the extracts. Due to the high chemical stability of the mercury-sulphur bond, the bioconjugate can be quantitatively extracted with a combination of tris(hydroxymethyl)aminomethane (TRIS) and sodium dodecyl sulphate (SDS). Using different separation techniques such as SEC and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) it can be shown that major binding occurs to a high-molecular weight protein (M(w) > 200 kDa). A potential target protein, skeletal muscle myosin heavy chain, could be identified after tryptic digestion and capillary LC-ESI-MS/MS.

Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.

PubMed

Karimi, Nasibeh; Cvjetkovic, Aleksander; Jang, Su Chul; Crescitelli, Rossella; Hosseinpour Feizi, Mohammad Ali; Nieuwland, Rienk; Lötvall, Jan; Lässer, Cecilia

2018-02-13

The isolation of extracellular vesicles (EVs) from blood is of great importance to understand the biological role of circulating EVs and to develop EVs as biomarkers of disease. Due to the concurrent presence of lipoprotein particles, however, blood is one of the most difficult body fluids to isolate EVs from. The aim of this study was to develop a robust method to isolate and characterise EVs from blood with minimal contamination by plasma proteins and lipoprotein particles. Plasma and serum were collected from healthy subjects, and EVs were isolated by size-exclusion chromatography (SEC), with most particles being present in fractions 8-12, while the bulk of the plasma proteins was present in fractions 11-28. Vesicle markers peaked in fractions 7-11; however, the same fractions also contained lipoprotein particles. The purity of EVs was improved by combining a density cushion with SEC to further separate lipoprotein particles from the vesicles, which reduced the contamination of lipoprotein particles by 100-fold. Using this novel isolation procedure, a total of 1187 proteins were identified in plasma EVs by mass spectrometry, of which several proteins are known as EV-associated proteins but have hitherto not been identified in the previous proteomic studies of plasma EVs. This study shows that SEC alone is unable to completely separate plasma EVs from lipoprotein particles. However, combining SEC with a density cushion significantly improved the separation of EVs from lipoproteins and allowed for a detailed analysis of the proteome of plasma EVs, thus making blood a viable source for EV biomarker discovery.

Integration of On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry: Cross-Path Reactive Chromatography.

PubMed

Pawlowski, Jake W; Carrick, Ian; Kaltashov, Igor A

2018-01-16

Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g,, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide-reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide-linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using β 2 -microglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as demonstrated in this work by oxidatively labeling mAb on the column, followed by reduction of its disulfide bonds and MS analysis of the extent of oxidation within each chain of the molecule).

Characterization of a protein conjugate using an asymmetrical-flow field-flow fractionation and a size-exclusion chromatography with multi-detection system.

PubMed

Rebolj, Katja; Pahovnik, David; Zagar, Ema

2012-09-04

In this study we present detailed characterization of a protein-PEG conjugate using two separation techniques, that is, asymmetrical-flow field-flow fractionation (AF4) and size-exclusion chromatography (SEC), which were online coupled to a series of successively connected detectors: an ultraviolet, a multiangle light-scattering, a quasi-elastic light-scattering, and a refractive-index detector (UV-MALS(QELS)-RI). Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a complementary characterization technique. The results of AF4 as well as SEC on two columns connected in series, with both separation techniques coupled to a multidetection system, indicate the uniform molar mass and chemical composition of the conjugate, that is, the molar ratio of protein to PEG is 1/1, the presence of minute amounts of residual unreacted protein and the aggregates with the same chemical composition as that of the conjugate. Since the portion of aggregated species is smaller in the acetate buffer solution containing 5% sorbitol than in the acetate buffer solution with 200-mM sodium chloride, the former buffer solution is more suitable for conjugate storage. The separation using only one SEC column results in poorly resolved peaks of the PEGylated protein conjugate and the aggregates, whereas MALDI-TOF MS analysis reveal the presence of the residual protein, but not the aggregates.

The importance of system band broadening in modern size exclusion chromatography.

PubMed

Goyon, Alexandre; Guillarme, Davy; Fekete, Szabolcs

2017-02-20

In the last few years, highly efficient UHP-SEC columns packed with sub-3μm particles were commercialized by several providers. Besides the particle size reduction, the dimensions of modern SEC stationary phases (150×4.6mm) was also modified compared to regular SEC columns (300×6 or 300×8mm). Because the analytes are excluded from the pores in SEC, the retention factors are very low, ranging from -1

Correlation of sensory bitterness in dairy protein hydrolysates: Comparison of prediction models built using sensory, chromatographic and electronic tongue data.

PubMed

Newman, J; Egan, T; Harbourne, N; O'Riordan, D; Jacquier, J C; O'Sullivan, M

2014-08-01

Sensory evaluation can be problematic for ingredients with a bitter taste during research and development phase of new food products. In this study, 19 dairy protein hydrolysates (DPH) were analysed by an electronic tongue and their physicochemical characteristics, the data obtained from these methods were correlated with their bitterness intensity as scored by a trained sensory panel and each model was also assessed by its predictive capabilities. The physiochemical characteristics of the DPHs investigated were degree of hydrolysis (DH%), and data relating to peptide size and relative hydrophobicity from size exclusion chromatography (SEC) and reverse phase (RP) HPLC. Partial least square regression (PLS) was used to construct the prediction models. All PLS regressions had good correlations (0.78 to 0.93) with the strongest being the combination of data obtained from SEC and RP HPLC. However, the PLS with the strongest predictive power was based on the e-tongue which had the PLS regression with the lowest root mean predicted residual error sum of squares (PRESS) in the study. The results show that the PLS models constructed with the e-tongue and the combination of SEC and RP-HPLC has potential to be used for prediction of bitterness and thus reducing the reliance on sensory analysis in DPHs for future food research. Copyright © 2014 Elsevier B.V. All rights reserved.

Size exclusion chromatography with evaporative light scattering detection as a method for speciation analysis of polydimethylsiloxanes. III. Identification and determination of dimeticone and simeticone in pharmaceutical formulations.

PubMed

Mojsiewicz-Pieńkowska, Krystyna

2012-01-25

The pharmaceutical industry is one of the more important sectors for the use of polydimethylsiloxanes (PDMS), which belong to the organosilicon polymers. In drugs for internal use, they are used as an active pharmaceutical ingredient (API) called dimeticone or simeticone. Due to their specific chemical nature, PDMS can have different degrees of polymerization, which determine the molecular weight and viscosity. The Pharmacopoeial monographs for dimeticone and simeticone, only give the permitted polymerization and viscosity range. It is, however, essential to know also the degree of polymerization or the specific molecular weight of PDMS that are present in pharmaceutical formulations. In the literature there is information about the impact of particle size, and thus molecular weight, on the toxicity, absorption and migration in living organisms. This study focused on the use of a developed method - the exclusion chromatography with evaporative light scattering detector (SEC-ELSD) - for identification and determination of dimeticone and simeticone in various pharmaceutical formulations. The method had a high degree of specificity and was suitable for speciation analysis of these polymers. So far the developed method has not been used in the control of medicinal products containing dimeticone or simeticone. Copyright © 2011 Elsevier B.V. All rights reserved.

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.

PubMed

Petzoldt, Svenja; Kahra, Dana; Kovermann, Michael; Dingeldein, Artur P G; Niemiec, Moritz S; Ådén, Jörgen; Wittung-Stafshede, Pernilla

2015-06-01

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Interaction chromatography for characterization and large-scale fractionation of chemically heterogeneous copolymers

NASA Astrophysics Data System (ADS)

Han, Junwon

The remarkable development of polymer synthesis techniques to make complex polymers with controlled chain architectures has inevitably demanded the advancement of polymer characterization tools to analyze the molecular dispersity in polymeric materials beyond size exclusion chromatography (SEC). In particular, man-made synthetic copolymers that consist of more than one monomer type are disperse mixtures of polymer chains that have distributions in terms of both chemical heterogeneity and chain length (molar mass). While the molecular weight distribution has been quite reliably estimated by the SEC, it is still challenging to properly characterize the chemical composition distribution in the copolymers. Here, I have developed and applied adsorption-based interaction chromatography (IC) techniques as a promising tool to characterize and fractionate polystyrene-based block, random and branched copolymers in terms of their chemical heterogeneity. The first part of this thesis is focused on the adsorption-desorption based purification of PS-b-PMMA diblock copolymers using nanoporous silica. The liquid chromatography analysis and large scale purification are discussed for the PS-b-PMMA block copolymers that have been synthesized by sequential anionic polymerization. SEC and IC are compared to critically analyze the contents of PS homopolymers in the as-synthesized block copolymers. In addition, I have developed an IC technique to provide faster and more reliable information on the chemical heterogeneity in the as-synthesized block copolymers. Finally, a large scale (multi-gram) separation technique is developed to obtain "homopolymer-free" block copolymers via a simple chromatographic filtration technique. By taking advantage of the large specific surface area of nanoporous silica (≈300m 2/g), large scale purification of neat PS-b-PMMA has successfully been achieved by controlling adsorption and desorption of the block copolymers on the silica gel surface using a gravity column. The second part of this thesis is focused on the liquid chromatography analysis and fractionation of RAFT-polymerized PS-b -PMMA diblock copolymers and AFM studies. In this study, PS- b-PMMA block copolymers were synthesized by a RAFT free radical polymerization process---the PMMA block with a phenyldithiobenzoate end group was synthesized first. The contents of unreacted PS and PMMA homopolymers in as-synthesized PS-b-PMMA block copolymers were quantitatively analyzed by solvent gradient interaction chromatography (SGIC) technique employing bare silica and C18-bonded silica columns, respectively. In addition, by 2-dimensional large-scale IC fractionation method, atomic force microscopy (AFM) study of these fractionated samples revealed various morphologies with respect to the chemical composition of each fraction. The third part of this thesis is to analyze random copolymers with tunable monomer sequence distributions using interaction chromatography. Here, IC was used for characterizing the composition and monomer sequence distribution in statistical copolymers of poly(styrene-co-4-bromostyrene) (PBrxS). The PBrS copolymers were synthesized by the bromination of monodisperse polystyrenes; the degree of bromination (x) and the sequence distribution were adjusted by varying the bromination time and the solvent quality, respectively. Both normal-phase (bare silica) and reversed-phase (C18-bonded silica) columns were used at different combinations of solvents and non-solvents to monitor the content of the 4-bromostyrene units in the copolymer and their average monomer sequence distribution. The fourth part of this thesis is to analyze and fractionate highly branched polymers such as dendronized polymers and star-shaped homo and copolymers. I have developed an interaction chromatography technique to separate polymers with nonlinear chain architecture. Specifically, the IC technique has been used to separate dendronized polymers and PS-based highly branched copolymers and to ultimately obtain well-defined dendronized or branched copolymers with a low polydispersity. The effects of excess arm-polymers on (1) the micellar self-assembly of dendronized polymers and (2) the regularity of the pore morphology in the low-k applications by the sol-gel process have been studied.

Molecular weight distribution characterization of hydrophobe-modified hydroxyethyl cellulose by size-exclusion chromatography.

PubMed

Li, Yongfu; Meunier, David M; Partain, Emmett M

2014-09-12

Size-exclusion chromatography (SEC) of hydrophobe-modified hydroxyethyl cellulose (HmHEC) is challenging because polymer chains are not isolated in solution due to association of hydrophobic groups and hydrophobic interaction with column packing materials. An approach to neutralize these hydrophobic interactions was developed by adding β-cyclodextrin (β-CD) to the aqueous eluent. SEC mass recovery, especially for the higher molecular weight chains, increased with increasing concentration of β-CD in the eluent. A β-CD concentration of 0.75wt% in the eluent was determined to be optimal for the HmHEC polymers studied. These conditions enabled precise determinations of apparent molecular weight distributions exhibiting less than 2% relative standard deviation in the measured weight-average molecular weight (MW) for five injections on three studied samples and showed no significant differences in MW determined on two different days. The developed technology was shown to be very robust for characterizing HmHEC having MW from 500kg/mol to 2000kg/mol, and it can be potentially applied to other hydrophobe-modified polymers. Copyright © 2014 Elsevier B.V. All rights reserved.

Application of total-reflection X-ray fluorescence spectrometry and high-performance liquid chromatography for the chemical characterization of xylem saps of nickel contaminated cucumber plants

NASA Astrophysics Data System (ADS)

Mihucz, Victor G.; Tatár, Eniko; Varga, Anita; Záray, Gyula; Cseh, Edit

2001-11-01

Total-reflection X-ray fluorescence (TXRF) spectrometry, reversed-phase (RP) and size-exclusion (SE) high-performance liquid chromatography (HPLC) methods were applied for the characterization of low-volume xylem sap of control and nickel contaminated cucumber plants growing in hydroponics containing urea as the sole nitrogen source. In these saps collected for 1 h, Ca, K, Fe, Mn, Ni, Zn, as well as malic, citric and fumaric acids were determined. The SEC measurements showed that macromolecules were not detectable in the samples. Nickel contamination had minimum impact on the organic acid transport, however, the transport of Zn, K and Fe was reduced by 50, 22 and 11%, respectively. This observation supports the results of our earlier experiments when nitrate ions were used as the sole nitrogen form. At the same time, the fresh root weight and the volume of the collected xylem sap increased by 36 and 85%, respectively. Therefore, nickel addition seemed to decrease the urea toxicity of the plants. By pooling the eluting fractions of the SEC column, which were 10-fold concentrated by freeze-drying, the series of the resulted samples were analyzed by the TXRF spectrometry and RP-HPLC. The three organic acids could be identified in only one of the fractions, which contained Fe and, in the case of the contaminated plants, Ni in detectable concentration. However, considerable parts of these two elements and Mn, as well as practically the total amounts of Cu may be transported by unidentified organic compounds in the xylem.

In vitro digestion method for estimation of copper bioaccessibility in Açaí berry.

PubMed

Ruzik, Lena; Wojcieszek, Justyna

Copper is an essential trace element for humans and its deficiency can lead to numerous diseases. A lot of mineral supplements are available to increase intake of copper. Unfortunately, only a part of the total concentration of elements is available for human body. Thus, the aim of the study was to determine bioaccessibility of copper in Açai berry, known as a "superfood" because of its antioxidant qualities. An analytical methodology was based on size exclusion chromatography (SEC) coupled to a mass spectrometer with inductively coupled plasma (ICP MS) and on capillary liquid chromatography coupled to tandem mass spectrometer with electrospray ionization (µ-HPLC-ESI MS/MS). To extract various copper compounds, berries were treated with the following buffers: ammonium acetate, Tris-HCl, and sodium dodecyl sulfate (SDS). The best extraction efficiency of copper was obtained for SDS extract (88 %), while results obtained for Tris-HCl and ammonium acetate were very similar (47 and 48 %, respectively). After SEC-ICP-MS analysis, main signal was obtained for all extracts in the region of molecular mass about 17 kDa. A two-step model simulated gastric (pepsin) and gastrointestinal (pancreatin) digestion was used to obtain the knowledge about copper bioaccessibility. Copper compounds present in Açai berry were found to be highly bioaccessible. The structures of five copper complexes with amino acids such as aspartic acid, tyrosine, phenylalanine, were proposed after µ-HPLC-ESI MS/MS analysis. Obtained results show that copper in enzymatic extracts is bound by amino acids and peptides what leads to better bioavailability of copper for human body.

Theory of chromatography of partially cyclic polymers: Tadpole-type and manacle-type macromolecules.

PubMed

Vakhrushev, Andrey V; Gorbunov, Alexei A

2016-02-12

A theory of chromatography is developed for partially cyclic polymers of tadpole- and manacle-shaped topological structures. We present exact equations for the distribution coefficient K at different adsorption interactions; simpler approximate formulae are also derived, relevant to the conditions of size-exclusion, adsorption, and critical chromatography. Theoretical chromatograms of heterogeneous partially cyclic polymers are simulated, and conditions for good separation by topology are predicted. According to the theory, an effective SEC-radius of tadpoles and manacles is mostly determined by the molar mass M, and by the linear-cyclic composition. In the interactive chromatography, the effect of molecular topology on the retention becomes significant. At the critical interaction point, partial dependences K(Mlin) and K(Mring) are qualitatively different: while being almost independent of Mlin, K increases with Mring. This behavior could be realized in critical chromatography-for separation of partially cyclic polymers by the number and molar mass of cyclic elements. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative characterization of solid epoxy resins using comprehensive two dimensional liquid chromatography coupled with electrospray ionization-time of flight mass spectrometry.

PubMed

Julka, Samir; Cortes, Hernan; Harfmann, Robert; Bell, Bruce; Schweizer-Theobaldt, Andreas; Pursch, Matthias; Mondello, Luigi; Maynard, Shawn; West, David

2009-06-01

A comprehensive multidimensional liquid chromatography system coupled to Electrospray Ionization-Mass Spectrometry (LCxLC-ESI-MS) was developed for detailed characterization and quantitation of solid epoxy resin components. The two orthogonal modes of separation selected were size exclusion chromatography (SEC) in the first dimension and liquid chromatography at critical conditions (LCCC) in the second dimension. Different components present in the solid epoxy resins were separated and quantitated for the first time based on the functional groups and molecular weight heterogeneity. Coupling LCxLC separations with mass spectrometry enabled the identification of components resolved in the two-dimensional space. Several different functional group families of compounds were separated and identified, including epoxy-epoxy and epoxy-alpha-glycol functional oligomers, and their individual molecular weight ranges were determined. Repeatability obtained ranged from 0.5% for the main product to 21% for oligomers at the 0.4% concentration level.

Identification of selenium-containing proteins in HEK 293 kidney cells using multiple chromatographies, LC-ICPMS and nano-LC-ESIMS.

PubMed

Chitta, Karnakar R; Landero-Figueroa, Julio A; Kodali, Phanichand; Caruso, Joseph A; Merino, Edward J

2013-09-30

Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS). Copyright © 2013 Elsevier B.V. All rights reserved.

Size exclusion chromatography for semipreparative scale separation of Au38(SR)24 and Au40(SR)24 and larger clusters.

PubMed

Knoppe, Stefan; Boudon, Julien; Dolamic, Igor; Dass, Amala; Bürgi, Thomas

2011-07-01

Size exclusion chromatography (SEC) on a semipreparative scale (10 mg and more) was used to size-select ultrasmall gold nanoclusters (<2 nm) from polydisperse mixtures. In particular, the ubiquitous byproducts of the etching process toward Au(38)(SR)(24) (SR, thiolate) clusters were separated and gained in high monodispersity (based on mass spectrometry). The isolated fractions were characterized by UV-vis spectroscopy, MALDI mass spectrometry, HPLC, and electron microscopy. Most notably, the separation of Au(38)(SR)(24) and Au(40)(SR)(24) clusters is demonstrated.

Size-exclusion chromatography of perfluorosulfonated ionomers.

PubMed

Mourey, T H; Slater, L A; Galipo, R C; Koestner, R J

2011-08-26

A size-exclusion chromatography (SEC) method in N,N-dimethylformamide containing 0.1 M LiNO(3) is shown to be suitable for the determination of molar mass distributions of three classes of perfluorosulfonated ionomers, including Nafion(®). Autoclaving sample preparation is optimized to prepare molecular solutions free of aggregates, and a solvent exchange method concentrates the autoclaved samples to enable the use of molar-mass-sensitive detection. Calibration curves obtained from light scattering and viscometry detection suggest minor variation in the specific refractive index increment across the molecular size distributions, which introduces inaccuracies in the calculation of local absolute molar masses and intrinsic viscosities. Conformation plots that combine apparent molar masses from light scattering detection with apparent intrinsic viscosities from viscometry detection partially compensate for the variations in refractive index increment. The conformation plots are consistent with compact polymer conformations, and they provide Mark-Houwink-Sakurada constants that can be used to calculate molar mass distributions without molar-mass-sensitive detection. Unperturbed dimensions and characteristic ratios calculated from viscosity-molar mass relationships indicate unusually free rotation of the perfluoroalkane backbones and may suggest limitations to applying two-parameter excluded volume theories for these ionomers. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel guanosine-cytidine dinucleoside that self-assembles into a trimeric supramolecule.

PubMed

Sessler, Jonathan L; Jayawickramarajah, Janarthanan; Sathiosatham, Muhunthan; Sherman, Courtney L; Brodbelt, Jennifer S

2003-07-24

[reaction: see text] Synthesis and assembly studies of a guanosine-cytidine dinucleoside 1 that self-assembles into a trimeric supramolecule (I) are presented. Dinucleoside 1 was obtained by utilizing two consecutive palladium-catalyzed cross-coupling reactions. Ensemble I was analyzed by ESI-MS, NMR spectroscopies, size exclusion chromatography (SEC), and vapor pressure osmometry (VPO).

Mineralization of pyrrole, a recalcitrant heterocyclic compound, by electrochemical method: Multi-response optimization and degradation mechanism.

PubMed

Hiwarkar, Ajay Devidas; Singh, Seema; Srivastava, Vimal Chandra; Mall, Indra Deo

2017-08-01

In this study, the electrochemical (EC) oxidation of a recalcitrant heterocyclic compound namely pyrrole has been reported using platinum coated titanium (Pt/Ti) electrodes. Response surface methodology (RSM) comprising of full factorial central composite design (CCD) with four factors and five levels has been used to examine the effects of different operating parameters such as current density (j), aqueous solution pH, conductivity (k) and treatment time (t) in an EC batch reactor. Pyrrole mineralization in aqueous solution was examined with multiple responses such as chemical oxygen demand (COD) (response, Y 1 ) and specific energy consumption (SEC) in kWh/kg of COD removed (response, Y 2 ). During multiple response optimization, the desirability function approach was employed to concurrently maximize Y 1 and minimize Y 2 . At the optimum condition, 82.9% COD removal and 7.7 kWh/kg of COD removed were observed. Degradation mechanism of pyrrole in wastewater was elucidated at the optimum condition of treatment by using UV-visible spectroscopy, Fourier transformed infra-red spectroscopy (FTIR), cyclic voltammetry (CV), ion chromatography (IC), higher performance liquid chromatography (HPLC) and gas chromatography-mass spectroscopy (GC-MS). The degradation pathway of pyrrole was proposed on the basis of the various analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Power of Ultra Performance Liquid Chromatography/Electrospray Ionization-MS Reconstructed Ion Chromatograms in the Characterization of Small Differences in Polymer Microstructure.

PubMed

Epping, Ruben; Panne, Ulrich; Falkenhagen, Jana

2018-03-06

From simple homopolymers to functionalized, 3-dimensional structured copolymers, the complexity of polymeric materials has become more and more sophisticated. With new applications, for instance, in the semiconductor or pharmaceutical industry, the requirements for the characterization have risen with the complexity of the used polymers. For each additional distribution, an additional dimension in analysis is needed. Small, often isomeric heterogeneities in topology or microstructure can usually not be simply separated chromatographically or distinguished by any common detector but affect the properties of materials significantly. For a drug delivery system, for example, the degree of branching and branching distribution is crucial for the formation of micelles. Instead of a complicated, time-consuming, and/or expensive 2D-chromatography or ion mobility spectrometry (IMS) method, that also has its limitations, in this work, a simple approach using size exclusion chromatography (SEC) coupled with electrospray ionization (ESI) mass spectrometry is proposed. The online coupling allows the analysis of reconstructed ion chromatograms (RICs) of each degree of polymerization. While a complete separation often cannot be achieved, the derived retention times and peak widths lead to information on the existence and dispersity of heterogeneities. Although some microstructural heterogeneities like short chain branching can for large polymers be characterized with methods such as light scattering, for oligomers where the heterogeneities just start to form and their influence is at the maximum, they are inaccessible with these methods. It is also shown that with a proper calibration even quantitative information can be obtained. This method is suitable to detect small differences in, e.g., branching, 3D-structure, monomer sequence, or tacticity and could potentially be used in routine analysis to quickly determine deviations.

Rate of hexabromocyclododecane decomposition and production of brominated polycyclic aromatic hydrocarbons during combustion in a pilot-scale incinerator.

PubMed

Miyake, Yuichi; Tokumura, Masahiro; Wang, Qi; Amagai, Takashi; Horii, Yuichi

2017-11-01

Here, we examined the incineration of extruded polystyrene containing hexabromocyclododecane (HBCD) in a pilot-scale incinerator under various combustion temperatures (800-950°C) and flue gas residence times (2-8sec). Rates of HBCD decomposition ranged from 99.996% (800°C, 2sec) to 99.9999% (950°C, 8sec); the decomposition of HBCD, except during the initial stage of combustion (flue gas residence time<2sec), followed a pseudo-first-order kinetics model. An Arrhenius plot revealed that the activation energy and frequency factor of the decomposition of HBCD by combustion were 14.2kJ/mol and 1.69sec -1 , respectively. During combustion, 11 brominated polycyclic aromatic hydrocarbons (BrPAHs) were detected as unintentional by-products. Of the 11 BrPAHs detected, 2-bromoanthracene and 1-bromopyrene were detected at the highest concentrations. The mutagenic and carcinogenic BrPAHs 1,5-dibromoanthracene and 1-bromopyrene were most frequently detected in the flue gases analyzed. The total concentration of BrPAHs exponentially increased (range, 87.8-2,040,000ng/m 3 ) with increasing flue gas residence time. Results from a qualitative analysis using gas chromatography/high-resolution mass spectrometry suggest that bromofluorene and bromopyrene (or fluoranthene) congeners were also produced during the combustion. Copyright © 2017. Published by Elsevier B.V.

Chromatographic study of highly methoxylated lime pectins deesterified by different pectin methyl-esterases.

PubMed

Ralet, M C; Bonnin, E; Thibault, J F

2001-03-25

The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). "Homogeneous" populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

Application of SEC-ICP-MS for comparative analyses of metal-containing species in cancerous and healthy human thyroid samples.

PubMed

Boulyga, Sergei F; Loreti, Valeria; Bettmer, Jörg; Heumann, Klaus G

2004-09-01

Size exclusion chromatography (SEC) was coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for speciation study of trace metals in cancerous thyroid tissues in comparison to healthy thyroids aimed to estimation of changes in metalloprotein speciation in pathological tissue. The study showed a presence of species binding Cu, Zn, Cd and Pb in healthy thyroid tissue with a good reproducibility of chromatographic results, whereas the same species could not be detected in cancerous tissues. Thus, remarkable differences with respect to metal-binding species were revealed between healthy and pathological thyroid samples, pointing out a completely different distribution of trace metals in cancerous tissues. The metal-binding species could not be identified in the frame of this work because of a lack of appropriate standards. Nevertheless, the results obtained confirm the suitability of SEC-ICP-MS for monitoring of changes in trace metal distribution in cancerous tissue and will help to better understand the role of metal-containing species in thyroid pathology.

Comparison of different methods for MP detection: What can we learn from them, and why asking the right question before measurements matters?

PubMed

Elert, Anna M; Becker, Roland; Duemichen, Erik; Eisentraut, Paul; Falkenhagen, Jana; Sturm, Heinz; Braun, Ulrike

2017-12-01

In recent years, an increasing trend towards investigating and monitoring the contamination of the environment by microplastics (MP) (plastic pieces < 5 mm) has been observed worldwide. Nonetheless, a reliable methodology that would facilitate and automate the monitoring of MP is still lacking. With the goal of selecting practical and standardized methods, and considering the challenges in microplastics detection, we present here a critical evaluation of two vibrational spectroscopies, Raman and Fourier transform infrared (FTIR) spectroscopy, and two extraction methods: thermal extraction desorption gas chromatography mass spectrometry (TED-GC-MS) and liquid extraction with subsequent size exclusion chromatography (SEC) using a soil with known contents of PE, PP, PS and PET as reference material. The obtained results were compared in terms of measurement time, technique handling, detection limits and requirements for sample preparation. The results showed that in designing and selecting the right methodology, the scientific question that determines what needs to be understood is significant, and should be considered carefully prior to analysis. Depending on whether the object of interest is quantification of the MP particles in the sample, or merely a quick estimate of sample contamination with plastics, the appropriate method must be selected. To obtain overall information about MP in environmental samples, the combination of several parallel approaches should be considered. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization and Antiproliferative Effect of Novel Acid Polysaccharides from the Spent Substrate of Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Cultivation.

PubMed

Zhang, Yong; Liu, Wei; Xu, Chunping; Huang, Wei; He, Peixin

2017-01-01

In this study, a high yield of crude polysaccharide (16.73 ± 0.756%) was extracted from the spent mushroom substrate of Lentinus edodes using a hot alkali extraction method. Two groups of polysaccharides (designated as LSMS-1 and LSMS-2) were obtained from the crude extract by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index detector system. The weight-average molar masses of LSMS-1 and LSMS-2 were determined to be 6.842 × 106 and 2.154 × 106 g/mol, respectively. The SEC/MALLS analysis revealed that the molecular shapes of LSMS-1 and LSMS-2 were sphere-like forms in aqueous solution. Carbohydrate composition analysis using chromatography--mass spectrometry revealed that they were both acid heteropolysaccharides. LSMS-1 comprised mainly glucose and galacturonic acid, whereas LSMS-2 mainly consisted of xylose and glucuronic acid. Fourier transform infrared spectral analysis of the purified fractions revealed typical characteristic polysaccharide groups. In addition, MTT assays with refined polysaccharide doses of 25, 50, 100, 200, and 400 µg/mL suggested that both of the polysaccharide fractions exhibited antiproliferative activity against 6 tested human tumor cell lines in a concentration-dependent manner, and LSMS-2 had better anticancer capacity in vitro than LSMS-1. The inhibition ratio of LSMS-2 against A549 human lung cancer cells, the SGC7901 gastric cancer cell line, MCF-7 breast cancer cells, the U937 histiocytic lymphoma cell line, and the MG-63 human osteosarcoma cell line reached 43.55%, 29.97%, 19.63%, 18.24%, and 17.93%, respectively, at a concentration of 400 µg/mL.

Separation of Poly(styrene-block-t-butyl methacrylate) Copolymers by Various Liquid Chromatography Techniques

PubMed Central

Šmigovec Ljubič, Tina; Pahovnik, David; Žigon, Majda; Žagar, Ema

2012-01-01

The separation of a mixture of three poly(styrene-block-t-butyl methacrylate) copolymers (PS-b-PtBMA), consisting of polystyrene (PS) blocks of similar length and t-butyl methacrylate (PtBMA) blocks of different lengths, was performed using various chromatographic techniques, that is, a gradient liquid chromatography on reversed-phase (C18 and C8) and normal-phase columns, a liquid chromatography under critical conditions for polystyrene as well as a fully automated two-dimensional liquid chromatography that separates block copolymers by chemical composition in the first dimension and by molar mass in the second dimension. The results show that a partial separation of the mixture of PS-b-PtBMA copolymers can be achieved only by gradient liquid chromatography on reversed-phase columns. The coelution of the two block copolymers is ascribed to a much shorter PtBMA block length, compared to the PS block, as well as a small difference in the length of the PtBMA block in two of these copolymers, which was confirmed by SEC-MALS and NMR spectroscopy. PMID:22489207

Assessing the heterogeneity level in lipid nanoparticles for siRNA delivery: size-based separation, compositional heterogeneity, and impact on bioperformance.

PubMed

Zhang, Jingtao; Pei, Yi; Zhang, Hangchun; Wang, Lei; Arrington, Leticia; Zhang, Ye; Glass, Angela; Leone, Anthony M

2013-01-07

A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and in vitro performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60-190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60-150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated, and fractions were studied in-depth for potential presence of polydispersity in size, composition, and in vitro silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semipreparative SEC fractionation that showed similar size, chemical composition, in vitro silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less of a challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the in vitro silencing activity. The impact of LNP size and formulation composition on in vitro performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality.

The Path of Carbon in Photosynthesis XXI. The Cyclic Regeneration of Carbon Dioxide Acceptor

DOE R&D Accomplishments Database

Bassham, J. A.; Benson, A. A.; Kay, Lorel D.; Harris, Anne Z.; Wilson, A. T.; Calvin, M.

1953-10-01

Photosynthesizing plants have been exposed to C{sup 14}O{sub 2} for short periods of time (0.4 to 15 sec.) and the products of carbon dioxide reduction analyzed by paper chromatography and radio autography. Methods have been developed for the degradation of ribulose and sedoheptulose. These sugars, obtained as their phosphate esters from the above C{sup 14}O{sub 2} exposures and from other experiments, have been degraded and their distribution of radiocarbon determined. The distribution of radiocarbon in these sugars, and other data, indicate that sedoheptulose phosphate and ribulose diphosphates are formed during photosynthesis from triose and hexose phosphates, the latter being synthesized, in turn, by the reduction of 3-phosphoglyceric acid.

Strain gage measurement errors in the transient heating of structural components

NASA Technical Reports Server (NTRS)

Richards, W. Lance

1993-01-01

Significant strain-gage errors may exist in measurements acquired in transient thermal environments if conventional correction methods are applied. Conventional correction theory was modified and a new experimental method was developed to correct indicated strain data for errors created in radiant heating environments ranging from 0.6 C/sec (1 F/sec) to over 56 C/sec (100 F/sec). In some cases the new and conventional methods differed by as much as 30 percent. Experimental and analytical results were compared to demonstrate the new technique. For heating conditions greater than 6 C/sec (10 F/sec), the indicated strain data corrected with the developed technique compared much better to analysis than the same data corrected with the conventional technique.

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 8 2011-10-01 2011-10-01 false Method of payment. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE TO BE FOLLOWED BY GENERAL... Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods.

PubMed

Gimbert, Frédéric; Geffard, Alain; Guédron, Stéphane; Dominik, Janusz; Ferrari, Benoit J D

2016-02-01

Along with the growing body of evidence that total internal concentration is not a good indicator of toxicity, the Critical Body Residue (CBR) approach recently evolved into the Tissue Residue Approach (TRA) which considers the biologically active portion of metal that is available to contribute to the toxicity at sites of toxic action. For that purpose, we examined total mercury (Hg) bioaccumulation and subcellular fractionation kinetics in fourth stage larvae of the midge Chironomus riparius during a four-day laboratory exposure to Hg-spiked sediments and water. The debris (including exoskeleton, gut contents and cellular debris), granule and organelle fractions accounted only for about 10% of the Hg taken up, whereas Hg concentrations in the entire cytosolic fraction rapidly increased to approach steady-state. Within this fraction, Hg compartmentalization to metallothionein-like proteins (MTLP) and heat-sensitive proteins (HSP), consisting mostly of enzymes, was assessed in a comparative manner by two methodologies based on heat-treatment and centrifugation (HT&C method) or size exclusion chromatography separation (SECS method). The low Hg recoveries obtained with the HT&C method prevented accurate analysis of the cytosolic Hg fractionation by this approach. According to the SECS methodology, the Hg-bound MTLP fraction increased linearly over the exposure duration and sequestered a third of the Hg flux entering the cytosol. In contrast, the HSP fraction progressively saturated leading to Hg excretion and physiological impairments. This work highlights several methodological and biological aspects to improve our understanding of Hg toxicological bioavailability in aquatic invertebrates. Copyright © 2015 Elsevier B.V. All rights reserved.

Studies on the primary structure of short polysaccharides using SEC MALDI mass spectroscopy.

PubMed

Garozzo, D; Spina, E; Cozzolino, R; Cescutti, P; Fett, W F

2000-01-12

The introduction of size-exclusion chromatography (SEC) analysis of polysaccharides prior to MALDI mass spectroscopy accounts for the determination of the molecular mass of the repeating unit when neutral homopolymers are investigated. In the case of natural polysaccharides characterised by more complicated structural features (presence of non-carbohydrate substituents, charged groups, etc.), this mass value usually is in agreement with more than one sugar composition. Therefore, it is not sufficient to give the correct monosaccharidic composition of the polysaccharide investigated. To solve this problem, MALDI spectra were recorded on the permethylated sample and post-source decay experiments were performed on precursor ions. In this way, the composition (in terms of Hex, HexNAc, etc.), size and sequence of the repeating unit were determined.

Use of elemental and molecular-mass spectrometry to assess the toxicological effects of inorganic mercury in the mouse Mus musculus.

PubMed

García-Sevillano, Miguel Angel; García-Barrera, Tamara; Navarro, Francisco; Gailer, Jürgen; Gómez-Ariza, José Luiz

2014-09-01

The biochemical response of mice (Mus musculus) to acute subcutaneous inorganic-mercury exposure was assessed over a 14-day period by analyzing cytosolic extracts of the liver, the kidneys, and blood plasma. Integrated metallomic and metabolomic approaches using elemental and molecular-mass spectrometry were used to obtain comprehensive insight into the toxicological effects of mercury regarding its distribution and possible perturbation of metabolic pathways. The metallomic approach involved the use of size-exclusion chromatography (SEC) coupled with multiaffinity chromatography inductively coupled plasma-mass spectrometry (ICP-MS) and isotopic-dilution analysis. The metabolomic approach involved the direct infusion of polar and lipophilic tissue extracts into a mass spectrometer (DIMS) in the positive and negative acquisition mode (ESI+and ESI-). The use of SEC-ICP-MS enabled us to detect changes in the metalloproteome in the liver and the kidneys during the exposure period, and revealed that interactions between Hg and endogenous Cu and Zn adversely affected the homeostasis of these essential metals. The detection of an Hg-Se detoxification product in mouse plasma substantiated the known interaction between Hg and Se in mammals. Use of DIMS in conjunction with partial-least-squares discriminant analysis (PLS-DA) uncovered time-dependent changes of endogenous metabolites over time, corroborated by histopathology investigation of specific mouse tissues. The perturbations of endogenous metabolic profiles were explained in terms of the adverse effect of mercury on energy metabolism (e.g. glycolysis, Krebs cycle), the degradation of membrane phospholipids (apoptosis), and increased levels of specific lipids in plasma. In summary, use of an SEC-ICP-MS-based metallomics approach in conjunction with molecular-mass-spectrometry-based metabolomics is revealed as a promising strategy to more comprehensively investigate the toxicological effects of harmful environmental pollutants and xenobiotics.

Qualitative and quantitative analysis of heparin and low molecular weight heparins using size exclusion chromatography with multiple angle laser scattering/refractive index and inductively coupled plasma/mass spectrometry detectors.

PubMed

Ouyang, Yilan; Zeng, Yangyang; Yi, Lin; Tang, Hong; Li, Duxin; Linhardt, Robert J; Zhang, Zhenqing

2017-11-03

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of chromatography and mass spectrometry to the characterization of cobalt, copper, manganese and molybdenum in Morinda citrifolia.

PubMed

Rybak, Justyna; Ruzik, Lena

2013-03-15

An analytical procedure was proposed to determine the manganese species and to study the fractionation of microelements such as copper, cobalt and molybdenum in Noni juice. Morinda citrifolia is known as a noni fruit, Indian mulberry, nunaakai, dog dumpling, mengkudu, beach mulberry, vomit fruit and cheese fruit. It is a tropical plant with a long tradition of medicinal use in Polynesia and tropical parts of eastern Asia and Australia. This article covers the determination of manganese species in Noni juice and established by fractionation by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC ICP MS) and next characterization of species by electrospray ionization mass spectrometry (ESI MS). Also presented the fractionation analysis of copper, cobalt and molybdenum in Noni juice sample using SEC ICP MS - juice was treated with buffer and enzymatic extraction media and analyzed. For the evaluation of the amounts of the metal fractions distinguished, the ICP MS was used off-line prior to the determination of copper, cobalt, molybdenum and manganese concentrations in the juice. It was established that elements are present in the analyzed samples in different species and their concentration is μg mL(-1) and ng mL(-1) range in fruit. The accuracy of the entire fractionation scheme and sample preparation procedures involved was verified by the performance of the recovery test. For the information about the bioavailability of these elements, in vitro bioavailability investigation was used by SEC ICP MS technique. Two step digestion model simulating gastric (pepsin digestion) and intestinal (pancreatin digestion) juices. In Noni juice, manganese is complexed from flavonoids - rutin, from dye like anthraquinone (alizarin) and glycosides - asperulosidic acid (ESI MS - characterization). The study shows that copper and molybdenum contained in Noni juice are complexed by peptides, and cobalt by organic acids (which are 3.6% of juice). Molybdenum in the sample is also bound by the polysaccharides (SEC ICP MS). In addition, compounds complexing manganese, copper and molybdenum are hydrophobic proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

Flow field-flow fractionation for the analysis of nanoparticles used in drug delivery.

PubMed

Zattoni, Andrea; Roda, Barbara; Borghi, Francesco; Marassi, Valentina; Reschiglian, Pierluigi

2014-01-01

Structured nanoparticles (NPs) with controlled size distribution and novel physicochemical features present fundamental advantages as drug delivery systems with respect to bulk drugs. NPs can transport and release drugs to target sites with high efficiency and limited side effects. Regulatory institutions such as the US Food and Drug Administration (FDA) and the European Commission have pointed out that major limitations to the real application of current nanotechnology lie in the lack of homogeneous, pure and well-characterized NPs, also because of the lack of well-assessed, robust routine methods for their quality control and characterization. Many properties of NPs are size-dependent, thus the particle size distribution (PSD) plays a fundamental role in determining the NP properties. At present, scanning and transmission electron microscopy (SEM, TEM) are among the most used techniques to size characterize NPs. Size-exclusion chromatography (SEC) is also applied to the size separation of complex NP samples. SEC selectivity is, however, quite limited for very large molar mass analytes such as NPs, and interactions with the stationary phase can alter NP morphology. Flow field-flow fractionation (F4) is increasingly used as a mature separation method to size sort and characterize NPs in native conditions. Moreover, the hyphenation with light scattering (LS) methods can enhance the accuracy of size analysis of complex samples. In this paper, the applications of F4-LS to NP analysis used as drug delivery systems for their size analysis, and the study of stability and drug release effects are reviewed. Copyright © 2013 Elsevier B.V. All rights reserved.

Three-dimensional fluorescence analysis of chernozem humic acids and their electrophoretic fractions

NASA Astrophysics Data System (ADS)

Trubetskoi, O. A.; Trubetskaya, O. E.

2017-09-01

Polyacrylamide gel electrophoresis in combination with size-exclusion chromatography (SEC-PAGE) has been used to obtain stable electrophoretic fractions of different molecular size (MS) from chernozem humic acids (HAs). Three-dimensional fluorescence charts of chernozem HAs and their fractions have been obtained for the first time, and all fluorescence excitation-emission maxima have been identified in the excitation wavelength range of 250-500 nm. It has been found that fractionation by the SEC-PAGE method results in a nonuniform distribution of protein- and humin-like fluorescence of the original HA preparation among the electrophoretic fractions. The electrophoretic fractions of the highest and medium MSs have only the main protein-like fluorescence maximum and traces of humin-like fluorescence. In the electrophoretic fraction of the lowest MS, the intensity of protein-like fluorescence is low, but the major part of humin-like fluorescence is localized there. Relationships between the intensity of protein-like fluorescence and the weight distribution of amino acids have been revealed, as well as between the degree of aromaticity and the intensity of humin-like fluorescence in electrophoretic fractions of different MSs. The obtained relationships can be useful in the interpretation of the spatial structural organization and ecological functions of soil HAs.

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

PubMed

Sandra, Koen; Steenbeke, Mieke; Vandenheede, Isabel; Vanhoenacker, Gerd; Sandra, Pat

2017-11-10

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

PubMed Central

Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

2013-01-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

PubMed

Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I

2013-12-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.

Aggregation factor analysis for protein formulation by a systematic approach using FTIR, SEC and design of experiments techniques.

PubMed

Feng, Yan Wen; Ooishi, Ayako; Honda, Shinya

2012-01-05

A simple systematic approach using Fourier transform infrared (FTIR) spectroscopy, size exclusion chromatography (SEC) and design of experiments (DOE) techniques was applied to the analysis of aggregation factors for protein formulations in stress and accelerated testings. FTIR and SEC were used to evaluate protein conformational and storage stabilities, respectively. DOE was used to determine the suitable formulation and to analyze both the main effect of single factors and the interaction effect of combined factors on aggregation. Our results indicated that (i) analysis at a low protein concentration is not always applicable to high concentration formulations; (ii) an investigation of interaction effects of combined factors as well as main effects of single factors is effective for improving conformational stability of proteins; (iii) with the exception of pH, the results of stress testing with regard to aggregation factors would be available for suitable formulation instead of performing time-consuming accelerated testing; (iv) a suitable pH condition should not be determined in stress testing but in accelerated testing, because of inconsistent effects of pH on conformational and storage stabilities. In summary, we propose a three-step strategy, using FTIR, SEC and DOE techniques, to effectively analyze the aggregation factors and perform a rapid screening for suitable conditions of protein formulation. Copyright © 2011 Elsevier B.V. All rights reserved.

SPR Biosensors in Direct Molecular Fishing: Implications for Protein Interactomics.

PubMed

Florinskaya, Anna; Ershov, Pavel; Mezentsev, Yuri; Kaluzhskiy, Leonid; Yablokov, Evgeniy; Medvedev, Alexei; Ivanov, Alexis

2018-05-18

We have developed an original experimental approach based on the use of surface plasmon resonance (SPR) biosensors, applicable for investigation of potential partners involved in protein⁻protein interactions (PPI) as well as protein⁻peptide or protein⁻small molecule interactions. It is based on combining a SPR biosensor, size exclusion chromatography (SEC), mass spectrometric identification of proteins (LC-MS/MS) and direct molecular fishing employing principles of affinity chromatography for isolation of potential partner proteins from the total lysate of biological samples using immobilized target proteins (or small non-peptide compounds) as ligands. Applicability of this approach has been demonstrated within the frame of the Human Proteome Project (HPP) and PPI regulation by a small non-peptide biologically active compound, isatin.

Mercury and selenium binding biomolecules in terrestrial mammals (Cervus elaphus and Sus scrofa) from a mercury exposed area.

PubMed

Ropero, M J Patiño; Fariñas, N Rodríguez; Krupp, E; Mateo, R; Nevado, J J Berzas; Martín-Doimeadios, R C Rodríguez

2016-06-01

Mercury (Hg) is likely bound to large biomolecules (e.g. proteins) in living organisms, and in order to assess Hg metabolic pathways and possible toxicological effects, it is essential to study these Hg containing biomolecules. However, the exact nature of most metal binding biomolecules is unknown. Such studies are still in their infancy and information on this topic is scarce because the analysis is challenging, mainly due to their lability upon digestion or extraction from the tissue. New analytical methods that allow complex Hg-biomolecules to be analysed intact are needed and only few very recent studies deal with this approach. Therefore, as an initial step towards the characterization of Hg containing biomolecules, an analytical procedure has been optimised using size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS) detection. We applied this technique to elucidate the distribution and elution profile of Hg and Se, and some physiological important elements such as Fe, Ni, Zn and Cu, to assess metal binding profiles in liver and kidney samples of red deer (Cervus elaphus) and wild boar (Sus scrofa) who roam freely within the largest Hg mining district on Earth, Almadén in Spain. Elemental fractionation profiles of the extracts from different tissues were obtained using two different SEC columns (BioSep-SEC-S2000 GL 300-1kDa and Superdex 75 10/300 GL 70-3kDa). Similar profiles of Hg were observed in red deer and wild boar; however, significant differences were evident for liver and kidney. Moreover, the profiles of Se showed a single peak at high-medium molecular weight in all investigated tissues, while co-elution of Hg with Fe, Ni, Zn and Cu was observed. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer.

PubMed

Hong, Chang-Sook; Funk, Sonja; Muller, Laurent; Boyiadzis, Michael; Whiteside, Theresa L

2016-01-01

Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications.

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor.

PubMed

Knight, Martha; Finn, Thomas M; Zehmer, John; Clayton, Adam; Pilon, Aprile

2011-09-09

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor. Copyright © 2011 Elsevier B.V. All rights reserved.

Compositions and methods for making selenocysteine containing polypeptides

DOEpatents

Soll, Dieter; Aldag, Caroline; Hohn, Michael

2016-10-11

Non-naturally occurring tRNA.sup.Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA.sup.Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA.sup.Sec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA.sup.Sec.

Identification of isopropylbiphenyl, alkyl diphenylmethanes, diisopropylnaphthalene, linear alkyl benzenes and other polychlorinated biphenyl replacement compounds in effluents, sediments and fish in the Fox River System, Wisconsin

USGS Publications Warehouse

Peterman, Paul H.; Delfino, Joseph J.

1990-01-01

Five polychlorinated biphenyl replacement dye solvents and a diluent present in carbonless copy paper were identified by gas chromatography/mass spectrometry in the following matrices: effluents from a de-inking–recycling paper mill and a municipal wastewater treatment plant receiving wastewaters from a carbonless copy paper manufacturing plant; sediments; and fish collected near both discharges in the Fox River System, Wisconsin. An isopropylbiphenyl dye solvent mixture included mono-, di- and triisopropylbiphenyls. Also identified were two dye solvent mixtures marketed under the trade name Santosol. Santosol 100 comprised ethyl-diphenylmethanes (DPMs), benzyl-ethyl-DPMs, and dibenzyl-ethyl-DPMs. Similarly, Santosol 150 comprised dimethyl-DPMs, benzyl-dimethyl-DPMs, and dibenzyl-dimethyl-DPMs. Diisopropylnaphthalenes, widely used as a dye solvent in Japan, were identified for the first time in the US environment. sec-Butylbiphenyls and di-sec-butylbiphenyls, likely constituents of a sec-butylbiphenyl dye solvent mixture, were tentatively identified. Linear alkyl benzenes (C10 to C13-LABs) constituted the Alkylate 215 diluent mixture. Although known to occur as minor constituents in linear alkyl sulfonate detergents, LAB residues have not been previously attributed to commercial use of LABs.

Simultaneous determination of chromones and coumarins in Radix Saposhnikoviae by high performance liquid chromatography with diode array and tandem mass detectors.

PubMed

Kim, Min Kyung; Yang, Dong-Hyug; Jung, Mihye; Jung, Eun Ha; Eom, Han Young; Suh, Joon Hyuk; Min, Jung Won; Kim, Unyong; Min, Hyeyoung; Kim, Jinwoong; Han, Sang Beom

2011-09-16

Methods using high performance liquid chromatography with diode array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) were developed and validated for the simultaneous determination of 5 chromones and 6 coumarins: prim-O-glucosylcimifugin (1), cimifugin (2), nodakenin (3), 4'-O-β-d-glucosyl-5-O-methylvisamminol (4), sec-O-glucosylhamaudol (5), psoralen (6), bergapten (7), imperatorin (8), phellopterin (9), 3'-O-angeloylhamaudol (10) and anomalin (11), in Radix Saposhnikoviae. The separation conditions for HPLC-DAD were optimized using an Ascentis Express C18 (4.6 mm×100 mm, 2.7 μm particle size) fused-core column. The mobile phase was composed of 10% aqueous acetonitrile (A) and 90% acetonitrile (B) and the elution was performed under a gradient mode at a flow rate of 1.0 mL/min. The detection wavelength was set at 300 nm. The HPLC-DAD method yielded a base line separation of the 11 components in 50% methanol extract of Radix Saposhnikoviae with no interfering peaks detected. The HPLC-DAD method was validated in terms of linearity, accuracy and precision (intra- and inter-day), limit of quantification (LOQ), recovery, and robustness. Specific determination of the 11 components was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. This HPLC-MS/MS method was also validated by determining the linearity, limit of quantification, accuracy, and precision. Quantification of the 11 components in 51 commercial Radix Saposhnikoviae samples was successfully performed using the developed HPLC-DAD method. The identity, batch-to-batch consistency, and authenticity of Radix Saposhnikoviae were successfully monitored by the proposed HPLC-DAD and HPLC-MS/MS methods. Copyright © 2011 Elsevier B.V. All rights reserved.

40 CFR 63.11 - Control device and work practice requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... basis, as measured for organics by Test Method 18 and measured for hydrogen and carbon monoxide by... and operated with an exit velocity less than 18.3 m/sec (60 ft/sec), except as provided in paragraphs... the method specified in paragraph (b)(7)(i) of this section, equal to or greater than 18.3 m/sec (60...

40 CFR 63.11 - Control device and work practice requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... basis, as measured for organics by Test Method 18 and measured for hydrogen and carbon monoxide by... and operated with an exit velocity less than 18.3 m/sec (60 ft/sec), except as provided in paragraphs... the method specified in paragraph (b)(7)(i) of this section, equal to or greater than 18.3 m/sec (60...

40 CFR 63.11 - Control device and work practice requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... basis, as measured for organics by Test Method 18 and measured for hydrogen and carbon monoxide by... and operated with an exit velocity less than 18.3 m/sec (60 ft/sec), except as provided in paragraphs... the method specified in paragraph (b)(7)(i) of this section, equal to or greater than 18.3 m/sec (60...

40 CFR 63.11 - Control device and work practice requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... basis, as measured for organics by Test Method 18 and measured for hydrogen and carbon monoxide by... and operated with an exit velocity less than 18.3 m/sec (60 ft/sec), except as provided in paragraphs... the method specified in paragraph (b)(7)(i) of this section, equal to or greater than 18.3 m/sec (60...

Release mechanisms of acetaminophen from polyethylene oxide/polyethylene glycol matrix tablets utilizing magnetic resonance imaging.

PubMed

Tajiri, Tomokazu; Morita, Shigeaki; Sakamoto, Ryosaku; Suzuki, Masazumi; Yamanashi, Shigeyuki; Ozaki, Yukihiro; Kitamura, Satoshi

2010-08-16

Release mechanism of acetaminophen (AAP) from extended-release tablets of hydrogel polymer matrices containing polyethylene oxide (PEO) and polyethylene glycol (PEG) were achieved using flow-through cell with magnetic resonance imaging (MRI). The hydrogel forming abilities are observed characteristically and the layer thickness which is corresponding to the diffusion length of AAP has a good correlation with the drug release profiles. In addition, polymeric erosion contribution to AAP releasing from hydrogel matrix tablets was directly quantified using size-exclusion chromatography (SEC). The matrix erosion profile indicates that the PEG erosion kinetic depends primarily on the composition ratio of PEG to PEO. The present study has confirmed that the combination of in situ MRI and SEC should be well suited to investigate the drug release mechanisms of hydrogel matrix such as PEO/PEG. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.

PubMed

Stressler, Timo; Tanzer, Coralie; Ewert, Jacob; Claaßen, Wolfgang; Fischer, Lutz

2017-03-01

The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His 6 -tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His 6 -tag harboring PepA; the K M value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

Miniaturized protein separation using a liquid chromatography column on a flexible substrate

NASA Astrophysics Data System (ADS)

Yang, Yongmo; Chae, Junseok

2008-12-01

We report a prototype protein separator that successfully miniaturizes existing technology for potential use in biocompatible health monitoring implants. The prototype is a liquid chromatography (LC) column (LC mini-column) fabricated on an inexpensive, flexible, biocompatible polydimethylsiloxane (PDMS) enclosure. The LC mini-column separates a mixture of proteins using size exclusion chromatography (SEC) with polydivinylbenzene beads (5-20 µm in diameter with 10 nm pore size). The LC mini-column is smaller than any commercially available LC column by a factor of ~11 000 and successfully separates denatured and native protein mixtures at ~71 psi of the applied fluidic pressure. Separated proteins are analyzed using NuPAGE-gel electrophoresis, high-performance liquid chromatography (HPLC) and an automated electrophoresis system. Quantitative HPLC results demonstrate successful separation based on intensity change: within 12 min, the intensity between large and small protein peaks changed by a factor of ~20. In further evaluation using the automated electrophoresis system, the plate height of the LC mini-column is between 36 µm and 100 µm. The prototype LC mini-column shows the potential for real-time health monitoring in applications that require inexpensive, flexible implant technology that can function effectively under non-laboratory conditions.

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches.

PubMed

Barnett, Gregory V; Perhacs, Julia M; Das, Tapan K; Kar, Sambit R

2018-02-08

Characterizing submicron protein particles (approximately 0.1-1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS). Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment. A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4 × 10 7 -1 × 10 11 particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering. RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

PubMed

Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

2017-07-01

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

Monomer Release from Resin Based Dental Materials Cured With LED and Halogen Lights

PubMed Central

Ak, Asli Topaloglu; Alpoz, A. Riza; Bayraktar, Oguz; Ertugrul, Fahinur

2010-01-01

Objectives: To measure the release of TEGDMA and BisGMA from two commercially available composite resins; Filtek Z 250 (3M ESPE, Germany), Leaddent (Leaddent, Germany) and two fissure sealants; Helioseal F (3M ESPE, Germany) Enamel Loc (Premiere Rev, USA) over 1, 3 and 7 days after polymerization with standard quartz-tungsten halogen Coltolux II (QHL) (Coltene Switzerland) and a standard blue light emitting diode Elipar Freelight 2 (3M ESPE, Germany). Methods: 9 samples of each material were placed in disc shaped specimens in 1 mm of thickness and 10 mm in diameter (n=36). Each material was polymerized using LED for 20 s (n=12), 40 s (n=12) and halogen for 40 s (n=12), respectively. High Performance Liquid Chromatography (HPLC) was used to measure the amount of monomers released over 1, 3 and 7 days. Data was analyzed using one way ANOVA and Bonferroni test for multiple comparisons with a significance level of .05. Results: LED 20 sec group showed the highest release of monomers at 1, 3 and 7 days in sealant groups. Halogen 40 sec group resulted highest release of monomers for Leaddent at all time intervals (P<.05) Conclusions: Efficiency of the curing unit and applying the recommended curing time of the light activated resin based dental materials is very important to protect the patient from potential hazards of residual monomers. PMID:20046478

Analysis of ecstasy tablets using capillary electrophoresis with capacitively coupled contactless conductivity detection.

PubMed

Porto, Suely K S S; Nogueira, Thiago; Blanes, Lucas; Doble, Philip; Sabino, Bruno D; do Lago, Claudimir L; Angnes, Lúcio

2014-11-01

A method for the identification of 3,4-methylenedioxymethamphetamine (MDMA) and meta-chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4) D). Sample extraction, separation, and detection of "Ecstasy" tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE-C(4) D and compared against routine gas chromatography-mass spectrometry (GC-MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C(4) D instead of traditional CE-UV methods for in-field analysis are also discussed. © 2014 American Academy of Forensic Sciences.

Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination.

PubMed

Goetz, H; Kuschel, M; Wulff, T; Sauber, C; Miller, C; Fisher, S; Woodward, C

2004-09-30

Protein analysis techniques are developing fast due to the growing number of proteins obtained by recombinant DNA techniques. In the present paper we compare selected techniques, which are used for protein sizing, quantitation and molecular weight determination: sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip or microfluidics technology (LoaC), size exclusion chromatography (SEC) and mass spectrometry (MS). We compare advantages and limitations of each technique in respect to different application areas, analysis time, protein sizing and quantitation performance.

Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity.

PubMed

Wei, Ziping; McEvoy, Matt; Razinkov, Vladimir; Polozova, Alla; Li, Elizabeth; Casas-Finet, Jose; Tous, Guillermo I; Balu, Palani; Pan, Alfred A; Mehta, Harshvardhan; Schenerman, Mark A

2007-09-01

Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2010 CFR

2010-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2014 CFR

2014-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2013 CFR

2013-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

46 CFR Sec. 4 - Method of payment.

Code of Federal Regulations, 2012 CFR

2012-10-01

... AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 4 Method of payment. The General Agent shall prepare check drawn on the NSA Special bank account...

Characterization of plant polysaccharides from Dendrobium officinale by multiple chromatographic and mass spectrometric techniques.

PubMed

Ma, Huiying; Zhang, Keke; Jiang, Qing; Dai, Diya; Li, Hongli; Bi, Wentao; Chen, David Da Yong

2018-04-27

Plant polysaccharides have numerous medicinal functions. Due to the differences in their origins, regions of production, and cultivation conditions, the quality and the functions of polysaccharides can vary significantly. They are macromolecules with large molecular weight (MW) and complex structure, and pose great challenge for the analytical technology used. Taking Dendrobium officinale (DO) from various origins and locations as model samples. In this investigation, mechanochemical extraction method was used to successfully extract polysaccharides from DO using water as solvent, the process is simple, fast (40 s) and with high yield. The MWs of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography (SEC). The large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry (LC-MS 2 ). Obvious differences were observed among LC-MS 2 chromatograms of digested products, and the chemical structures for the products were proposed based on accurate mass values. More importantly, isomeric digested carbohydrate compounds were explored and characterized. All the chromatographic and mass spectrometric results in this study provided a multi-dimensional characterization, fingerprint analysis, and molecular structure level assessment of plant polysaccharides. Copyright © 2018 Elsevier B.V. All rights reserved.

Storage effects on quantity and composition of dissolved organic carbon and nitrogen of lake water, leaf leachate and peat soil water.

PubMed

Heinz, Marlen; Zak, Dominik

2018-03-01

This study aimed to evaluate the effects of freezing and cold storage at 4 °C on bulk dissolved organic carbon (DOC) and nitrogen (DON) concentration and SEC fractions determined with size exclusion chromatography (SEC), as well as on spectral properties of dissolved organic matter (DOM) analyzed with fluorescence spectroscopy. In order to account for differences in DOM composition and source we analyzed storage effects for three different sample types, including a lake water sample representing freshwater DOM, a leaf litter leachate of Phragmites australis representing a terrestrial, 'fresh' DOM source and peatland porewater samples. According to our findings one week of cold storage can bias DOC and DON determination. Overall, the determination of DOC and DON concentration with SEC analysis for all three sample types were little susceptible to alterations due to freezing. The findings derived for the sampling locations investigated here may not apply for other sampling locations and/or sample types. However, DOC size fractions and DON concentration of formerly frozen samples should be interpreted with caution when sample concentrations are high. Alteration of some optical properties (HIX and SUVA 254 ) due to freezing were evident, and therefore we recommend immediate analysis of samples for spectral analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Geosynthesis of organic compounds: I. Alkylphenols

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

Ioppolo-Armanios, M.; Alexander, R.; Kagi, R.I.

1995-07-01

Methylation, isopropylation, and sec-butylation are proposed as geosynthetic processes to account for the alkylphenol compositions of crude oils with phenol distributions dominated by ortho and para substituted compounds. Phenol distributions in eleven crude oils and four kerogen pyrolysates were analysed using GC-MS (gas chromatography-mass spectrometry). Ten of the crude oils show high relative abundances of ortho and para substituted phenol isomers and some were also enriched in C{sub 3}-C{sub 5} alkylphenols compared to the kerogen pyrolysates. Because the distributions of products obtained from the laboratory alkylation of cresols closely resemble those of phenols in these crude oils, we propose thatmore » similar alkylation processes occur in source rocks. Alkylation ratios reflecting the degree of methylation, isopropylation, and sec-butylation, which were based on the relative abundance of the dominant alkylation products compared to their likely precursor ortho-cresol, indicate that high levels of methylation occurred in crude oils over a wide range of maturities, whereas high levels of isopropylation and sec-butylation were observed only in mature samples. Dissolution of the phenols in crude oils by water contact was discounted as an explanation for the observed phenol distributions based on the relative distribution coefficients of phenols between a hydrocarbon phase and water.« less

An efficient protocol for the production of tRNA-free recombinant Selenocysteine Synthase (SELA) from Escherichia coli and its biophysical characterization.

PubMed

Manzine, Livia Regina; Cassago, Alexandre; da Silva, Marco Túlio Alves; Thiemann, Otavio Henrique

2013-03-01

Selenocysteine Synthase (SELA, E.C. 2.9.1.1) from Escherichia coli is a homodecamer pyridoxal-5'-phosphate containing enzyme responsible for the conversion of seryl-tRNA(sec) into selenocysteyl-tRNA(sec) in the biosynthesis of the 21th amino acid, selenocysteine (Sec or U). This paper describes the cloning of the E. coli selA gene into a modified pET29a(+) vector and its expression in E. coli strain WL81460, a crucial modification allowing SELA expression without bound endogenous tRNA(sec). This expression strategy enabled the purification and additional biochemical and biophysical characterization of the SELA decamer. The homogeneous SELA protein was obtained using three chromatographic steps. Size Exclusion Chromatography and Native Gel Electrophoresis showed that SELA maintains a decameric state with molecular mass of approximately 500 kDa with an isoelectric point of 6,03. A predominance of α-helix structures was detected by circular dichroism with thermal stability up to 45 °C. The oligomeric assemblage of SELA was investigated by glutaraldehyde crosslinking experiments indicate that SELA homodecameric structure is the result of a stepwise addition of intermediate oligomeric states and not a direct monomer to homodecamer transition. Our results have contributed to the establishment of a robust expression model for the enzyme free of bound RNA and are of general interest to be taken into consideration in all cases of heterologous/homologous expressions of RNA-binding proteins avoiding the carryover of endogenous RNAs, which may interfere with further biochemical characterizations. Copyright © 2012 Elsevier Inc. All rights reserved.

Technology of an adhesive silicone film as drug carrier in transdermal therapy. I: Analytical methods used for characterization and design of the universal elastomer layers.

PubMed

Mojsiewicz-Pieńkowska, Krystyna; Jamrógiewicz, Marzena; Zebrowska, Maria; Sznitowska, Małgorzata; Centkowska, Katarzyna

2011-08-25

Silicone polymers possess unique properties, which make them suitable for many different applications, for example in the pharmaceutical and medical industry. To create an adhesive silicone film, the appropriate silicone components have to be chosen first. From these components two layers were made: an adhesive elastomer applied on the skin, and a non-adhesive elastomer on the other side of the film. The aim of this study was to identify a set of analytical methods that can be used for detailed characterization of the elastomer layers, as needed when designing new silicone films. More specifically, the following methods were combined to detailed identification of the silicone components: Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (¹H NMR) and size exclusion chromatography with evaporative light scattering detector (SEC-ELSD). It was demonstrated that these methods together with a rheological analysis are suitable for controlling the cross-linking reaction, thus obtaining the desired properties of the silicone film. Adhesive silicone films can be used as universal materials for medical use, particularly for effective treatment of scars and keloids or as drug carriers in transdermal therapy.

De facto molecular weight distributions of glucans by size-exclusion chromatography combined with mass/molar-detection of fluorescence labeled terminal hemiacetals.

PubMed

Praznik, Werner; Huber, Anton

2005-09-25

A major capability of polysaccharides in aqueous media is their tendency for aggregation and dynamic formation of supermolecular structures. Even extended dissolution processes will not eliminate these structures which dominate many analytical approaches, in particular absolute molecular weight determinations referring to light scattering data. An alternative approach for determination of de facto molecular weight for glucans with free terminal hemiacetal functionality (reducing end group) has been adjusted from carbohydrates for midrange and high-dp glucans: quantitative and stabilized labeling as aminopyridyl-derivatives (AP-glucans) and subsequent analysis of SEC-separated elution profiles based on simultaneously monitored mass and molar fractions by refractive index and fluorescence detection. SEC-DRI/FL of AP-glucans proved as an appropriate approach for determination of de facto molecular weight of constituting glucan molecules even in the presence of supermolecular structures for non-branched (pullulan), branched (dextran), narrow distributed and broad distributed and for mixes of compact and loose packed polymer coils (starch glucan hydrolizate).

Virus Characterization by FFF-MALS Assay

NASA Astrophysics Data System (ADS)

Razinkov, Vladimer

2009-03-01

Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

Integrated protein analysis platform based on column switch recycling size exclusion chromatography, microenzymatic reactor and microRPLC-ESI-MS/MS.

PubMed

Yuan, Huiming; Zhou, Yuan; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

2009-10-30

An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by microRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.

Ozonation effects on emerging micropollutants and effluent organic matter in wastewater: characterization using changes of three-dimensional HP-SEC and EEM fluorescence data.

PubMed

Liu, Chen; Li, Penghui; Tang, Xiangyu; Korshin, Gregory V

2016-10-01

The degradation of effluent organic matter (EfOM) in a municipal wastewater treated by ozonation was characterized using the methods of high-performance size-exclusion chromatography (HP-SEC) and excitation/emission matrix (EEM) fluorescence combined with parallel factor analysis (PARAFAC). The removal of 40 diverse trace-level contaminants of emerging concern (CEC) present in the wastewater was determined as well. Ozonation caused a rapid decrease of the absorbance and fluorescence of the wastewater, which was associated primarily with the oxidation of high and low apparent molecular weight (AMW) EfOM fractions. PARAFAC analysis also showed that components C1 and C2 decreased prominently in these conditions. The EfOM fraction of intermediate molecular weight ascribable to a terrestrial humic-like component (C3) tended to be less reactive toward ozone. Relative changes of EEM fluorescence were quantified using F max values of PARAFAC-identified components (∆F/F 0 max ). Unambiguous relationships between ∆F/F 0 max values and the extent of the degradation of the examined CECs (∆C/C 0 ) were established. This allowed correlating main parameters of the ∆C/C 0 vs. ∆F/F 0 max relationships with the rates of oxidation of these CECs. The results demonstrate the potential of online measurements of EEM fluorescence for quantitating effects of ozonation on EfOM and micropollutants in wastewater effluents.

Characterization of complete particles (VSV-G/SIN-GFP) and empty particles (VSV-G/EMPTY) in human immunodeficiency virus type 1-based lentiviral products for gene therapy: potential applications for improvement of product quality and safety.

PubMed

Zhao, Yuan; Keating, Kenneth; Dolman, Carl; Thorpe, Robin

2008-05-01

Lentiviral vectors persist in the host and are therefore ideally suited for long-term gene therapy. To advance the use of lentiviral vectors in humans, improvement of their production, purification, and characterization has become increasingly important and challenging. In addition to cellular contaminants derived from packaging cells, empty particles without therapeutic function are the major impurities that compromise product safety and efficacy. Removal of empty particles is difficult because of their innate similarity in particle size and protein composition to the complete particles. We propose that comparison of the properties of lentiviral products with those of purposely expressed empty particles may reveal potential differences between empty and complete particles. For this, three forms of recombinant lentiviral samples, that is, recombinant vesicular stomatitis virus glycoprotein (VSV-G) proteins, empty particles (VSV-G/Empty), and complete particles (VSV-G/SIN-GFP) carrying viral RNA, were purified by size-exclusion chromatography (SEC). The SEC-purified samples were further analyzed by immunoblotting with six antibodies to examine viral and cellular proteins associated with the particles. This study has demonstrated, for the first time, important differences between VSV-G/Empty particles and complete VSV-G/SIN-GFP particles. Differences include the processing of Gag protein and the inclusion of cellular proteins in the particles. Our findings support the development of improved production, purification, and characterization methods for lentiviral products.

Reductive amination derivatization for the quantification of garlic components by isotope dilution analysis.

PubMed

Lin, Yi-Reng; Huang, Mei-Fang; Wu, You-Ying; Liu, Meng-Chieh; Huang, Jing-Heng; Chen, Ziyu; Shiue, Yow-Ling; Wu, Chia-En; Liang, Shih-Shin

2017-09-01

In this work, we synthesized internal standards for four garlic organosulfur compounds (OSCs) by reductive amination with 13 C, D 2 -formaldehyde, and developed an isotope dilution analysis method to quantitate these organosulfur components in garlic samples. Internal standards were synthesized for internal absolute quantification of S-allylcysteine (SAC), S-allylcysteine sulfoxide (alliin), S-methylcysteine (SMC), and S-ethylcysteine (SEC). We used a multiple reaction monitoring (MRM) to detect 13 C, D 2 -formaldehyde-modified OSCs by ultrahigh-performance liquid phase chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and obtained MS spectra showing different ratios of 13 C, D 2 -formaldehyde-modified and H 2 -formaldehyde-modified compounds. The resulting labeled and unlabeled OSCs were exhibited correlation coefficient (R 2 ) ranged from 0.9989 to 0.9994, respectively. The average recoveries for four OSCs at three concentration levels ranged from 89% to 105%. By 13 C, D 2 -formaldehyde and sodium cyanoborohydride, the reductive amination-based method can be utilized to generate novel internal standard for isotope dilution and to extend the quantitative application. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exploiting the User: Adapting Personas for Use in Security Visualization Design

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

Stoll, Jennifer C.; McColgin, David W.; Gregory, Michelle L.

It has long been noted that visual representations of complex information can facilitate rapid understanding of data {citation], even with respect to ComSec applications {citation]. Recognizing that visualizations can increase usability in ComSec applications, [Zurko, Sasse] have argued that there is a need to create more usable security visualizations. (VisSec) However, usability of applications generally fall into the domain of Human Computer Interaction (HCI), which generally relies on heavy-weight user-centered design (UCD) processes. For example, the UCD process can involve many prototype iterations, or an ethnographic field study that can take months to complete. The problem is that VisSec projectsmore » generally do not have the resources to perform ethnographic field studies, or to employ complex UCD methods. They often are running on tight deadlines and budgets that can not afford standard UCD methods. In order to help resolve the conflict of needing more usable designs in ComSec, but not having the resources to employ complex UCD methods, in this paper we offer a stripped-down lighter weight version of a UCD process which can help with capturing user requirements. The approach we use is personas which a user requirements capturing method arising out of the Participatory Design philosophy [Grudin02].« less

Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry

PubMed Central

Martín-Ortiz, A.; Salcedo, J.; Barile, D.; Bunyatratchata, A.; Moreno, F.J.; Martin-García, I.; Clemente, A.; Sanz, M.L.; Ruiz-Matute, A.I.

2016-01-01

A detailed qualitative and quantitative characterization of goat colostrum oligosaccharides (GCO) has been carried out for the first time. Defatted and deproteinized colostrum samples, previously treated by size exclusion chromatography (SEC) to remove lactose, were analyzed by nanoflow liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip-Q-TOF MS). Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or N-glycolylneuraminic acid monomeric units were identified in the samples, some of them detected for the first time in goat colostra. As a second step, a hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) methodology was developed for the separation and quantitation of the main GCO, both acidic and neutral carbohydrates. Among other experimental chromatographic conditions, mobile phase additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry of target carbohydrates. Narrow peaks (wh: 0.2–0.6 min) and good symmetry (As: 0.8–1.4) were obtained for GCO using an acetonitrile:water gradient with 0.1% ammonium hydroxide at 40 °C. These conditions were selected to quantify the main oligosaccharides in goat colostrum samples. Values ranging from 140 to 315 mg L−1 for neutral oligosaccharides and from 83 to 251 mg L−1 for acidic oligosaccharides were found. The combination of both techniques resulted to be useful to achieve a comprehensive characterization of GCO. PMID:26427327

77 FR 46289 - Technical Corrections to Organizational Names, Addresses, and OMB Control Numbers

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-03

...]795.232 Inhalation and dermal pharmacokinetics of commercial hexane. * * * * * (c) * * * (2) * * * (i... to read as follows: Sec. 799.6755 TSCA partition coefficient (n-octanol/water), shake flask method... read as follows: Sec. 799.6756 TSCA partition coefficient (n-octanol/water), generator column method...

Impaired Behavior Regulation under Conditions of Concurrent Variable Schedules of Reinforcement in Children with ADHD

ERIC Educational Resources Information Center

Taylor, David; Lincoln, Alan J.; Foster, Sharon L.

2010-01-01

Objective: To bridge theory of response inhibition and learning in children with ADHD. Method: Thirty ADHD and 30 non-ADHD children (ages 9-12) were compared under concurrent variable interval (VI-15 sec., VI-30 sec. and VI- 45 sec.) reinforcement schedules that required the child to switch between the three schedules under conditions of…

Eco-friendly streamlined process for sporopollenin exine capsule extraction

PubMed Central

Mundargi, Raghavendra C.; Potroz, Michael G.; Park, Jae Hyeon; Seo, Jeongeun; Tan, Ee-Lin; Lee, Jae Ho; Cho, Nam-Joon

2016-01-01

Sporopollenin exine capsules (SECs) extracted from Lycopodium clavatum spores are an attractive biomaterial possessing a highly robust structure suitable for microencapsulation strategies. Despite several decades of research into SEC extraction methods, the protocols commonly used for L. clavatum still entail processing with both alkaline and acidolysis steps at temperatures up to 180 °C and lasting up to 7 days. Herein, we demonstrate a significantly streamlined processing regimen, which indicates that much lower temperatures and processing durations can be used without alkaline lysis. By employing CHN elemental analysis, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and dynamic image particle analysis (DIPA), the optimum conditions for L. clavatum SEC processing were determined to include 30 hours acidolysis at 70 °C without alkaline lysis. Extending these findings to proof-of-concept encapsulation studies, we further demonstrate that our SECs are able to achieve a loading of 0.170 ± 0.01 g BSA per 1 g SECs by vacuum-assisted loading. Taken together, our streamlined processing method and corresponding characterization of SECs provides important insights for the development of applications including drug delivery, cosmetics, personal care products, and foods. PMID:26818918

Eco-friendly streamlined process for sporopollenin exine capsule extraction

NASA Astrophysics Data System (ADS)

Mundargi, Raghavendra C.; Potroz, Michael G.; Park, Jae Hyeon; Seo, Jeongeun; Tan, Ee-Lin; Lee, Jae Ho; Cho, Nam-Joon

2016-01-01

Sporopollenin exine capsules (SECs) extracted from Lycopodium clavatum spores are an attractive biomaterial possessing a highly robust structure suitable for microencapsulation strategies. Despite several decades of research into SEC extraction methods, the protocols commonly used for L. clavatum still entail processing with both alkaline and acidolysis steps at temperatures up to 180 °C and lasting up to 7 days. Herein, we demonstrate a significantly streamlined processing regimen, which indicates that much lower temperatures and processing durations can be used without alkaline lysis. By employing CHN elemental analysis, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and dynamic image particle analysis (DIPA), the optimum conditions for L. clavatum SEC processing were determined to include 30 hours acidolysis at 70 °C without alkaline lysis. Extending these findings to proof-of-concept encapsulation studies, we further demonstrate that our SECs are able to achieve a loading of 0.170 ± 0.01 g BSA per 1 g SECs by vacuum-assisted loading. Taken together, our streamlined processing method and corresponding characterization of SECs provides important insights for the development of applications including drug delivery, cosmetics, personal care products, and foods.

Removal of Fe3+ and Zn2+ from plasma metalloproteins by iron chelating therapeutics depicted with SEC-ICP-AES.

PubMed

Sooriyaarachchi, Melani; Gailer, Jürgen

2010-08-28

The iron chelation therapy drugs desferrioxamine B (DFO) and deferiprone (DFP) are used to treat iron overload patients, but not much is known about their adverse effects on other essential metals in vivo. After the addition of a clinically relevant dose of DFP or an equimolar dose of DFO to human plasma in vitro, the mixtures were analyzed by size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES). Simultaneous detection of the emission lines of copper, iron and zinc allowed the visualization of changes that these drugs exerted at the metalloprotein level. After the addition of DFP, a <10 kDa novel Fe-peak was detected and identified as (DFP)(3)Fe, whereas DFO resulted in the elution of a much smaller amount of Fe in this elution range. In fact, DFP was approximately 8-times more efficient than DFO regarding the removal of Fe from plasma proteins. The addition of both iron chelators also resulted in the elution of a <10 kDa novel Zn-peak. DFP abstracted twice as much Zn from plasma proteins compared to DFO. The identification of one of these peaks as (DFP)(2)Zn establishes a feasible biomolecular basis for the etiology of Zn-deficiency in patients that undergo long-term treatment with these drugs. Our results demonstrate that the analysis of plasma by SEC-ICP-AES can simultaneously provide insight into the efficacy of chelation therapy drugs and their adverse health effects at the metalloprotein level. Thus, SEC-ICP-AES emerges as a useful analytical tool to visualize health-relevant bioinorganic chemistry-related reactions of medicinal drugs in blood plasma in vitro.

Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection

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

Qu, Yi; Feng, Ju; Deng, Shuang

2014-11-19

Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation,) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we employed SEC, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), and ZIC-HILIC coupled with dialysis strategies to enrich the glycopeptides from the pronase E digests of RNase B, followed by NMR analysis ofmore » the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is the most efficient, which was thus applied to the analysis of biological complex sample, the pronase E digest of the secreted proteins from the fungi Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled to dialysis is superior to the commonly used SEC separation to prepare glycopeptides for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.« less

Advanced Numerical Methods for Simulating Nonlinear Multirate Lumped Parameter Models

DTIC Science & Technology

1991-05-01

defining a Waveform: typedef struct Waveform char *name; /* character string of the name of the variable */ double tn; /* time of the beginning of the...A State-Space Approach, Reprinted from Proc. Third Ann. Allerton Conf. Circuits and Systems Thoery , 659-668, in Computer-Aided Circuit Design...kg/n 3) 1025.9 kg/m3 @ 15* C. v Kinematic Viscosity of Water (m2/sec) 1.19x10-6 m2/sec @ 15* C. G Acceleration of Gravity (m/sec2) 9.80665 m/sec 2 L

Analytical strategies for controlling polysorbate-based nanomicelles in fruit juice.

PubMed

Krtkova, Veronika; Schulzova, Vera; Lacina, Ondrej; Hrbek, Vojtech; Tomaniova, Monika; Hajslova, Jana

2014-06-01

This study focused on the detection and quantification of organic micelle-type nanoparticles (NPs) with polysorbate components (polysorbate 20 and polysorbate 80) in their micelle shells that could be used to load biologically active compounds into fruit juice. Several advanced analytical techniques were applied in the stepwise method development strategy used. In the first phase, a system consisting of ultrahigh-performance liquid chromatography employing a size exclusion column coupled with an evaporative light scattering detector (UHPLC-SEC-ELSD) was used for the fractionation of micelle assemblies from other, lower molecular weight sample components. The limit of detection (LoD) of these polysorbate micelles in spiked apple juice was 500 μg mL(-1). After this screening step, mass spectrometric (MS) detection was utilized to confirm the presence of polysorbates in the detected micelles. Two alternative MS techniques were tested: (i) ambient high-resolution mass spectrometry employing a direct analysis in real time ion source coupled with an Orbitrap MS analyzer (DART-Orbitrap MS) enabled fast and simple detection of the polysorbates present in the samples, with a lowest calibration level (LCL) of 1000 μg mL(-1); (ii) ultrahigh-performance reversed-phase liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (UHPLC-HRTOF-MS) provided highly selective and sensitive detection and quantification of polysorbates with an LCL of 0.5 μg mL(-1).

A log-normal distribution model for the molecular weight of aquatic fulvic acids

USGS Publications Warehouse

Cabaniss, S.E.; Zhou, Q.; Maurice, P.A.; Chin, Y.-P.; Aiken, G.R.

2000-01-01

The molecular weight of humic substances influences their proton and metal binding, organic pollutant partitioning, adsorption onto minerals and activated carbon, and behavior during water treatment. We propose a lognormal model for the molecular weight distribution in aquatic fulvic acids to provide a conceptual framework for studying these size effects. The normal curve mean and standard deviation are readily calculated from measured M(n) and M(w) and vary from 2.7 to 3 for the means and from 0.28 to 0.37 for the standard deviations for typical aquatic fulvic acids. The model is consistent with several types of molecular weight data, including the shapes of high- pressure size-exclusion chromatography (HP-SEC) peaks. Applications of the model to electrostatic interactions, pollutant solubilization, and adsorption are explored in illustrative calculations.The molecular weight of humic substances influences their proton and metal binding, organic pollutant partitioning, adsorption onto minerals and activated carbon, and behavior during water treatment. We propose a log-normal model for the molecular weight distribution in aquatic fulvic acids to provide a conceptual framework for studying these size effects. The normal curve mean and standard deviation are readily calculated from measured Mn and Mw and vary from 2.7 to 3 for the means and from 0.28 to 0.37 for the standard deviations for typical aquatic fulvic acids. The model is consistent with several type's of molecular weight data, including the shapes of high-pressure size-exclusion chromatography (HP-SEC) peaks. Applications of the model to electrostatic interactions, pollutant solubilization, and adsorption are explored in illustrative calculations.

Identification of protein components and quantitative immunoassay for SEC2 in staphylococcin injection.

PubMed

Ding, Ding; Huang, Peng; Sun, Hongying; Xue, Qiao; Pan, Yingqiu; Chen, Shuqing

2009-08-15

In China, staphylococcin injection has been commonly used in combined cancer therapy to enhance the systemic immune response and reduce the toxicities associated with chemotherapy or radiation therapy in the last decade. It is claimed that the main effective component is staphylococcal enterotoxin C2 (SEC2). However, no standard method based on the concentration of SEC2 has been established for quality control of the injection products. In this study, a sensitive and reliable biotin-streptavidin-ELISA (BS-ELISA) method was established for detection and quantification of SEC2. In addition, 1-D SDS-PAGE coupled with nano-LC-MS/MS was performed to identify the protein components in the injection products from one manufacturing company. The results of the BS-ELISA showed that SEC2 only accounted for less than 0.1% of the total protein in the injection products, and the nano-LC-MS/MS results showed that fifty-five proteins of Staphylococcus aureus were confidently identified in the injection solution. Seventeen out of these proteins, including SEC2, were well-known virulence factors. In addition, eighteen proteins of other Gram-positive bacteria were also confidently identified. Thus, the results indicated that SEC2 is of very low concentration in the injection products and the process of the injection preparation should be improved for health and safety consideration.

78 FR 25129 - Self-Regulatory Organizations; Fixed Income Clearing Corporation; Notice of Filing of Proposed...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-29

... provide guaranteed settlement and central counterparty services,\\10\\ which among other things established... following methods: Electronic Comments Use the Commission's Internet comment form ( http://www.sec.gov/rules... method. The Commission will post all comments on the Commission's Internet Web site ( http://www.sec.gov...

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry.

PubMed

Wang, Guanbo; de Jong, Rob N; van den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

2017-05-02

The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.

Size-Dependency of the Surface Ligand Density of Liposomes Prepared by Post-insertion.

PubMed

Lee, Shang-Hsuan; Sato, Yusuke; Hyodo, Mamoru; Harashima, Hideyoshi

2017-01-01

In the active targeting of a drug delivery system (DDS), the density of the ligand on the functionalized liposome determines its affinity for binding to the target. To evaluate these densities on the surface of different sized liposomes, 4 liposomes with various diameters (188, 137, 70, 40 nm) were prepared and their surfaces were modified with fluorescently labeled ligand-lipid conjugates by the post-insertion method. Each liposomal mixture was fractionated into a series of fractions using size exclusion chromatography (SEC), and the resulting liposome fractions were precisely analyzed and the surface ligand densities calculated. The data collected using this methodology indicate that the density of the ligand on a particle is greatly dependent on the size of the liposome. This, in turn, indicates that smaller liposomes (75-40 nm) tend to possess higher densities. For developing active targeting systems, size and the density of the ligands are two important and independent factors that can affect the efficiency of a system as it relates to medical use.

Recognizing ancient papyri by a combination of spectroscopic, diffractional and chromatographic analytical tools

PubMed Central

Łojewska, J.; Rabin, I.; Pawcenis, D.; Bagniuk, J.; Aksamit-Koperska, M. A.; Sitarz, M.; Missori, M.; Krutzsch, M.

2017-01-01

Ancient papyri are a written heritage of culture that flourished more than 3000 years ago in Egypt. One of the most significant collections in the world is housed in the Egyptian Museum and Papyrus Collection in Berlin, from where the samples for our investigation come. The papyrologists, curators and conservators of such collections search intensely for the analytical detail that would allow ancient papyri to be distinguished from modern fabrications, in order to detect possible forgeries, assess papyrus deterioration state, and improve the design of storage conditions and conservation methods. This has become the aim of our investigation. The samples were studied by a number of methods, including spectroscopic (FTIR, fluorescent-FS, Raman) diffractional (XRD) and chromatographic (size exclusion chromatography-SEC), selected in order to determine degradation parameters: overall oxidation of lignocellulosic material, degree of polymerization and crystallinity of cellulose. The results were correlated with those obtained from carefully selected model samples including modern papyri and paper of different composition aged at elevated temperature in humid air. The methods were classified in the order SEC > FS > FTIR > XRD, based on their effectiveness in discriminating the state of papyri degradation. However, the most trustworthy evaluation of the age of papyri samples should rely on several methods. PMID:28382971

Recognizing ancient papyri by a combination of spectroscopic, diffractional and chromatographic analytical tools.

PubMed

Łojewska, J; Rabin, I; Pawcenis, D; Bagniuk, J; Aksamit-Koperska, M A; Sitarz, M; Missori, M; Krutzsch, M

2017-04-06

Ancient papyri are a written heritage of culture that flourished more than 3000 years ago in Egypt. One of the most significant collections in the world is housed in the Egyptian Museum and Papyrus Collection in Berlin, from where the samples for our investigation come. The papyrologists, curators and conservators of such collections search intensely for the analytical detail that would allow ancient papyri to be distinguished from modern fabrications, in order to detect possible forgeries, assess papyrus deterioration state, and improve the design of storage conditions and conservation methods. This has become the aim of our investigation. The samples were studied by a number of methods, including spectroscopic (FTIR, fluorescent-FS, Raman) diffractional (XRD) and chromatographic (size exclusion chromatography-SEC), selected in order to determine degradation parameters: overall oxidation of lignocellulosic material, degree of polymerization and crystallinity of cellulose. The results were correlated with those obtained from carefully selected model samples including modern papyri and paper of different composition aged at elevated temperature in humid air. The methods were classified in the order SEC > FS > FTIR > XRD, based on their effectiveness in discriminating the state of papyri degradation. However, the most trustworthy evaluation of the age of papyri samples should rely on several methods.

Characterization of goat colostrum oligosaccharides by nano-liquid chromatography on chip quadrupole time-of-flight mass spectrometry and hydrophilic interaction liquid chromatography-quadrupole mass spectrometry.

PubMed

Martín-Ortiz, A; Salcedo, J; Barile, D; Bunyatratchata, A; Moreno, F J; Martin-García, I; Clemente, A; Sanz, M L; Ruiz-Matute, A I

2016-01-08

A detailed qualitative and quantitative characterization of goat colostrum oligosaccharides (GCO) has been carried out for the first time. Defatted and deproteinized colostrum samples, previously treated by size exclusion chromatography (SEC) to remove lactose, were analyzed by nanoflow liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip-Q-TOF MS). Up to 78 oligosaccharides containing hexose, hexosamine, fucose, N-acetylneuraminic acid or N-glycolylneuraminic acid monomeric units were identified in the samples, some of them detected for the first time in goat colostra. As a second step, a hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS) methodology was developed for the separation and quantitation of the main GCO, both acidic and neutral carbohydrates. Among other experimental chromatographic conditions, mobile phase additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry of target carbohydrates. Narrow peaks (wh: 0.2-0.6min) and good symmetry (As: 0.8-1.4) were obtained for GCO using an acetonitrile:water gradient with 0.1% ammonium hydroxide at 40°C. These conditions were selected to quantify the main oligosaccharides in goat colostrum samples. Values ranging from 140 to 315mgL(-1) for neutral oligosaccharides and from 83 to 251mgL(-1) for acidic oligosaccharides were found. The combination of both techniques resulted to be useful to achieve a comprehensive characterization of GCO. Copyright © 2015 Elsevier B.V. All rights reserved.

Operationally defined species characterization and bioaccessibility evaluation of cobalt, copper and selenium in Cape gooseberry (Physalis Peruviana L.) by SEC-ICP MS.

PubMed

Wojcieszek, Justyna; Ruzik, Lena

2016-03-01

Physalis peruviana could attract great interest because of its nutritional and industrial properties. It is an excellent source of vitamins, minerals, essential fatty acids and carotenoids. Physalis Peruviana is also known to have a positive impact on human health. Unfortunately, still little is known about trace elements present in Physalis Peruviana and their forms available for the human body. Thus, the aim of this study was to estimate bioaccessibility and characterization of species of cobalt, copper and selenium in Physalis Peruviana fruits. Total and extractable contents of elements were determined by mass spectrometer with inductively coupled plasma (ICP MS). In order to separate the different types of metal complexes Physalis peruviana fruits were treated with the following solvents: Tris-HCl (pH 7.4), sodium dodecyl sulfate (SDS) (pH 7.4) and ammonium acetate (pH 5.5). The best efficiency of extraction of: cobalt was obtained for ammonium acetate (56%) and Tris-HCl (60%); for copper was obtained for SDS (66%), for selenium the best extraction efficiency was obtained after extraction with SDS (48%). To obtain information about bioaccessibility of investigated elements, enzymatic extraction based on in vitro simulation of gastric (pepsin) and intestinal (pancreatin) digestion was performed. For copper and selenium the simulation of gastric digestion leads to the extraction yield above 90%, while both steps of digestion method were necessary to obtain satisfactory extraction yield in the case of cobalt. Size exclusion chromatography (SEC) coupled to on-line ICP MS detection was used to investigate collected metal species. The main fraction of metal compounds was found in the 17 kDa region. Cobalt and copper create complexes mostly with compounds extracted by means of ammonium acetate and SDS, respectively. Cobalt, copper and selenium were found to be highly bioaccessible from Physalis Peruviana. Investigation of available standards of cobalt and selenium allows confirming the presence of vitamin B12 and probably selenomethionine in the fraction bioaccessible by human body (obtained during enzymatic extraction). It should be noted that the presence of small seleno-compounds in Cape gooseberry was performed for the first time. The results show that the combination of SEC and ICP MS could provide a simple method for separating of soluble element species. Copyright © 2015 Elsevier GmbH. All rights reserved.

27 CFR 19.454 - Gauge for denaturation.

Code of Federal Regulations, 2010 CFR

2010-04-01

... dumped from previously gauged containers or spirits transferred directly to mixing tanks from gauge tanks... devices or methods. (Sec. 201, Pub. L. 85-859, 72 Stat. 1358, as amended (26 U.S.C. 5204); sec. 807, Pub...

Influence of l-pyroglutamic acid on the color formation process of non-enzymatic browning reactions.

PubMed

Wegener, Steffen; Kaufmann, Martin; Kroh, Lothar W

2017-10-01

Heating aqueous d-glucose model reactions with l-glutamine and l-alanine yielded similar colored solutions. However, size-exclusion chromatography (SEC) revealed that both non-enzymatic browning reactions proceeded differently. Due to a fast occurring cyclization of l-glutamine to pyroglutamic acid, the typical amino-carbonyl reaction was slowed down. However, l-glutamine and l-alanine model reactions showed the same browning index. Closer investigations could prove that l-pyroglutamic acid was able to influence non-enzymatic browning reactions. SEC analyses of d-glucose model reactions with and without l-pyroglutamic acid revealed an increase of low molecular colored compounds in the presence of l-pyroglutamic acid. Polarimetric measurements showed a doubling of d-glucose mutarotation velocity and HPLC analyses of d-fructose formation during thermal treatment indicated a tripling of aldose-ketose transformation in the presence of l-pyroglutamic acid, which are signs of a faster proceeding non-enzymatic browning process. 2-Pyrrolidone showed no such behavior, thus the additional carboxylic group should be responsible for the observed effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fusion of sensor geometry into additive strain fields measured with sensing skin

NASA Astrophysics Data System (ADS)

Downey, Austin; Sadoughi, Mohammadkazem; Laflamme, Simon; Hu, Chao

2018-07-01

Recently, numerous studies have been conducted on flexible skin-like membranes for the cost effective monitoring of large-scale structures. The authors have proposed a large-area electronic consisting of a soft elastomeric capacitor (SEC) that transduces a structure’s strain into a measurable change in capacitance. Arranged in a network configuration, SECs deployed onto the surface of a structure could be used to reconstruct strain maps. Several regression methods have been recently developed with the purpose of reconstructing such maps, but all these algorithms assumed that each SEC-measured strain located at its geometric center. This assumption may not be realistic since an SEC measures the average strain value of the whole area covered by the sensor. One solution is to reduce the size of each SEC, but this would also increase the number of required sensors needed to cover the large-scale structure, therefore increasing the need for the power and data acquisition capabilities. Instead, this study proposes an algorithm that accounts for the sensor’s strain averaging feature by adjusting the strain measurements and constructing a full-field strain map using the kriging interpolation method. The proposed algorithm fuses the geometry of an SEC sensor into the strain map reconstruction in order to adaptively adjust the average kriging-estimated strain of the area monitored by the sensor to the signal. Results show that by considering the sensor geometry, in addition to the sensor signal and location, the proposed strain map adjustment algorithm is capable of producing more accurate full-field strain maps than the traditional spatial interpolation method that considered only signal and location.

Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

PubMed

Frahm, Grant E; Smith, Daryl G S; Kane, Anita; Lorbetskie, Barry; Cyr, Terry D; Girard, Michel; Johnston, Michael J W

2014-01-01

The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.

40 CFR 63.11 - Control device and work practice requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... in ppmv on a wet basis, as measured for organics by Test Method 18 and measured for hydrogen and... be designed for and operated with an exit velocity less than 18.3 m/sec (60 ft/sec), except as... determined by the method specified in paragraph (b)(7)(i) of this section, equal to or greater than 18.3 m...

Molar mass characterization of sodium carboxymethyl cellulose by SEC-MALLS.

PubMed

Shakun, Maryia; Maier, Helena; Heinze, Thomas; Kilz, Peter; Radke, Wolfgang

2013-06-05

Two series of sodium carboxymethyl celluloses (NaCMCs) derived from microcrystalline cellulose (Avicel samples) and cotton linters (BWL samples) with average degrees of substitution (DS) ranging from DS=0.45 to DS=1.55 were characterized by size exclusion chromatography with multi-angle laser light scattering detection (SEC-MALLS) in 100 mmol/L aqueous ammonium acetate (NH4OAc) as vaporizable eluent system. The application of vaporizable NH4OAc allows future use of the eluent system in two-dimensional separations employing evaporative light scattering detection (ELSD). The losses of samples during filtration and during the chromatographic experiment were determined. The scaling exponent as of the relation [Formula: see text] was approx. 0.61, showing that NaCMCs exhibit an expanded coil conformation in solution. No systematic dependencies of as on DS were observed. The dependences of molar mass on SEC-elution volume for samples of different DS can be well described by a common calibration curve, which is of advantage, as it allows the determination of molar masses of unknown samples by using the same calibration curve, irrespective of the DS of the NaCMC sample. Since no commercial NaCMC standards are available, correction factors were determined allowing converting a pullulan based calibration curve into a NaCMC calibration using the broad calibration approach. The weight average molar masses derived using the so established calibration curve closely agree with the ones determined by light scattering, proving the accuracy of the correction factors determined. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of retention models for polymers 1. Poly(ethylene glycol)s.

PubMed

Bashir, Mubasher A; Radke, Wolfgang

2006-10-27

The suitability of three different retention models to predict the retention times of poly(ethylene glycol)s (PEGs) in gradient and isocratic chromatography was investigated. The models investigated were the linear (LSSM) and the quadratic solvent strength model (QSSM). In addition, a model describing the retention behaviour of polymers was extended to account for gradient elution (PM). It was found that all models are suited to properly predict gradient retention volumes provided the extraction of the analyte specific parameters is performed from gradient experiments as well. The LSSM and QSSM on principle cannot describe retention behaviour under critical or SEC conditions. Since the PM is designed to cover all three modes of polymer chromatography, it is therefore superior to the other models. However, the determination of the analyte specific parameters, which are needed to calibrate the retention behaviour, strongly depend on the suitable selection of initial experiments. A useful strategy for a purposeful selection of these calibration experiments is proposed.

Quality control considerations for size exclusion chromatography with online ICP-MS: a powerful tool for evaluating the size dependence of metal-organic matter complexation.

PubMed

McKenzie, Erica R; Young, Thomas M

2013-01-01

Size exclusion chromatography (SEC), which separates molecules based on molecular volume, can be coupled with online inductively coupled plasma mass spectrometry (ICP-MS) to explore size-dependent metal-natural organic matter (NOM) complexation. To make effective use of this analytical dual detector system, the operator should be mindful of quality control measures. Al, Cr, Fe, Se, and Sn all exhibited columnless attenuation, which indicated unintended interactions with system components. Based on signal-to-noise ratio and peak reproducibility between duplicate analyses of environmental samples, consistent peak time and height were observed for Mg, Cl, Mn, Cu, Br, and Pb. Al, V, Fe, Co, Ni, Zn, Se, Cd, Sn, and Sb were less consistent overall, but produced consistent measurements in select samples. Ultrafiltering and centrifuging produced similar peak distributions, but glass fiber filtration produced more high molecular weight (MW) peaks. Storage in glass also produced more high MW peaks than did plastic bottles.

Modulation of Phosphorylation of Tocopherol and Phosphatidylinositol by hTAP1/SEC14L2-Mediated Lipid Exchange

PubMed Central

Zingg, Jean-Marc; Libinaki, Roksan; Meydani, Mohsen; Azzi, Angelo

2014-01-01

The vitamin E derivative, alpha-tocopheryl phosphate (αTP), is detectable in cultured cells, plasma and tissues in small amounts, suggesting the existence of enzyme(s) with α-tocopherol (αT) kinase activity. Here, we characterize the production of αTP from αT and [γ-32P]-ATP in primary human coronary artery smooth muscle cells (HCA-SMC) using separation by thin layer chromatography (TLC) and subsequent analysis by Ultra Performance Liquid Chromatography (UPLC). In addition to αT, although to a lower amount, also γT is phosphorylated. In THP-1 monocytes, γTP inhibits cell proliferation and reduces CD36 scavenger receptor expression more potently than αTP. Both αTP and γTP activate the promoter of the human vascular endothelial growth factor (VEGF) gene with similar potency, whereas αT and γT had no significant effect. The recombinant human tocopherol associated protein 1 (hTAP1, hSEC14L2) binds both αT and αTP and stimulates phosphorylation of αT possibly by facilitating its transport and presentation to a putative αT kinase. Recombinant hTAP1 reduces the in vitro activity of the phosphatidylinositol-3-kinase gamma (PI3Kγ) indicating the formation of a stalled/inactive hTAP1/PI3Kγ heterodimer. The addition of αT, βT, γT, δT or αTP differentially stimulates PI3Kγ, suggesting facilitated egress of sequestered PI from hTAP1 to the enzyme. It is suggested that the continuous competitive exchange of different lipophilic ligands in hTAPs with cell enzymes and membranes may be a way to make these lipophiles more accessible as substrates for enzymes and as components of specific membrane domains. PMID:24983950

Continuous processing of recombinant proteins: integration of refolding and purification using simulated moving bed size-exclusion chromatography with buffer recycling.

PubMed

Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

2014-04-11

Continuous processing of recombinant proteins was accomplished by combining continuous matrix-assisted refolding and purification by tandem simulated moving bed (SMB) size-exclusion chromatography (SEC). Recombinant proteins, N(pro) fusion proteins from inclusion bodies were dissolved with NaOH and refolded in the SMB system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling of the refolding buffer of the raffinate by tangential flow filtration. For further purification of the refolded proteins, a second SMB operation also based on SEC was added. The whole system could be operated isocratically with refolding buffer as the desorbent buffer, and buffer recycling could also be applied in the purification step. Thus, a significant reduction in buffer consumption was achieved. The system was evaluated with two proteins, the N(pro) fusion pep6His and N(pro) fusion MCP-1. Refolding solution, which contained residual N(pro) fusion peptide, the cleaved autoprotease N(pro), and the cleaved target peptide was used as feed solution. Full separation of the cleaved target peptide from residual proteins was achieved at a purity and recovery in the raffinate and extract, respectively, of approximately 100%. In addition, more than 99% of the refolding buffer of the raffinate was recycled. A comparison of throughput, productivity, and buffer consumption of the integrated continuous process with two batch processes demonstrated that up to 60-fold higher throughput, up to 180-fold higher productivity, and at least 28-fold lower buffer consumption can be obtained by the integrated continuous process, which compensates for the higher complexity. Copyright © 2014 Elsevier B.V. All rights reserved.

Implementation of the Single European Code in a Multi-Tissue Bank

PubMed Central

Schroeter, Jan; Schulz, Tino; Schroeter, Bernard; Fleischhauer, Katrin; Pruß, Axel

2017-01-01

Introduction The traceability of tissue and cells transplants is important to ensure a high level of safety for the recipients. With the final introduction of the Single European Code (SEC) in April 2017 in the EU a consistent system among all member states became mandatory. Methods The regulations for the SEC on EU and national level were evaluated. An overview on the different parts of the SEC with detailed explanations is given. Our own experiences with the implementation of the SEC in our multi-tissue bank are reported in addition. Results The implementation of the SEC in our multi-tissue bank could be successfully realized. However, it revealed a number of difficulties, especially the sterile labeling of certain tissue transplants and the complex update of the existing database. Conclusion The introduction of the SEC has made a contribution to the safety of recipients of tissue and cells transplants through a system of comprehensive and transparent traceability. PMID:29344015

Validation of a portable nitric oxide analyzer for screening in primary ciliary dyskinesias.

PubMed

Harris, Amanda; Bhullar, Esther; Gove, Kerry; Joslin, Rhiannon; Pelling, Jennifer; Evans, Hazel J; Walker, Woolf T; Lucas, Jane S

2014-02-10

Nasal nitric oxide (nNO) levels are very low in primary ciliary dyskinesia (PCD) and it is used as a screening test. We assessed the reliability and usability of a hand-held analyser in comparison to a stationary nitric oxide (NO) analyser in 50 participants (15 healthy, 13 PCD, 22 other respiratory diseases; age 6-79 years). Nasal NO was measured using a stationary NO analyser during a breath-holding maneuver, and using a hand-held analyser during tidal breathing, sampling at 2 ml/sec or 5 ml/sec. The three methods were compared for their specificity and sensitivity as a screen for PCD, their success rate in different age groups, within subject repeatability and acceptability. Correlation between methods was assessed. Valid nNO measurements were obtained in 94% of participants using the stationary analyser, 96% using the hand-held analyser at 5 ml/sec and 76% at 2 ml/sec. The hand-held device at 5 ml/sec had excellent sensitivity and specificity as a screening test for PCD during tidal breathing (cut-off of 30 nL/min,100% sensitivity, >95% specificity). The cut-off using the stationary analyser during breath-hold was 38 nL/min (100% sensitivity, 95% specificity). The stationary and hand-held analyser (5 ml/sec) showed reasonable within-subject repeatability(% coefficient of variation = 15). The hand-held NO analyser provides a promising screening tool for PCD.

A robust signal processing method for quantitative high-cycle fatigue crack monitoring using soft elastomeric capacitor sensors

NASA Astrophysics Data System (ADS)

Kong, Xiangxiong; Li, Jian; Collins, William; Bennett, Caroline; Laflamme, Simon; Jo, Hongki

2017-04-01

A large-area electronics (LAE) strain sensor, termed soft elastomeric capacitor (SEC), has shown great promise in fatigue crack monitoring. The SEC is able to monitor strain changes over a mesoscale structural surface and endure large deformations without being damaged under cracking. Previous tests verified that the SEC is able to detect, localize, and monitor fatigue crack activities under low-cycle fatigue loading. In this paper, to examine the SEC's capability of monitoring high-cycle fatigue cracks, a compact specimen is tested under cyclic tension, designed to ensure realistic crack opening sizes representative of those in real steel bridges. To overcome the difficulty of low signal amplitude and relatively high noise level under high-cycle fatigue loading, a robust signal processing method is proposed to convert the measured capacitance time history from the SEC sensor to power spectral densities (PSD) in the frequency domain, such that signal's peak-to-peak amplitude can be extracted at the dominant loading frequency. A crack damage indicator is proposed as the ratio between the square root of the amplitude of PSD and load range. Results show that the crack damage indicator offers consistent indication of crack growth.

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis.

PubMed

Li, Lian; Li, Yan; Feng, Danyang; Xu, Linghua; Yin, Fengxin; Zang, Hengchang; Liu, Chunhui; Wang, Fengshan

2016-10-11

Chondroitin sulfate (CS) plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs) and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV) spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O) had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA) was investigated by destabilization of the medial meniscus (DMM) model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system.

Modeling and simulation of Charpy impact test of maraging steel 300 using Abaqus

NASA Astrophysics Data System (ADS)

Madhusudhan, D.; Chand, Suresh; Ganesh, S.; Saibhargavi, U.

2018-03-01

This work emphasizes the modeling and simulation of Charpy impact test to evaluate fracture energy at different pendulum velocities of armor maraging steel 300 using ABAQUS. To evaluate the fracture energy, V-notch specimen is fractured using the Johnson and Cook Damage model. The Charpy impact tests are of great importance related to fracture properties of steels. The objective of this work is to present absorbed energy variation at pendulum velocities of 5 m/sec, 6 m/sec, 7 m/sec and 9 m/sec in addition to stress distribution at v-notch. Finite Element Method of modeling for three dimensional specimens is used for simulation in commercial software of ABAQUS.

Hydrodynamic chromatography of macromolecules using polymer monolithic columns.

PubMed

Edam, Rob; Eeltink, Sebastiaan; Vanhoutte, Dominique J D; Kok, Wim Th; Schoenmakers, Peter J

2011-12-02

The selectivity window of size-based separations of macromolecules was tailored by tuning the macropore size of polymer monolithic columns. Monolithic materials with pore sizes ranging between 75 nm and 1.2 μm were prepared in situ in large I.D. columns. The dominant separation mechanism was hydrodynamic chromatography in the flow-through pores. The calibration curves for synthetic polymers matched with the elution behavior by HDC separations in packed columns with 'analyte-to-pore' aspect ratios (λ) up to 0.2. For large-macropore monoliths, a deviation in retention behavior was observed for small polystyrene polymers (M(r)<20 kDa), which may be explained by a combined HDC-SEC mechanism for λ<0.02. The availability of monoliths with very narrow pore sizes allowed investigation of separations at high λ values. For high-molecular weight polymers (M(r)>300,000 Da) confined in narrow channels, the separation strongly depended on flow rate. Flow-rate dependent elution behavior was evaluated by calculation of Deborah numbers and confirmed to be outside the scope of classic shear deformation or slalom chromatography. Shear-induced forces acting on the periphery of coiled polymers in solution may be responsible for flow-rate dependent elution. Copyright © 2011 Elsevier B.V. All rights reserved.

Synthesis and characterization of poly(phenylacetylene)s with Ru(II) bis-terpyridine complexes in the side-chain.

PubMed

Breul, Alexander M; Kübel, Joachim; Häupler, Bernhard; Friebe, Christian; Hager, Martin D; Winter, Andreas; Dietzek, Benjamin; Schubert, Ulrich S

2014-04-01

An alkyne-functionalized ruthenium(II) bis-terpyridine complex is directly copolymerized with phenylacetylene by alkyne polymerization. The polymer is characterized by size-exclusion chromatography (SEC), (1) H NMR spectroscopy, cyclic voltammetry (CV) measurements, and thermal analysis. The photophysical properties of the polymer are studied by UV-vis absorption spectroscopy. In addition, spectro-electrochemical measurements are carried out. Time-resolved luminescence lifetime decay curves show an enhanced lifetime of the metal complex attached to the conjugated polymer backbone compared with the Ru(tpy)2 (2+) model complex. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1.

PubMed

Huang, Yukun; Chen, Xiujuan; Duan, Nuo; Wu, Shijia; Wang, Zhouping; Wei, Xinlin; Wang, Yuanfeng

2015-01-01

Enterotoxins from pathogenic bacteria are known as the main reason that can cause the bacterial foodborne diseases. In this study, aptamers that bound to Staphylococcus aureus enterotoxin C1 (SEC1) with high affinity and selectivity were generated in vitro by twelve rounds of selection based on magnetic separation technology, with a low-level dissociation constant (Kd) value of 65.14 ± 11.64 nmol/L of aptamer C10. Aptamer-based quantification of SEC1 in the food sample by a graphene oxide (GO)-based method was implemented to investigate the potential of the aptamer against SEC1 with a limit of detection of 6 ng/mL. On the basis of this work, biosensors using the selected SEC1 aptamers as new molecular recognition elements could be applied for innovative determinations of SEC1. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PubMed

Zhao, F; Stein, D J; Paborji, M; Cash, P W; Root, B J; Wei, Z; Knupp, C J

2001-01-01

BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.

Effects of different sonic activation protocols on debridement efficacy in teeth with single-rooted canals

PubMed Central

Niu, Li-na; Luo, Xiao-juan; Li, Guo-hua; Bortoluzzi, Eduardo A.; Mao, Jing; Chen, Ji-hua; Gutmann, James L.; Pashley, David H.; Tay, Franklin R.

2014-01-01

Objectives The effects of different EndoActivator® (EA) sonic activation protocols on root canal debridement efficacy were examined. Methods Root canals in 48 single-rooted teeth were instrumented, irrigated initially with NaOCl and divided into 6 groups (N=8) based on the application time of QMix (antimicrobial calcium-chelating irrigant), and the time and sequence of EA irrigant activation - Positive Control: 90 sec QMix; Negative Control: 90 sec saline; Group 1A: 15 sec QMix + 15 sec QMix with EA-activation; Group 1B: 30 sec QMix + 30 sec of QMix with EA-activation; Group 2A: 15 sec QMix with EA-activation + 15 sec QMix; Group 2B: 30 sec QMix with EA-activation + 30 sec QMix. Split roots were examined with scanning electron microscopy for assignment of smear and debris scores in locations along the coronal, middle and apical thirds of the canals. The overall cleanliness of pooled canal locations in the Positive Control and the 4 experimental groups were compared with chi-square tests. Results Significant differences were detected among the 5 groups (p<0.001). Post-hoc pairwise comparisons indicated that the overall canal cleanliness was in the order (from best to worst): 1B = 2B > 2A > 1A > Positive Control. Completely clean canals could not be achieved due to the absence of continuous irrigant flow for EA to clear intraradicular debris. Conclusions Irrespective of the sonic activation sequence, irrigant activation for 30 seconds during a 60-second period of QMix application appears to maximize the smear layer and debris removal potential of the EndoActivator® system. PMID:24878251

46 CFR Sec. 7 - Disbursements at foreign ports.

Code of Federal Regulations, 2010 CFR

2010-10-01

...-agent shall pay invoices from such advances and shall make proper accounting to the agent for all... RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Disbursements Sec. 7 Disbursements at foreign... or by any other method outlined to and approved by the owner in advance of its use: (a) The agent may...

One-pot synthesis of star-shaped macromolecules containing polyglycidol and poly(ethylene oxide) arms.

PubMed

Lapienis, Grzegorz; Penczek, Stanislaw

2005-01-01

Synthesis of fully hydrophilic star-shaped macromolecules with different kinds of arms (A(x)B(y)C(z)) based on polyglycidol (PGL, A(x)) and poly(ethylene oxide) (PEO, C(z)) arms and diepoxy compounds (diglycidyl ethers of ethylene glycol (DGEG) or neopentyl glycol (DGNG) in the core, B(y)) forming the core is described. Precursors of arms were prepared by polymerization of glycidol with protected -OH groups. The first-generation stars were formed in the series of consecutive-parallel reactions of arms A(x) with diepoxy compounds (B). These first-generation stars (A(x)B(y)), having approximately O-, Mt+ groups on the cores, were used as multianionic initiators for the second generation of arms (C(z)) built by polymerization of ethylene oxide. The products with M(n) up to 10(5) and having up to approximately 40 arms were obtained. The number of arms (f) was determined by direct measurements of M(n) of the first-generation stars (M(n) of arms A(x) is known), compared with f calculated from the branching index g, determined from R(g) measured with size-exclusion chromatography (SEC) triple detection with TriSEC software. The progress of the star formation was monitored by 1H NMR and SEC. These novel water-soluble stars, having a large number of hydroxyl groups, both at the ends of PEO arms as well as within the PGL arms, can be functionalized and further used for attaching compounds of interest. This approach opens, therefore, a new way of "multiPEGylation".

Liver Cell-Derived Microparticles Activate Hedgehog Signaling and Alter Gene Expression in Hepatic Endothelial Cells

PubMed Central

Witek, Rafal P.; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S.; Cheong, Yeiwon; Fearing, Caitlin M.; Agboola, Kolade M.; Chen, Wei; Diehl, Anna Mae

2013-01-01

Background & Aims Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). Methods MF-HSCs and cholangiocytes were exposed to platelet-derived growth factor (PDGF) to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy (TEM) and immunoblots, and applied to Hh-reporter containing cells. Microparticles were also obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, a Hh signaling inhibitor. Effects on SEC gene expression were evaluated by QRT-PCR and immunoblotting. Finally, Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Results PDGF-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically active Hh ligands. BDL also increased release of Hh-containing exosome-enriched microparticles into plasma and bile. TEM and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Conclusions Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy. PMID:19013163

Effect of in vitro enzymatic degradation on 3D printed poly(ε-caprolactone) scaffolds: morphological, chemical and mechanical properties.

PubMed

Ferreira, Joana; Gloria, Antonio; Cometa, Stefania; Coelho, Jorge F J; Domingos, Marco

2017-07-27

In recent years, the tissue engineering (TE) field has significantly benefited from advanced techniques such as additive manufacturing (AM), for the design of customized 3D scaffolds with the aim of guided tissue repair. Among the wide range of materials available to biomanufacture 3D scaffolds, poly(ε-caprolactone) (PCL) clearly arises as the synthetic polymer with the greatest potential, due to its unique properties - namely, biocompatibility, biodegradability, thermal and chemical stability and processability. This study aimed for the first time to investigate the effect of pore geometry on the in vitro enzymatic chain cleavage mechanism of PCL scaffolds manufactured by the AM extrusion process. Methods: Morphological properties of 3D printed PCL scaffolds before and after degradation were evaluated using Scanning Electron Microscopy (SEM) and micro-computed tomography (μ-CT). Differential Scanning Calorimetry (DSC) was employed to determine possible variations in the crystallinity of the scaffolds during the degradation period. The molecular weight was assessed using Size Exclusion Chromatography (SEC) while the mechanical properties were investigated under static compression conditions. Morphological results suggested a uniform reduction of filament diameter, while increasing the scaffolds' porosity. DSC analysis revealed and increment in the crystallinity degree while the molecular weight, evaluated through SEC, remained almost constant during the incubation period (25 days). Mechanical analysis highlighted a decrease in the compressive modulus and maximum stress over time, probably related to the significant weight loss of the scaffolds. All of these results suggest that PCL scaffolds undergo enzymatic degradation through a surface erosion mechanism, which leads to significant variations in mechanical, physical and chemical properties, but which has little influence on pore geometry.

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

S. Dong; N. Paterson; S.G. Kazarian

A suite of tuyere-level coke samples have been withdrawn from a working blast furnace during coal injection, using the core-drilling technique. The samples have been characterized by size exclusion chromatography (SEC), Fourier transform Raman spectroscopy (FT-RS), and X-ray powder diffraction (XRD) spectroscopy. The 1-methyl-2-pyrrolidinone (NMP) extracts of the cokes sampled from the 'bosh', the rear of the 'bird's nest', and the 'dead man' zones were found by SEC to contain heavy soot-like materials (ca. 10{sup 7}-10{sup 8} apparent mass units). In contrast, NMP extracts of cokes taken from the raceway and the front of the 'bird's nest' only contained amore » small amount of material of relatively lower apparent molecular mass (up to ca. 10{sup 5} u). Since the feed coke contained no materials extractable by the present method, the soot-like materials are thought to have formed during the reactions of volatile matter released from the injectant coal, probably via dehydrogenation and repolymerization of the tars. The Raman spectra of the NMP-extracted core-drilled coke samples showed variations reflecting their temperature histories. Area ratios of D-band to G-band decreased as the exposure temperature increased, while intensity ratios of D to G band and those of 2D to G bands increased with temperature. The graphitic (G), defect (D), and random (R) fractions of the carbon structure of the cokes were also derived from the Raman spectra. The R fractions decreased with increasing temperature, whereas G fractions increased, while the D fractions showed a more complex variation with temperature. These data appear to give clues regarding the graphitization mechanism of tuyere-level cokes in the blast furnace. 41 refs., 9 figs., 6 tabs.« less

Polysaccharide characterization by hollow-fiber flow field-flow fractionation with on-line multi-angle static light scattering and differential refractometry.

PubMed

Pitkänen, Leena; Striegel, André M

2015-02-06

Accurate characterization of the molar mass and size of polysaccharides is an ongoing challenge, oftentimes due to architectural diversity but also to the broad molar mass (M) range over which a single polysaccharide can exist and to the ultra-high M of many polysaccharides. Because of the latter, many of these biomacromolecules experience on-column, flow-induced degradation during analysis by size-exclusion and, even, hydrodynamic chromatography (SEC and HDC, respectively). The necessity for gentler fractionation methods has, to date, been addressed employing asymmetric flow field-flow fractionation (AF4). Here, we introduce the coupling of hollow-fiber flow field-flow fractionation (HF5) to multi-angle static light scattering (MALS) and differential refractometry (DRI) detection for the analysis of polysaccharides. In HF5, less stresses are placed on the macromolecules during separation than in SEC or HDC, and HF5 can offer a higher sensitivity, with less propensity for system overloading and analyte aggregation, than generally found in AF4. The coupling to MALS and DRI affords the determination of absolute, calibration-curve-independent molar mass averages and dispersities. Results from the present HF5/MALS/DRI experiments with dextrans, pullulans, and larch arabinogalactan were augmented with hydrodynamic radius (RH) measurements from off-line quasi-elastic light scattering (QELS) and by RH distribution calculations and fractogram simulations obtained via a finite element analysis implementation of field-flow fractionation theory by commercially available software. As part of this study, we have investigated analyte recovery in HF5 and also possible reasons for discrepancies between calculated and simulated results vis-à-vis experimentally determined data. Published by Elsevier B.V.

Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating.

PubMed

Nishikawa, Shuh-ichi; Hirata, Aiko; Endo, Toshiya

2008-11-01

During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

Characterization of subvisible particle formation during the filling pump operation of a monoclonal antibody solution.

PubMed

Nayak, Arpan; Colandene, James; Bradford, Victor; Perkins, Melissa

2011-10-01

Characterization and control of aggregate and subvisible particle formation during fill-finish process steps are important for biopharmaceutical products. The filling step is of key importance as there is no further filtration of the drug product beyond sterile filtration. Filling processes can impact product quality by introducing physical stresses such as shear, friction, and cavitation. Other detrimental factors include temperature generated in the process of filling, foaming, and contact with filling system materials, including processing aids such as silicone oil. Certain pumps may shed extrinsic particles that may lead to heterogeneous nucleation-induced aggregation. In this work, microflow imaging, size-exclusion chromatography (SEC), and turbidimetry were utilized to quantify subvisible particles, aggregation, and opalescence, respectively. The filling process was performed using several commonly used filling systems, including rotary piston pump, rolling diaphragm pump, peristaltic pump, and time-pressure filler. The rolling diaphragm pump, peristaltic pump, and time-pressure filler generated notably less protein subvisible particles than the rotary piston pump, although no change in aggregate content by SEC was observed by any pump. An extreme increase in subvisible particles was also reflected in an increase in turbidity. Copyright © 2011 Wiley-Liss, Inc.

Process development for robust removal of aggregates using cation exchange chromatography in monoclonal antibody purification with implementation of quality by design.

PubMed

Xu, Zhihao; Li, Jason; Zhou, Joe X

2012-01-01

Aggregate removal is one of the most important aspects in monoclonal antibody (mAb) purification. Cation-exchange chromatography (CEX), a widely used polishing step in mAb purification, is able to clear both process-related impurities and product-related impurities. In this study, with the implementation of quality by design (QbD), a process development approach for robust removal of aggregates using CEX is described. First, resin screening studies were performed and a suitable CEX resin was chosen because of its relatively better selectivity and higher dynamic binding capacity. Second, a pH-conductivity hybrid gradient elution method for the CEX was established, and the risk assessment for the process was carried out. Third, a process characterization study was used to evaluate the impact of the potentially important process parameters on the process performance with respect to aggregate removal. Accordingly, a process design space was established. Aggregate level in load is the critical parameter. Its operating range is set at 0-3% and the acceptable range is set at 0-5%. Equilibration buffer is the key parameter. Its operating range is set at 40 ± 5 mM acetate, pH 5.0 ± 0.1, and acceptable range is set at 40 ± 10 mM acetate, pH 5.0 ± 0.2. Elution buffer, load mass, and gradient elution volume are non-key parameters; their operating ranges and acceptable ranges are equally set at 250 ± 10 mM acetate, pH 6.0 ± 0.2, 45 ± 10 g/L resin, and 10 ± 20% CV respectively. Finally, the process was scaled up 80 times and the impurities removal profiles were revealed. Three scaled-up runs showed that the size-exclusion chromatography (SEC) purity of the CEX pool was 99.8% or above and the step yield was above 92%, thereby proving that the process is both consistent and robust.

Properties Characterization of Chemically Modified Hemp Hurds

PubMed Central

Stevulova, Nadezda; Cigasova, Julia; Estokova, Adriana; Terpakova, Eva; Geffert, Anton; Kacik, Frantisek; Singovszka, Eva; Holub, Marian

2014-01-01

The effect of chemical treatment of hemp hurds slices in three solutions (EDTA (Ethylenediaminetetraacetic acid), NaOH and Ca(OH)2) on the properties of natural material was discussed in this paper. Changes in the morphology, chemical composition and structure as well as thermal stability of hemp hurds before and after their modification were investigated by using FTIR (Fourier transform infrared spectroscopy), XRD (X-ray powder diffraction analysis) and TG (thermogravimetry)/DSC (differential scanning calorimetry). Size exclusion chromatography (SEC) measurements were used for determination of degree of cellulose polymerization of hemp hurd samples. Chemical modification is related to the partial removal of non-cellulosic components of lignin, hemicellulose and pectin as well as waxes from the surface of hemp hurd slices. Another effect of the chemical treatment applied is connected with increasing the crystallinity index of cellulose determined by FTIR and XRD methods. Decrease in degree of cellulose polymerization and polydispersity index in chemically modified hemp hurds compared to the original sample was observed. Increase in thermal stability of treated hemp hurd was found. The most significant changes were observed in alkaline treated hemp hurds by NaOH. PMID:28788294

Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris.

PubMed

Wee, May S M; Matia-Merino, Lara; Carnachan, Susan M; Sims, Ian M; Goh, Kelvin K T

2014-09-01

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ∼1.9×10(6) Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure. Copyright © 2014 Elsevier B.V. All rights reserved.

Light airplane crash tests at impact velocities of 13 and 27 m/sec

NASA Technical Reports Server (NTRS)

Alfaro-Bou, E.; Vaughan, V. L., Jr.

1977-01-01

Two similar general aviation airplanes were crash tested at the Langley impact dynamics research facility at velocities of 13 and 27 m/sec. Other flight parameters were held constant. The facility, instrumentation, tests specimens, and test method are briefly described. Structural damage and accelerometer data are discussed.

77 FR 31551 - Hazardous Materials; Miscellaneous Amendments Pertaining to DOT Specification Cylinders (RRR)

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-29

... comment. To avoid duplication, please use only one of these four methods. All comments received will be... Sec. 180.205(f) (visual inspection) to permit the shot blasting \\d\\ of cylinders to remove surface... conducted by an authorized facility in accordance with Sec. 180.212. \\d\\ Shot blasting aluminum cylinders...

The self-disproportionation of enantiomers (SDE): a menace or an opportunity?

PubMed Central

Kitagawa, Osamu; Wzorek, Alicja

2018-01-01

Herein we report on the well-documented, yet not widely known, phenomenon of the self-disproportionation of enantiomers (SDE): the spontaneous fractionation of scalemic material into enantioenriched and -depleted fractions when any physicochemical process is applied. The SDE has implications ranging from the origins of prebiotic homochirality to unconventional enantiopurification methods, though the risks of altering the enantiomeric excess (ee) unintentionally, regrettably, remain greatly unappreciated. While recrystallization is well known as an SDE process, occurrences of the SDE in other processes are much less recognized, e.g. sublimation and even distillation. But the most common process that many workers seem to be completely ignorant of is SDE via chromatography and reports have included all manner of structures, all types of interactions, and all forms of chromatography, including GC. The SDE can be either a blessing – as a means to obtain enantiopure samples from scalemates – or a curse, as unwitting alteration of the ee leads to errors in the reporting of results and/or misinterpretation of the system under study. Thus the ramifications of the SDE are relevant to any area involving chirality – natural products, asymmetric synthesis, etc. Moreover, there is grave concern regarding errors in the literature, in addition to the possible occurrence of valid results which may have been overlooked and thus remain unreported, as well as the potential for the SDE to alter the ee, particularly via chromatography, and the following concepts will be conveyed: (1) the SDE occurs under totally achiral conditions of (a) precipitation, (b) centrifugation, (c) evaporation, (d) distillation, (e) crystallization, (f) sublimation, and (g) achiral chromatography (e.g. column, flash, MPLC, HPLC, SEC, GC, etc.). (2) The SDE cannot be controlled simply by experimental accuracy and ignorance of the SDE unavoidably leads to mistakes in the recorded and reported stereochemical outcome of enantioselective transformations. (3) The magnitude of the SDE (the difference between the extremes of enantioenrichment and -depletion) can be controlled and used to: (a) minimize mistakes in the recorded experimental values and (b) to develop unconventional and preparatively superior methods for enantiopurification. (4) The magnitude of the SDE cannot be predicted but can be expected for compounds possessing SDE-phoric groups or which have a general tendency for strong hydrogen or halogen bonds or dipole–dipole or aromatic π–π interactions. (5) An SDE test and the rigorous reporting and description of applied physicochemical processes should become part of standard experimental practice to prevent the erroneous reporting of the stereochemical outcome of enantioselective catalytic reactions and the chirooptical properties of scalemates. New directions in the study of the SDE, including halogen bonding-based interactions and novel, unconventional enantiopurification methods such as pseudo-SDE (chiral selector-assisted SDE resolution of racemates), are also reported. PMID:29675218

Structural characterization of sulfated arabinans extracted from Cladophora glomerata Kützing and their macrophage activation.

PubMed

Surayot, Utoomporn; Hun Lee, Ju; Kanongnuch, Chartchai; Peerapornpisal, Yuwadee; Park, WooJung; You, SangGuan

2016-05-01

Water-soluble sulfated heteropolysaccharides were extracted from Cladophora glomerata Kützing and fractionated by ion-exchange chromatography, which yielded two subfractions, F1 and F2. The crude and fractionated polysaccharides (F1 and F2) mostly consisted of carbohydrates (62.8-74.5%) with various amounts of proteins (9.00-17.3%) and sulfates (16.5-23.5%), including different levels of arabinose (41.7-54.4%), galactose (13.5-39.0%), glucose (0.80-10.6%), xylose (6.84-13.4%), and rhamnose (0.20-2.83%). Based on the size exclusion chromatography (SEC) profiles, the crude and fractions mainly contained one peak with shoulders having molecular weight (Mw) ranges of 358-1,501 × 10(3). The F1 fraction stimulated RAW264.7 cells to produce considerable amounts of nitric oxide and cytokines compared to the crude and F2 fraction. The backbone of the most potent immunostimulating fraction (F1) was α-(1→4)-L-arabinopyranoside with galactose and xylose residues as branches at O-2 position, and sulfates mainly at O-2 position as well.

Comparison of exhalation time methods (6 sec vs. 10 sec) of a hand-held exhaled nitric oxide analyzer.

PubMed

Ito, Yasunori; Adachi, Yuichi; Itazawa, Toshiko; Okabe, Yoshie; Adachi, Yoko S; Katsumuma, Toshio; Miyawaki, Toshio

2010-10-01

Standard exhalation time for measuring fractional exhaled nitric oxide (FeNO) is 10 sec, but this is not easy for younger children. We aimed to investigate the agreement between FeNO values during 10-sec (FeNO-10) and 6-sec (FeNO-6) exhalation and the feasibility of measuring FeNO-6, using a hand-held analyzer, NIOX-MINO®. FeNO values measured during 10- and 6-sec (random order) were compared. Success rates of the two different time modes were also evaluated. In 119 asthmatic children (median age 8 years [range 4-15]) who had been already accustomed to NIOX-MINO®, median FeNO-10 (29 ppb [IQR 15.2-42.0]) and FeNO-6 (27 ppb [IQR 16.0-43.5]) did not differ significantly (P = 0.90), and there was a good correlation between both values (r = 0.984, P < 0.001). Mean difference (FeNO-10-FeNO-6) was -0.151 ppb (95% CI: -0.95 to 0.65, limits of agreement: -8.8 to 8.5). In 46 asthmatic children (median age 7 years [range 4-15]) who had never used any FeNO analyzers, all the children aged 8 years and more (n = 21) succeeded in measuring FeNO on both time modes, whereas for children aged younger than 8 years (n = 25) success rates of the 10- and 6-sec mode were 60.0% and 92.0%, respectively. In conclusion, we showed good agreement between FeNO-10 and FeNO-6, and the 6-sec mode of NIOX-MINO® is more feasible than 10-sec mode for measuring FeNO in younger children. © 2010 Wiley-Liss, Inc.

MO-FG-BRA-02: A Feasibility Study of Integrating Breathing Audio Signal with Surface Surrogates for Respiratory Motion Management

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

Lei, Y; Zhu, X; Zheng, D

Purpose: Tracking the surrogate placed on patient skin surface sometimes leads to problematic signals for certain patients, such as shallow breathers. This in turn impairs the 4D CT image quality and dosimetric accuracy. In this pilot study, we explored the feasibility of monitoring human breathing motion by integrating breathing sound signal with surface surrogates. Methods: The breathing sound signals were acquired though a microphone attached adjacently to volunteer’s nostrils, and breathing curve were analyzed using a low pass filter. Simultaneously, the Real-time Position Management™ (RPM) system from Varian were employed on a volunteer to monitor respiratory motion including both shallowmore » and deep breath modes. The similar experiment was performed by using Calypso system, and three beacons taped on volunteer abdominal region to capture breath motion. The period of each breathing curves were calculated with autocorrelation functions. The coherence and consistency between breathing signals using different acquisition methods were examined. Results: Clear breathing patterns were revealed by the sound signal which was coherent with the signal obtained from both the RPM system and Calypso system. For shallow breathing, the periods of breathing cycle were 3.00±0.19 sec (sound) and 3.00±0.21 sec (RPM); For deep breathing, the periods were 3.49± 0.11 sec (sound) and 3.49±0.12 sec (RPM). Compared with 4.54±0.66 sec period recorded by the calypso system, the sound measured 4.64±0.54 sec. The additional signal from sound could be supplement to the surface monitoring, and provide new parameters to model the hysteresis lung motion. Conclusion: Our preliminary study shows that the breathing sound signal can provide a comparable way as the RPM system to evaluate the respiratory motion. It’s instantaneous and robust characteristics facilitate it possibly to be a either independently or as auxiliary methods to manage respiratory motion in radiotherapy.« less

Middle cerebral artery vasospasm: transcranial color-coded duplex sonography versus conventional nonimaging transcranial Doppler sonography.

PubMed

Swiat, Maciej; Weigele, John; Hurst, Robert W; Kasner, Scott E; Pawlak, Mikolaj; Arkuszewski, Michal; Al-Okaili, Riyadh N; Swiercz, Miroslaw; Ustymowicz, Andrzej; Opala, Grzegorz; Melhem, Elias R; Krejza, Jaroslaw

2009-03-01

To prospectively compare accuracies of transcranial color-coded duplex sonography (TCCS) and transcranial Doppler sonography (TCD) in the diagnosis of middle cerebral artery (MCA) vasospasm. Prospective blinded head-to-head comparison TCD and TCCS methods using digital subtraction angiography (DSA) as the reference standard. Department of Radiology in a tertiary university health center in a metropolitan area. Eighty-one consecutive patients (mean age, 53.9 +/- 13.9 years; 48 women). The indication for DSA was subarachnoid hemorrhage in 71 patients (87.6%), stroke or transient ischemic attack in five patients (6.2%), and other reasons in five patients (6.2%). The MCA was graded as normal, narrowed <50%, and >50% using DSA. The accuracy of ultrasound methods was estimated by total area (Az) under receiver operator characteristic curve. To compare sensitivities of ultrasound methods, McNemar's test was used with mean velocity thresholds of 120 cm/sec for the detection of less advanced, and 200 cm/sec for the more advanced MCA narrowing. Angiographic MCA narrowing 50% in 10 of 135 arteries. Accuracy of TCCS was insignificantly higher than that of TCD in the detection of 50% narrowing, total Az for mean velocity being 0.83 +/- 0.05, 0.77 +/- 0.05, and 0.95 +/- 0.02, 0.86 +/- 0.08, respectively. Sensitivity of TCCS at commonly used threshold of 120 cm/sec for less advanced MCA spasm was significantly better than that of TCD at similar specificity, 55% vs. 39%, p = 0.038, whereas at a threshold of 200 cm/sec used for more advanced spasm, sensitivities and specificities of both methods were not different. The accuracy of TCCS and TCD is similar, but TCCS is more sensitive than TCD in the detection of MCA spasm. Sensitivity of both techniques in the detection of mild and more advanced spasm using 120 cm/sec and 200 cm/sec thresholds, respectively, is poor; however, a larger sample is required to increase precision of our sensitivity estimates.

Flux agreement above a Scots pine plantation

NASA Astrophysics Data System (ADS)

Gay, L. W.; Vogt, R.; Bernhofer, Ch.; Blanford, J. H.

1996-03-01

The surface energy exchange of 12m high Scots pine plantation at Hartheim, Germany, was measured with a variety of methods during a 11-day period of fine weather in mid-May 1992. Net radiation and rate of thermal storage were measured with conventional net radiometers, soil heat flux discs and temperature-based storage models. The turbulent fluxes discussed in this report were obtained with an interchanging Bowen ratio energy budget system (BREB, at 14 m), two one-propeller eddy correlation systems (OPEC systems 1 and 2 at 17m), a 1-dimensional sonic eddy correlation system (SEC system 3) at 15 m, all on one “low” tower, and a 3-dimensional sonic eddy correlation system (SEC system 22) at 22 m on the “high” tower that was about 46 m distant. All systems measured sensible and latent heat (H and LE) directly, except for OPEC systems 1 and 2 which estimated LE as a residual term in the surface energy balance. Closure of turbulent fluxes from the two SEC systems was around 80% for daytime and 30% for night, with closure of 1-dimensional SEC system 3 exceeding that of 3-dimensional SEC system 22. The night measurements of turbulent fluxes contained considerable uncertainty, especially with the BREB system where measured gradients often yielded erroneous fluxes due to problems inherent in the method (i.e., computational instability as Bowen's ratio approaches -1). Also, both eddy correlation system designs (OPEC and SEC) appeared to underestimate |H| during stable conditions at night. In addition, both sonic systems (1- and 3-dimensional) underestimated |LE| during stable conditions. The underestimate of |H| at night generated residual estimates of OPEC LE containing a “phantom dew” error that erroneously decreased daily LE totals by about 10 percent. These special night problems are circumvented here by comparing results for daytime periods only, rather than for full days. To summarize, turbulent fluxes on the low tower from OPEC system 2 and the adjacent SEC system 3 were in reasonable agreement, while the BREB system appeared to overestimate H and underestimate LE; H and LE measured by SEC system 22 on the high tower were lower than from OPEC and SEC3 on the low tower. The turbulent flux measurements tended to converge, but the data exhibit unexplained differences between days, between systems, and between locations.

[Progresses in screening active compounds from herbal medicine by affinity chromatography].

PubMed

Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

2015-03-01

Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis

PubMed Central

Li, Lian; Li, Yan; Feng, Danyang; Xu, Linghua; Yin, Fengxin; Zang, Hengchang; Liu, Chunhui; Wang, Fengshan

2016-01-01

Chondroitin sulfate (CS) plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs) and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV) spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O) had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA) was investigated by destabilization of the medial meniscus (DMM) model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system. PMID:27727159

Particle size effects on protein and virus-like particle adsorption on perfusion chromatography media.

PubMed

Wu, Yige; Abraham, Dicky; Carta, Giorgio

2015-01-02

The resin structure, chromatographic behavior, and adsorption kinetics of proteins and virus-like-particles (VLPs) are studied for POROS HS 20 and POROS HS 50 (23 and 52 μm mean diameter, respectively) to determine the effects of particle size on perfusion chromatography and to determine the predictive ability of available models. Transmission electron microscopy (TEM) and inverse size-exclusion chromatography (iSEC) show similar structures for the two resins, both containing 200-1000 nm pores that transect a network of much smaller pores. For non-binding conditions, trends of the height equivalent to a theoretical plate (HETP) as a function of reduced velocity are consistent with perfusion. The estimated intraparticle flow fractions for these conditions are 0.0018 and 0.00063 for POROS HS 20 and HS 50, respectively. For strong binding conditions, confocal laser scanning microscopy (CLSM) shows asymmetrical intraparticle concentrations profiles and enhanced rates of IgG adsorption on POROS HS 20 at 1000 cm/h. The corresponding effective diffusivity under flow is 2-3 times larger than for non-flow conditions and much larger than observed for POROS HS 50, consistent with available models. For VLPs, however, adsorption is confined to a thin layer near the particle surface for both resins, suggesting that the bound VLPs block the pores. Copyright © 2014 Elsevier B.V. All rights reserved.

An alternative method for determining daily bladder perception.

PubMed

Erdem, Erim; Karazindiyanoglu, Sinan; Ulger, Suleyman

2010-01-01

To avoid the unphysiologic nature of cystometry, we searched a new tool for evaluating bladder perceptions. The study group consisted of 25 (14 girls and 11 boys) primary monosymptomatic enuretic children with a mean age of 11 (range 8-16). Four children were excluded due to neuromuscular dysfunctions of the bladder, which was demonstrated with the help of cystometry. All children filled a voiding chart 3 times daily to record the duration elapsed till normal desire (ND(daily)) and strong desire (SD(daily)). During cystometry, the amounts of infused medium (cystometric ND(ml) and cystometric SD(ml)) and the duration (cystometric ND(sec) and cystometric SD(sec)), till ND and SD were perceived and recorded. Mean cystometric ND(ml) was 209.9 +/- 107.2 and ND(sec), 318.1 +/- 135.5, whereas mean cystometric SD(ml) was 273.0 +/- 103.1 and SD(sec), 415.7 +/- 136.8. To evaluate the reliability of elapsed time instead of milliliters, as a parameter, cystometric ND/SD values were calculated and a strong correlation was found between the 2 (ND/SD(sec) = 0.77 +/- 0.19 and ND/SD(ml) = 0.77 +/- 0.19, r = 0.9795, P = .000). Although there was a strong correlation between 3 ND(daily) (r = 0.9576, P = .000), between 3 SD(daily) (r = 0.9706, P = .000), and 3 ND/SD(daily) (r = 0.8706, P = .000), no significant correlation was determined between mean ND(daily) and cystometric ND(sec) (r = 0.3410, P = .2032), and also between mean SD(daily) and cystometric SD(sec) (r = 0.2740, P = .2402). Daily durations of sensations do not correlate with those perceived during cystometry. However, as the results of 3 consecutive daily recordings have a strong correlation, comparison of the reliability of these methods is still needed. 2010 Elsevier Inc. All rights reserved.

Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy as an Analytical Method to Investigate the Secondary Structure of a Model Protein Embedded in Solid Lipid Matrices.

PubMed

Zeeshan, Farrukh; Tabbassum, Misbah; Jorgensen, Lene; Medlicott, Natalie J

2018-02-01

Protein drugs may encounter conformational perturbations during the formulation processing of lipid-based solid dosage forms. In aqueous protein solutions, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can investigate these conformational changes following the subtraction of spectral interference of solvent with protein amide I bands. However, in solid dosage forms, the possible spectral contribution of lipid carriers to protein amide I band may be an obstacle to determine conformational alterations. The objective of this study was to develop an ATR FT-IR spectroscopic method for the analysis of protein secondary structure embedded in solid lipid matrices. Bovine serum albumin (BSA) was chosen as a model protein, while Precirol AT05 (glycerol palmitostearate, melting point 58 ℃) was employed as the model lipid matrix. Bovine serum albumin was incorporated into lipid using physical mixing, melting and mixing, or wet granulation mixing methods. Attenuated total reflection FT-IR spectroscopy and size exclusion chromatography (SEC) were performed for the analysis of BSA secondary structure and its dissolution in aqueous media, respectively. The results showed significant interference of Precirol ATO5 with BSA amide I band which was subtracted up to 90% w/w lipid content to analyze BSA secondary structure. In addition, ATR FT-IR spectroscopy also detected thermally denatured BSA solid alone and in the presence of lipid matrix indicating its suitability for the detection of denatured protein solids in lipid matrices. Despite being in the solid state, conformational changes occurred to BSA upon incorporation into solid lipid matrices. However, the extent of these conformational alterations was found to be dependent on the mixing method employed as indicated by area overlap calculations. For instance, the melting and mixing method imparted negligible effect on BSA secondary structure, whereas the wet granulation mixing method promoted more changes. Size exclusion chromatography analysis depicted the complete dissolution of BSA in the aqueous media employed in the wet granulation method. In conclusion, an ATR FT-IR spectroscopic method was successfully developed to investigate BSA secondary structure in solid lipid matrices following the subtraction of lipid spectral interference. The ATR FT-IR spectroscopy could further be applied to investigate the secondary structure perturbations of therapeutic proteins during their formulation development.

75 FR 8239 - Federal Home Loan Bank Housing Associates, Core Mission Activities and Standby Letters of Credit

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-24

... part 950 of this chapter. title. Sec. 1264.3(a), Sec. 926.4........ Sec. 1264.4. Introductory text. Sec...).. Sec. 1264.3(a)(2). Introductory text. Sec. 1264.4(c)(1)......... Sec. 926.3(a)(3).. Sec. 1264.3(a)(3...)......... Sec. 950.7 of this Sec. 950.7 of this chapter. title. Sec. 1269.3(a), Sec. Sec. Sec. Sec. Introductory...

Computerized Orders with Standardized Concentrations Decrease Dispensing Errors of Continuous Infusion Medications for Pediatrics

PubMed Central

Sowan, Azizeh K.; Vaidya, Vinay U.; Soeken, Karen L.; Hilmas, Elora

2010-01-01

OBJECTIVES The use of continuous infusion medications with individualized concentrations may increase the risk for errors in pediatric patients. The objective of this study was to evaluate the effect of computerized prescriber order entry (CPOE) for continuous infusions with standardized concentrations on frequency of pharmacy processing errors. In addition, time to process handwritten versus computerized infusion orders was evaluated and user satisfaction with CPOE as compared to handwritten orders was measured. METHODS Using a crossover design, 10 pharmacists in the pediatric satellite within a university teaching hospital were given test scenarios of handwritten and CPOE order sheets and asked to process infusion orders using the pharmacy system in order to generate infusion labels. Participants were given three groups of orders: five correct handwritten orders, four handwritten orders written with deliberate errors, and five correct CPOE orders. Label errors were analyzed and time to complete the task was recorded. RESULTS Using CPOE orders, participants required less processing time per infusion order (2 min, 5 sec ± 58 sec) compared with time per infusion order in the first handwritten order sheet group (3 min, 7 sec ± 1 min, 20 sec) and the second handwritten order sheet group (3 min, 26 sec ± 1 min, 8 sec), (p<0.01). CPOE eliminated all error types except wrong concentration. With CPOE, 4% of infusions processed contained errors, compared with 26% of the first group of handwritten orders and 45% of the second group of handwritten orders (p<0.03). Pharmacists were more satisfied with CPOE orders when compared with the handwritten method (p=0.0001). CONCLUSIONS CPOE orders saved pharmacists' time and greatly improved the safety of processing continuous infusions, although not all errors were eliminated. pharmacists were overwhelmingly satisfied with the CPOE orders PMID:22477811

GLC analysis of base composition of RNA and DNA hydrolysates

NASA Technical Reports Server (NTRS)

Lakings, D. B.; Gehreke, C. W.

1971-01-01

Various methods used for the analysis of the base composition of RNA and DNA hydrolysates are presented. The methods discussed are: (1) ion-exchange chromatography, (2) paper chromatography, (3) paper electrophoresis, (4) thin layer chromatography, (5) paper chromatography and time of flight mass spectrometry, and (6) gas-liquid chromatography. The equipment required and the conditions for obtaining the best results with each method are described.

Predicting protein aggregation during storage in lyophilized solids using solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS).

PubMed

Moorthy, Balakrishnan S; Schultz, Steven G; Kim, Sherry G; Topp, Elizabeth M

2014-06-02

Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products.

Sternal Route More Effective than Tibial Route for Intraosseous Amiodarone Administration in a Swine Model of Ventricular Fibrillation.

PubMed

Burgert, James M; Martinez, Andre; O'Sullivan, Mara; Blouin, Dawn; Long, Audrey; Johnson, Arthur D

2018-01-01

The pharmacokinetics of IO administered lipid soluble amiodarone during ventricular fibrillation (VF) with ongoing CPR are unknown. This study measured mean plasma concentration over 5 minutes, maximum plasma concentration (Cmax), and time to maximum concentration (Tmax) of amiodarone administered by the sternal IO (SIO), tibial IO (TIO), and IV routes in a swine model of VF with ongoing CPR. Twenty-one Yorkshire-cross swine were randomly assigned to three groups: SIO, TIO, and IV. Ventricular fibrillation was induced under general anesthesia. After 4 minutes in VF, 300 mg amiodarone was administered as indicated by group assignment. Serial blood specimens collected at 30, 60, 90, 120, 150, 180, 240, and 300 seconds were analyzed using high performance liquid chromatography with tandem mass spectrometry. The mean plasma concentration of IV amiodarone over 5 minutes was significantly higher than the TIO group at 60 seconds (P = 0.02) and 90 seconds (P = 0.017) post-injection. No significant differences in Cmax between the groups were found (P <0.05). The Tmax of amiodarone was significantly shorter in the SIO (99 secs) and IV (86 secs) groups compared to the TIO group (215 secs); P = 0.002 and P = 0.002, respectively. The SIO and IV routes of amiodarone administration were comparable. The TIO group took nearly three times longer to reach Tmax than the SIO and IV groups, likely indicating depot of lipid-soluble amiodarone in adipose-rich tibial yellow bone marrow. The SIO route was more effective than the TIO route for amiodarone delivery in a swine model of VF with ongoing CPR. Further investigations are necessary to determine if the kinetic differences found between the SIO and TIO routes in this study affect survival of VF in humans.

Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine.

PubMed

Monguió-Tortajada, Marta; Roura, Santiago; Gálvez-Montón, Carolina; Pujal, Josep Maria; Aran, Gemma; Sanjurjo, Lucía; Franquesa, Marcel la; Sarrias, Maria-Rosa; Bayes-Genis, Antoni; Borràs, Francesc E

2017-01-01

Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect.

Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional expression.

PubMed Central

Brown, J D; Hann, B C; Medzihradszky, K F; Niwa, M; Burlingame, A L; Walter, P

1994-01-01

The signal recognition particle (SRP) is an evolutionarily conserved ribonucleoprotein (RNP) complex that functions in protein targeting to the endoplasmic reticulum (ER) membrane. Only two protein subunits of the SRP, Srp54p and Sec65p, and the RNA subunit, scR1, were previously known in the yeast Saccharomyces cerevisiae. Purification of yeast SRP by immunoaffinity chromatography revealed five additional proteins. Amino acid sequencing and cloning of the genes encoding four of these proteins demonstrated that the yeast SRP contains homologs (termed Srp14p, Srp68p and Srp72p) of the SRP14, SRP68 and SRP72 subunits found in mammalian SRP. The yeast SRP also contains a 21 kDa protein (termed Srp21p) that is not homologous to any protein in mammalian SRP. An additional 7 kDa protein may correspond to the mammalian SRP9. Disruption of any one of the four genes encoding the newly identified SRP proteins results in slow cell growth and inefficient protein translocation across the ER membrane. These phenotypes are indistinguishable from those resulting from the disruption of genes encoding SRP components identified previously. These data indicate that a lack of any of the analyzed SRP components results in loss of SRP function. ScR1 RNA and SRP proteins are at reduced levels in cells lacking any one of the newly identified proteins. In contrast, SRP components are present at near wild type levels and SRP subparticles are present in cells lacking either Srp54p or Sec65p. Thus Srp14p, Srp21p, Srp68p and Srp72p, but not Sec65p or Srp54p, are required for stable expression of the yeast SRP. Images PMID:7925282

Structural characterization of human general transcription factor TFIIF in solution

PubMed Central

Akashi, Satoko; Nagakura, Shinjiro; Yamamoto, Seiji; Okuda, Masahiko; Ohkuma, Yoshiaki; Nishimura, Yoshifumi

2008-01-01

Human general transcription factor IIF (TFIIF), a component of the transcription pre-initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size-exclusion chromatography (SEC), electrospray ionization mass spectrometry (ESI-MS), and chemical cross-linking. Recombinant TFIIF, composed of an equimolar ratio of α and β subunits, was bacterially expressed, purified to homogeneity, and found to have a transcription activity similar to a natural one in the human in vitro transcription system. SEC of purified TFIIF, as previously reported, suggested that this protein has a size >200 kDa. In contrast, ESI-MS of the purified sample gave a molecular size of 87 kDa, indicating that TFIIF is an αβ heterodimer, which was confirmed by matrix-assisted laser desorption/ionization (MALDI) MS of the cross-linked TFIIF components. Recent electron microscopy (EM) and photo-cross-linking studies showed that the yeast TFIIF homolog containing Tfg1 and Tfg2, corresponding to the human α and β subunits, exists as a heterodimer in the PIC, so the human TFIIF is also likely to exist as a heterodimer even in the PIC. In the yeast PIC, EM and photo-cross-linking studies showed different results for the mutual location of TFIIE and TFIIF along DNA. We have examined the direct interaction between human TFIIF and TFIIE by ESI-MS, SEC, and chemical cross-linking; however, no direct interaction was observed, at least in solution. This is consistent with the previous photo-cross-linking observation that TFIIF and TFIIE flank DNA separately on both sides of the Pol II central cleft in the yeast PIC. PMID:18218714

Progress in Y-00 physical cipher for Giga bit/sec optical data communications (intensity modulation method)

NASA Astrophysics Data System (ADS)

Hirota, Osamu; Futami, Fumio

2014-10-01

To guarantee a security of Cloud Computing System is urgent problem. Although there are several threats in a security problem, the most serious problem is cyber attack against an optical fiber transmission among data centers. In such a network, an encryption scheme on Layer 1(physical layer) with an ultimately strong security, a small delay, and a very high speed should be employed, because a basic optical link is operated at 10 Gbit/sec/wavelength. We have developed a quantum noise randomied stream cipher so called Yuen- 2000 encryption scheme (Y-00) during a decade. This type of cipher is a completely new type random cipher in which ciphertext for a legitimate receiver and eavesdropper are different. This is a condition to break the Shannon limit in theory of cryptography. In addition, this scheme has a good balance on a security, a speed and a cost performance. To realize such an encryption, several modulation methods are candidates such as phase-modulation, intensity-modulation, quadrature amplitude modulation, and so on. Northwestern university group demonstrated a phase modulation system (α=η) in 2003. In 2005, we reported a demonstration of 1 Gbit/sec system based on intensity modulation scheme(ISK-Y00), and gave a design method for quadratic amplitude modulation (QAM-Y00) in 2005 and 2010. An intensity modulation scheme promises a real application to a secure fiber communication of current data centers. This paper presents a progress in quantum noise randomized stream cipher based on ISK-Y00, integrating our theoretical and experimental achievements in the past and recent 100 Gbit/sec(10Gbit/sec × 10 wavelengths) experiment.

Selenium protein identification and profiling by mass spectrometry: A tool to assess progression of cardiomyopathy in a whale model.

PubMed

Bryan, Colleen E; Bossart, Gregory D; Christopher, Steven J; Davis, W Clay; Kilpatrick, Lisa E; McFee, Wayne E; O'Brien, Terrence X

2017-12-01

Non-ischemic cardiomyopathy is a leading cause of congestive heart failure and sudden cardiac death in humans and in some cases the etiology of cardiomyopathy can include the downstream effects of an essential element deficiency. Of all mammal species, pygmy sperm whales (Kogia breviceps) present the greatest known prevalence of cardiomyopathy with more than half of examined individuals indicating the presence of cardiomyopathy from gross and histo-pathology. Several factors such as genetics, infectious agents, contaminants, biotoxins, and inappropriate dietary intake (vitamins, selenium, mercury, and pro-oxidants), may contribute to the development of idiopathic cardiomyopathy in K. breviceps. Due to the important role Se can play in antioxidant biochemistry and protein formation, Se protein presence and relative abundance were explored in cardiomyopathy related cases. Selenium proteins were separated and detected by multi-dimension liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS), Se protein identification was performed by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS), and Se protein profiles were examined in liver (n=30) and heart tissue (n=5) by SEC/UV/ICP-MS detection. Data collected on selenium proteins was evaluated in the context of individual animal trace element concentration, life history, and histological information. Selenium containing protein peak profiles varied in presence and intensity between animals with no pathological findings of cardiomyopathy and animals exhibiting evidence of cardiomyopathy. In particular, one class of proteins, metallothioneins, was found to be associated with Se and was in greater abundance in animals with cardiomyopathy than those with no pathological findings. Profiling Se species with SEC/ICP-MS proved to be a useful tool to identify Se protein pattern differences between heart disease stages in K. breviceps and an approach similar to this may be applied to other species to study Se protein associations with cardiomyopathy. Published by Elsevier GmbH.

Interpretation of size-exclusion chromatography for the determination of molecular-size distribution of human immunoglobulins.

PubMed

Christians, S; Schluender, S; van Treel, N D; Behr-Gross, M-E

2016-01-01

Molecular-size distribution by size-exclusion chromatography (SEC) [1] is used for the quantification of unwanted aggregated forms in therapeutic polyclonal antibodies, referred to as human immunoglobulins (Ig) in the European Pharmacopoeia. Considering not only the requirements of the monographs for human normal Ig (0338, 0918 and 2788) [2-4], but also the general chapter on chromatographic techniques (2.2.46) [5], several chromatographic column types are allowed for performing this test. Although the EDQM knowledge database gives only 2 examples of suitable columns as a guide for the user, these monographs permit the use of columns with different lengths and diameters, and do not prescribe either particle size or pore size, which are considered key characteristics of SEC columns. Therefore, the columns used may differ significantly from each other with regard to peak resolution, potentially resulting in ambiguous peak identity assignment. In some cases, this may even lead to situations where the manufacturer and the Official Medicines Control Laboratory (OMCL) in charge of Official Control Authority Batch Release (OCABR) have differing molecular-size distribution profiles for aggregates of the same batch of Ig, even though both laboratories follow the requirements of the relevant monograph. In the present study, several formally acceptable columns and the peak integration results obtained therewith were compared. A standard size-exclusion column with a length of 60 cm and a particle size of 10 µm typically detects only 3 Ig fractions, namely monomers, dimers and polymers. This column type was among the first reliable HPLC columns on the market for this test and very rapidly became the standard for many pharmaceutical manufacturers and OMCLs for batch release testing. Consequently, the distribution of monomers, dimers and polymers was established as the basis for the interpretation of the results of the molecular-size distribution test in the relevant monographs. However, modern columns with a smaller particle size provide better resolution and also reveal a class of components designated here as oligomers. This publication addresses the interpretation of the SEC test for Ig with respect to the following questions: - how can molecular-size distribution tests benefit from the use of the most recent column technology without changing the sense of well-established quality parameters? - is it possible to mathematically define a way to interpret chromatograms generated with various column types with the same fractionation range but different resolution power? - how should oligomers be considered regarding compliance with compendial specifications?

Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

PubMed Central

Swanson, Stephanie; Ioerger, Thomas R.; Rigel, Nathan W.; Miller, Brittany K.; Braunstein, Miriam

2015-01-01

ABSTRACT While SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure of Mycobacterium tuberculosis SecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in how MtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negative secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG. IMPORTANCE SecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 of Mycobacterium tuberculosis (the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative contacts with the SecYEG channel. Several structural differences are also identified that could relate to the unique function and selectivity of SecA2. Suppressor mutations of a secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG. PMID:26668263

Headspace Gas Chromatography Method for Studies of Reaction and Permeation of Volatile Agents with Solid Materials

DTIC Science & Technology

2015-01-01

HEADSPACE GAS CHROMATOGRAPHY METHOD FOR STUDIES OF REACTION AND PERMEATION OF...TITLE AND SUBTITLE Headspace Gas Chromatography Method for Studies of Reaction and Permeation of Volatile Agents with Solid Materials 5a...method is described for measuring the reactivity and permeability of fabrics, films, and other solid materials. Headspace gas chromatography (GC)

75 FR 16222 - Self-Regulatory Organizations; Notice of Filing and Immediate Effectiveness of Proposed Rule...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-31

... liability for system outages. The text of the proposed rule change is attached as Exhibit 5 to the 19b-4...: Electronic Comments Use the Commission's Internet comment form ( http://www.sec.gov/rules/sro.shtml ); or... method. The Commission will post all comments on the Commission's Internet Web site ( http://www.sec.gov...

A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract.

PubMed

Tripathi, Vinayak R; Kumar, Shailendra; Garg, Satyendra K

2011-07-12

Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools. The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD) of protease inhibitor (PI) was detected in dialyzed and SEC elute. The conidial germination of Aspergillus flavus was moderately inhibited (30%) by the dialyzed seed extract. Cassia tora seed extract has strong protease inhibitory activity against trypsin, Aspergillus flavus and Bacillus sp. proteases. The inhibitor in Cassia tora may attenuate microbial proteases and also might be used as phytoprotecting agent. © 2011 Tripathi et al; licensee BioMed Central Ltd.

Electromagnetic PIC modeling with a background gas

NASA Astrophysics Data System (ADS)

Verboncoeur, J. P.; Cooperberg, D.

1997-02-01

Modeling the interaction of relativistic electromagnetic plasmas with a background gas is described. The timescales range over many orders of magnitude, from the electromagnetic Courant condition (˜10-12 sec) to electron-neutral collision times (˜10-7 sec) to ion transit times (˜10-5 sec). For this work, the traditional Monte Carlo algorithm [1] is described for relativistic electrons. Subcycling is employed to improve efficiency, and smoothing is employed to reduce particle noise. Applications include plasma-focused electron guns, gas-filled microwave tubes, surface wave discharges driven at microwave frequencies, and electron-cyclotron resonance discharges. The method is implemented in the OOPIC code [2].

Study of monopropellants for electrothermal thrusters

NASA Technical Reports Server (NTRS)

Kuenzly, J. D.

1974-01-01

A 333 mN electrothermal thruster designed to use MIL-grade hydrazine was demonstrated to be suitable for operation with low freezing point monopropellants containing hydrazine azide, monomethylhydrazine, unsymmetrical-dimethylhydrazine and ammonia. The steady-state specific impulse was greater than 200 sec for all propellants. The pulsed-mode specific impulse for an azide blend exceeded 175 sec for pulse widths greater than 50 msec; propellants containing carbonaceous species delivered 175 sec pulsed-mode specific impulses for pulse widths greater than 100 msec. Longer thrust chamber residence times were required for the carbonaceous propellants; the original thruster design was modified by increasing the characteristic chamber length and screen packing density. Specific recommendations were made for the work required to design and develop flight worthy thrusters, including methods to increase propellant dispersal at injection, thruster geometry changes to reduce holding power levels and methods to initiate the rapid decomposition of the carbonaceous propellants.

Microstructure of the IMF turbulences at 2.5 AU

NASA Technical Reports Server (NTRS)

Mavromichalaki, H.; Vassilaki, A.; Marmatsouri, L.; Moussas, X.; Quenby, J. J.; Smith, E. J.

1995-01-01

A detailed analysis of small period (15-900 sec) magnetohydrodynamic (MHD) turbulences of the interplanetary magnetic field (IMF) has been made using Pioneer-11 high time resolution data (0.75 sec) inside a Corotating Interaction Region (CIR) at a heliocentric distance of 2.5 AU in 1973. The methods used are the hodogram analysis, the minimum variance matrix analysis and the cohenrence analysis. The minimum variance analysis gives evidence of linear polarized wave modes. Coherence analysis has shown that the field fluctuations are dominated by the magnetosonic fast modes with periods 15 sec to 15 min. However, it is also shown that some small amplitude Alfven waves are present in the trailing edge of this region with characteristic periods (15-200 sec). The observed wave modes are locally generated and possibly attributed to the scattering of Alfven waves energy into random magnetosonic waves.

Staphylococcal enterotoxin C2 promotes osteogenesis of mesenchymal stem cells and accelerates fracture healing

PubMed Central

Wu, T.; Wang, B.; Sun, Y.; Liu, Y.; Li, G.

2018-01-01

Objectives As one of the heat-stable enterotoxins, Staphylococcal enterotoxin C2 (SEC2) is synthesized by Staphylococcus aureus, which has been proved to inhibit the growth of tumour cells, and is used as an antitumour agent in cancer immunotherapy. Although SEC2 has been reported to promote osteogenic differentiation of human mesenchymal stem cells (MSCs), the in vivo function of SCE2 in animal model remains elusive. The aim of this study was to further elucidate the in vivo effect of SCE2 on fracture healing. Materials and Methods Rat MSCs were used to test the effects of SEC2 on their proliferation and osteogenic differentiation potentials. A rat femoral fracture model was used to examine the effect of local administration of SEC2 on fracture healing using radiographic analyses, micro-CT analyses, biomechanical testing, and histological analyses. Results While SEC2 was found to have no effect on rat MSCs proliferation, it promoted the osteoblast differentiation of rat MSCs. In the rat femoral fracture model, the local administration of SEC2 accelerated fracture healing by increasing fracture callus volumes, bone volume over total volume (BV/TV), and biomechanical recovery. The SEC2 treatment group has superior histological appearance compared with the control group. Conclusion These data suggest that local administration of SEC2 may be a novel therapeutic approach to enhancing bone repair such as fracture healing. Cite this article: T. Wu, J. Zhang, B. Wang, Y. Sun, Y. Liu, G. Li. Staphylococcal enterotoxin C2 promotes osteogenesis of mesenchymal stem cells and accelerates fracture healing. Bone Joint Res 2018;7:179–186. DOI: 10.1302/2046-3758.72.BJR-2017-0229.R1. PMID:29682284

Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells.

PubMed

Yokomori, Hiroaki; Oda, Masaya; Yoshimura, Kazunori; Nagai, Toshihiro; Ogi, Mariko; Nomura, Masahiko; Ishii, Hiromasa

2003-12-01

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and vascular permeability. Hepatic sinusoidal endothelial cells (SECs) possess sieve-like pores that form an anastomosing labyrinth structure by the deeply invaginated plasma membrane. Caveolin is the principal structural protein in caveolae. In this study, we examined the role of VEGF on the fenestration and permeability of SECs and the relation with caveolin-1. SECs isolated from rat livers by collagenase infusion method were cultured for 24 h with (10 or 100 ng/ml) or without VEGF. The cells were then examined by transmission and scanning electron microscopy (EM). The expression of caveolin was investigated by confocal immunofluorescence, immunogold EM, and Western blot. Endocytosis and intracellular traffic was studied using horseradish peroxidase (HRP) reaction as a marker of fluid phase transport in SECs. Both transmission and scanning EM showed an increased number of sinusoidal endothelial fenestrae (SEF) in SECs cultured with VEGF. By confocal immunofluorescence, SECs cultured with VEGF displayed prominent caveolin-l-positive aggregates in the cytoplasm, especially surrounding the nucleus region. Immunogold EM depicted increased caveolin-1 reactivity on vesicles and vacuoles of VEGF-treated SECs compared with VEGF-nontreated cells. However, there was no change in the level of caveolin-1 protein expression on Western blot. After HRP injection, an increase of electron-dense tracer filled the SEF in cells treated with VEGF. Our results suggested that VEGF induced fenestration in SECs, accompanied by an increased number of caveolae-like vesicles. Increased caveolin-1 might be associated with vesicle formation but not with fenestration. Increased fenestration may augment hepatic sinusoidal permeability and transendothelial transport.

Optimization of a two stage light gas gun. M.S. Thesis

NASA Technical Reports Server (NTRS)

Rynearson, R. J.; Rand, J. L.

1972-01-01

Performance characteristics of the Texas A&M University light gas gun are presented along with a review of basic gun theory and popular prediction methods. A computer routine based on the simple isentropic compression method is discussed. Results from over 60 test shots are given which demonstrate an increase in gun muzzle velocity from 9.100 ft/sec. to 19,000 ft/sec. The data gathered indicated the Texas A&M light gas gun more closely resembles an isentropic compression gun rather than a shock compression gun.

Fungal treatment of humic-rich industrial wastewater: application of white rot fungi in remediation of food-processing wastewater.

PubMed

Zahmatkesh, Mostafa; Spanjers, Henri; van Lier, Jules B

2017-11-01

This paper presents the results of fungal treatment of a real industrial wastewater (WW), providing insight into the main mechanisms involved and clarifying some ambiguities and uncertainties in the previous reports. In this regard, the mycoremediation potentials of four strains of white rot fungi (WRF): Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus and Pleurotus pulmonarius were tested to remove humic acids (HA) from a real humic-rich industrial treated WW of a food-processing plant. The HA removal was assessed by color measurement and size-exclusion chromatography (SEC) analysis. T. versicolor showed the best decolorization efficiency of 90% and yielded more than 45% degradation of HA, which was the highest among the tested fungal strains. The nitrogen limitation was studied and results showed that it affected the fungal extracellular laccase and manganese peroxidase (MnP) activities. The results of the SEC analysis revealed that the mechanism of HA removal by WRF involves degradation of large HA molecules to smaller molecules, conversion of HA to fulvic acid-like molecules and also biosorption of HA by fungal mycelia. The effect of HS on the growth of WRF was investigated and results showed that the inhibition or stimulation of growth differs among the fungal strains.

Effect of frozen storage on molecular weight, size distribution and conformation of gluten by SAXS and SEC-MALLS.

PubMed

Zhao, Lei; Li, Lin; Liu, Guo-Qin; Liu, Xing-Xun; Li, Bing

2012-06-12

In this study, the effects of frozen (-18 °C) storage time on molecular weight, size distribution, conformation, free amino groups and free sulfhydryl groups of gluten were studied by small-angle X-ray scattering (SAXS), multi-angle laser light scattering (MALLS) in conjunction with a size exclusion chromatography (SEC) and spectrophotometrically. The results showed that the gluten dissolved in 50 mM acetic acid appeared to be similar to quasi-spherical of the chain conformation and the slope of the conformation plot decreased during the storage. Both the molecular weight and radius of gyration of the frozen gluten decreased with the storage time showing a depolymerization in the high molecular weight fraction of gluten (10(5) Da ~ 10(9) Da). Therefore, at constant molecular weight the change of the chain conformation did not show a clear correlation with the storage time. The free amino groups content changed little and the free sulfhydryl groups content of the gluten increased from 9.8 μmol/g for the control to 12.87 μmol/g for 120-day-stored gluten, indicating that the water redistribution and ice recrystallization lead to the breakage of the disulphide bonds and may be one of the reasons for the depolymerization of gluten polymer.

Extraction and Isolation of Antineoplastic Pristimerin from Mortonia greggii (Celastraceae).

PubMed

Mejia-Manzano, Luis Alberto; Barba-Dávila, Bertha A; Gutierrez-Uribe, Janet A; Escalante-Vázquez, Edgardo J; Serna-Saldivar, Sergio O

2015-11-01

The aim of this research was to identify, extract and isolate pristimerin in leaves, stems and roots of the Mexican plant Mortonia greggii (Celastraceae). The principal objective was to determine the best laboratory experimental conditions for the extraction and isolation of this powerful natural anticancer agent from the root tissue. Six experimental factors in solid-liquid pristimerin extraction were analyzed: solvent systems, number of extractions, ratio of plant weight (g)/solvent volume (mL) used, time of extraction, temperature and agitation. A mathematical model was generated for pristimerin purity and yield. Ethanol, first extraction, 0.5 ratio of plant weight/solvent volume (g/mL), 0.5 h, 200 rpm and 49.7°C were optimal conditions for the extraction of this phytochemical. The degree of purification of pristimerin root extract was studied by size-exclusion chromatography (SEC) using Sephadex LH-20 reaching fractions with purification indexes (PI) greater than 2 and recoveries of 28.3%. When fractions with purification indices higher than 1 and less than 2 were accumulated, the recovery of pristimerin increased by about 73.6%. By combining the optimum extracts and SEC purification protocols, an enriched fraction containing 245.6 mg pristimerin was obtained from 100 g of root bark, representing about 14.4%, w/w, pristimerin from the total solids presented in the fraction.

Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

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

Prakash, Aishwarya; Moharana, Kedar; Wallace, Susan S.

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis andmore » mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.« less

Comparative investigation of low-molecular-weight fulvic acids of different origin by SEC-Q-TOF-ms: new insights into structure and formation.

PubMed

Reemtsma, Thorsten; These, Anja

2005-05-15

Size exclusion chromatography (SEC) coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) was used to analyze the elemental composition and structure of low-molecular-weight fulvic acid molecules. It is shown that the set of hundreds of individual molecules form a homogeneous and structurally unique class of compounds that can be clearly differentiated from any other class of biogenic matter investigated to date. The molecular composition of low-molecular-weight fulvic acids in isolates of very different origin (surface water, groundwater, peat) is virtually indistinguishable. Significant and characteristic differences are, however, recognized when qualitative information and quantitative information provided by ESI-Q-TOF-MS are linked to each other. The relative frequency of the various molecules in each mixture can differ significantly, with the peat showing higher intensity of the aromatic and less carboxylated molecules of this set, whereas the aquatic fulvic acids show a strong contribution of the molecules with less aromaticity and a higher carboxylate content. The identity of fulvic acid molecules in isolates of different origin implies that no specific source material is required forfulvic acid formation but that they may be formed from different sources by different oxidative processes.

Evaluating Activated Carbon Adsorption of Dissolved Organic Matter and Micropollutants Using Fluorescence Spectroscopy.

PubMed

Shimabuku, Kyle K; Kennedy, Anthony M; Mulhern, Riley E; Summers, R Scott

2017-03-07

Dissolved organic matter (DOM) negatively impacts granular activated carbon (GAC) adsorption of micropollutants and is a disinfection byproduct precursor. DOM from surface waters, wastewater effluent, and 1 kDa size fractions were adsorbed by GAC and characterized using fluorescence spectroscopy, UV-absorption, and size exclusion chromatography (SEC). Fluorescing DOM was preferentially adsorbed relative to UV-absorbing DOM. Humic-like fluorescence (peaks A and C) was selectively adsorbed relative to polyphenol-like fluorescence (peaks T and B) potentially due to size exclusion effects. In the surface waters and size fractions, peak C was preferentially removed relative to peak A, whereas the reverse was found in wastewater effluent, indicating that humic-like fluorescence is associated with different compounds depending on DOM source. Based on specific UV-absorption (SUVA), aromatic DOM was preferentially adsorbed. The fluorescence index (FI), if interpreted as an indicator of aromaticity, indicated the opposite but exhibited a strong relationship with average molecular weight, suggesting that FI might be a better indicator of DOM size than aromaticity. The influence of DOM intermolecular interactions on adsorption were minimal based on SEC analysis. Fluorescence parameters captured the impact of DOM size on the fouling of 2-methylisoborneol and warfarin adsorption and correlated with direct competition and pore blockage indicators.

A programmable palm-size gas analyzer for use in micro-autonomous systems

NASA Astrophysics Data System (ADS)

Gordenker, Robert J. M.; Wise, Kensall D.

2012-06-01

Gas analysis systems having small size, low power, and high selectivity are badly needed for defense (detection of explosives and chemical warfare agents), homeland security, health care, and environmental applications. This paper presents a palm-size gas chromatography system having analysis times of 5-50sec, detection limits less than 1ppb, and an average power dissipation less than one watt. It uses no consumables. The three-chip fluidic system consists of a preconcentrator, a 25cm-3m separation column, and a chemi-resistive detector and is supported by a microcomputer and circuitry for programmable temperature control. The entire system, including the mini-pump and battery, occupies less than 200cc and is configured for use on autonomous robotic vehicles.

Three-point Dixon method enables whole-body water and fat imaging of obese subjects.

PubMed

Berglund, Johan; Johansson, Lars; Ahlström, Håkan; Kullberg, Joel

2010-06-01

Dixon imaging techniques derive chemical shift-separated water and fat images, enabling the quantification of fat content and forming an alternative to fat suppression. Whole-body Dixon imaging is of interest in studies of obesity and the metabolic syndrome, and possibly in oncology. A three-point Dixon method is proposed where two solutions are found analytically in each voxel. The true solution is identified by a multiseed three-dimensional region-growing scheme with a dynamic path, allowing confident regions to be solved before unconfident regions, such as background noise. 2 pi-Phase unwrapping is not required. Whole-body datasets (256 x 184 x 252 voxels) were collected from 39 subjects (body mass index 19.8-45.4 kg/m(2)), in a mean scan time of 5 min 15 sec. Water and fat images were reconstructed offline, using the proposed method and two reference methods. The resulting images were subjectively graded on a four-grade scale by two radiologists, blinded to the method used. The proposed method was found superior to the reference methods. It exclusively received the two highest grades, implying that only mild reconstruction failures were found. The computation time for a whole-body dataset was 1 min 51.5 sec +/- 3.0 sec. It was concluded that whole-body water and fat imaging is feasible even for obese subjects, using the proposed method. (c) 2010 Wiley-Liss, Inc.

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1.

PubMed

Morgera, Francesca; Sallah, Margaret R; Dubuke, Michelle L; Gandhi, Pallavi; Brewer, Daniel N; Carr, Chavela M; Munson, Mary

2012-01-01

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

PubMed Central

Ligon, Lauren S.; Rigel, Nathan W.; Romanchuk, Artur; Jones, Corbin D.

2013-01-01

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins. PMID:23913320

Systematic biochemical characterization of the SAM domains in Eph receptor family from Mus Musculus.

PubMed

Wang, Yue; Li, Qingxia; Zheng, Yunhua; Li, Gang; Liu, Wei

2016-05-13

The Eph receptor family is the largest subfamily of receptor tyrosine kinases and well-known for their pivotal roles in axon guidance, synaptogenesis, artery/venous differentiation and tumorigenesis, etc. Activation of the Eph receptor needs multimerization of the receptors. The intracellular C-terminal SAM domain of Eph receptor was reported to mediate self-association of Eph receptors via the homo SAM-SAM interaction. In this study, we systematically expressed and purified the SAM domain proteins of all fourteen Eph receptors of Mus musculus in Escherichia coli. The FPLC (fast protein liquid chromatography) results showed the recombinant SAM domains were highly homogeneous. Using CD (circular dichroism) spectrometry, we found that the secondary structure of all the SAM domains was typically alpha helical folded and remarkably similar. The thermo-stability tests showed that they were quite stable in solution. SEC-MALS (size exclusion chromatography coupled with multiple angle light scattering) results illustrated 200 μM Eph SAM domains behaved as good monomers in the size-exclusion chromatography. More importantly, DLS (dynamic light scattering) results revealed the overwhelming majority of SAM domains was not multimerized in solution either at 200 μM or 2000 μM protein concentration, which indicating the SAM domain alone was not sufficient to mediate the polymerization of Eph receptor. In summary, our studies provided the systematic biochemical characterizations of the Eph receptor SAM domains and implied their roles in Eph receptor mediated signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling.

PubMed

Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

2013-12-06

An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from N(pro) fusion pep6His and N(pro) fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes - one using urea for IB dissolution, the other one using NaOH for IB dissolution - and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of quality assurance methods for epoxy graphite prepreg

NASA Technical Reports Server (NTRS)

Chen, J. S.; Hunter, A. B.

1982-01-01

Quality assurance methods for graphite epoxy/prepregs were developed. Liquid chromatography, differential scanning calorimetry, and gel permeation chromatography were investigated. These methods were applied to a second prepreg system. The resin matrix formulation was correlated with mechanical properties. Dynamic mechanical analysis and fracture toughness methods were investigated. The chromatography and calorimetry techniques were all successfully developed as quality assurance methods for graphite epoxy prepregs. The liquid chromatography method was the most sensitive to changes in resin formulation. The were also successfully applied to the second prepreg system.

PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

PubMed Central

Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

2012-01-01

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

Biosynthesis of Selenocysteine on Its tRNA in Eukaryotes

PubMed Central

Mix, Heiko; Zhang, Yan; Saira, Kazima; Glass, Richard S; Berry, Marla J; Gladyshev, Vadim N; Hatfield, Dolph L

2007-01-01

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA[Ser]Sec as substrates to generate selenocysteyl-tRNA[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins. PMID:17194211

Assessment of Fecal Near-infrared Spectroscopy to Predict Feces Chemical Composition and Apparent Total Tract Digestibility of Nutrients in Pigs.

PubMed

Nirea, K G; Pérez de Nanclares, M; Skugor, A; Afseth, N K; Meuwissen, T H E; Hansen, J Ø; Mydland, L T; Øverland, M

2018-05-08

Apparent total tract digestibility (ATTD) of nutrients could be an alternative measure of feed efficiency when breeding for robust animals that are fed fiber-rich diets. Apparent total tract digestibility of nutrients requires measuring individual feed intake of a large number of animals which is expensive and complex. Alternatively, ATTD of nutrients and feces chemical composition can be predicted using fecal near-infrared spectroscopy (FNIRS). The objective of this study was to assess if the feces chemical composition and ATTD of nutrients can be predicted using FNIRS that originate from various pig experimental datasets. Fecal samples together with detailed information on the feces chemical composition and ATTD of nutrients were obtained from four different pig experiments. Feces near-infrared spectroscopy were analyzed from fecal samples of a complete dataset. The model was calibrated using the FNIRS and reference samples of feces chemical composition and ATTD of nutrients. The robustness and predictability of the model was evaluated by the r2 and the closeness between SE of calibration (SEC) and SE of cross-validation (SECV). Prediction of the feces chemical components and ATTD of nutrients was successful as SEC and SECV were equivalent. Calibration model was developed to estimate the ATTD of nutrients and fecal chemical composition from the FNIRS and worked well for OM (r2 = 0.94; SEC = 48.5; SECV = 56.6), CP ( r2 = 0.89; SEC = 18.1; SECV = 18.8), GE ( r2 = 0.92; SEC = 1.2; SECV = 1.4), NDF (r2 = 0.94 ; SEC = 55; SECV = 60.2), OM digestibility (r2 = 0.94; SEC = 5.5; SECV = 6.7), GE digestibility (r2 = 0.88; SEC = 2.3; SECV = 2.6) and fat digestibility (r2 = 0.79 ; SEC = 6, SECV = 6.8). However, the SE of prediction was slightly higher than what has been reported in another study. The prediction of feces chemical composition for fat (r2 = 0.69; SEC = 11.7, SECV = 12.3), CP digestibility (r2 = 0.63; SEC = 2.3; SECV = 2.7) and NDF digestibility (r2 = 0.64, SEC = 7.7, SECV = 8.8) was moderate. We conclude that the FNIRS accurately predicts the chemical composition of feces and ATTD of nutrients for OM, CP and GE. The approach of FNIRS is a cost-effective method for measuring digestibility and feed efficiency in a large-scale pig breeding programs.

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC×LC-IT-TOF MS.

PubMed

Xu, Yu; Wang, DanDan; Tang, Lan; Wang, Jian

2017-10-25

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC×LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8mm×30cm, 5μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6×150mm, 3.5μm) with ammonium formate solution (10mM) and ammonium formate (8mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MS n data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability. Copyright © 2017. Published by Elsevier B.V.

Interactions between sodium dodecyl sulphate and non-ionic cellulose derivatives studied by size exclusion chromatography with online multi-angle light scattering and refractometric detection.

PubMed

Wittgren, Bengt; Stefansson, Morgan; Porsch, Bedrich

2005-08-05

The novel approach described allows to characterise the surfactant-polymer interaction under several sodium dodecyl sulphate (SDS) concentrations (0-20 mM) using size exclusion chromatography (SEC) with online multi-angle light scattering (MALS) and refractometric (RI) detection. Three different cellulose derivatives, hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC) and hydroxyethyl cellulose (HEC), have been studied in solution containing 10 mM NaCl and various concentrations of sodium dodecyl sulphate. It is shown that this approach is well suited for successful application of both Hummel-Dreyer and multi-component light scattering principles and yields reliable molecular masses of both the polymer complex and the polymer itself within the complex, the amount of surfactant bound into the complex as well as appropriate values of the refractive index increment (dn/dc)micro, of both the complex and the polymer in question. The more hydrophobic derivatives HPC and HPMC adsorbed significantly more SDS than HEC. The inter-chain interactions close to critical aggregation concentration (cac) were clearly seen for HPC and HPMC as an almost two-fold average increase in polymer molecular mass contained in the complex.

Controlling of free radical copolymerization of styrene and maleic anhydride via RAFT process for the preparation of acetaminophen drug conjugates

NASA Astrophysics Data System (ADS)

Sütekin, S. Duygu; Atıcı, Ayşe Bakar; Güven, Olgun; Hoffman, Allan S.

2018-07-01

The presence of maleic anhydride moiety in styrene-maleic anhydride (SMA) copolymer makes it a versatile substrate for conjugation of drugs. In this study biocompatible styrene-maleic anhydride (SMA) copolymer with alternating structure was synthesized by gamma irradiation at room temperature in the presence of 2-phenyl-2-propyl benzodithioate (PPB). The poly(styrene-alt-maleic anhydride) (poly(St-alt-MA)) with narrow molecular weight distribution (Đ: 1.1-1.3) was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The synthesized poly(St-alt-MA) structure was characterized by ATR-FTIR spectroscopy, elemental analysis and 1H NMR spectroscopy and molecular weight and dispersity were determined by size exclusion chromatography (SEC). SMA copolymers were further conjugated with acetaminophen via ester linkage and FT-IR, 1H NMR investigation indicated that the acetaminophen was attached to poly(St-alt-MA). Drug release profile of the polymer-drug conjugate was followed by high performance liquid chromatography (HPLC). The drug-conjugate system was found to follow first order release kinetics with Hixson-Crowell model while drug release mechanism was found as non-Fickian diffusion after testing various kinetic models.

Structure and conformation of α-glucan extracted from Agaricus blazei Murill by high-speed shearing homogenization.

PubMed

Zhang, Anqiang; Deng, Jiaying; Liu, Xiaoqing; He, Pengfei; He, Liang; Zhang, Fuming; Linhardt, Robert J; Sun, Peilong

2018-07-01

Agaricus blazei Murill is an edible and medicinal mushroom favored in many countries, by virtue of both its delicious taste and its potential health benefits such as its purported anticancer activity. A neutral α-glucan (ABM40-1) with a carbohydrate content of 96% was purified from the high-speed shearing homogenization extracts of A. Blazei Murill by ethanol precipitation and column chromatography. Methylation analysis along with nuclear magnetic resonance spectroscopy revealed that ABM40-1 was an α-(1→4)-d-glucopyranan with O-6 position occasionally occupied with α-Glcp-(1→or α-Glcp-(1→6)-β-Glcp-(1→side chains. A weight-average molecular weight of 7.34×10 6 Da was determined for ABM40-1 and its chain in solution was revealed as a compact sphere by size exclusion chromatography (SEC) coupled with a laser light scattering. This spherical conformation was also further confirmed by Congo red test and using atom force microscopy. These results suggest it would be worthwhile to further study the potential bioactivities of ABM40-1. Copyright © 2018 Elsevier B.V. All rights reserved.

Physico-chemical characterization and pharmacological activities of sulfated polysaccharide from sea urchin, Paracentrotus lividus.

PubMed

Salem, Yosra Ben; Amri, Safa; Hammi, Khaoula Mkadmini; Abdelhamid, Amal; Cerf, Didier Le; Bouraoui, Abderrahman; Majdoub, Hatem

2017-04-01

Sulfated polysaccharide (SP) from the eggs of sea urchin Paracentrotus lividus, extracted by papain digestion, was characterized by size exclusion chromatography coupling on-line with light scattering and viscosity detectors (SEC/MALS/VD/DRI), gas chromatography coupled to mass spectrometer (GC-MS), and Fourier transform infrared spectroscopy (FTIR) analysis. The native molecular mass of the extracted polysaccharide is high (≥22 000 KDa) and it is composed mainly of arabinose, accompanied by other monosaccharides (mostly galactose, glucose and fucose), significant amounts of uronic acids (18.4%) and relatively high proportions of sulfate (22.4%). The pharmacological evaluation of SP showed a significant in vivo anti-inflammatory activity (p<0.001), 3h after injection, the edema inhibition was 75.8% at the dose of 100mg/Kg; a significant peripheral analgesic activity (p<0.001), with 64.9% of writhing inhibition, and a significant increase in the hot plate reaction time in mice indicating central analgesic activity. In addition, an interesting gastroprotective effect was observed with this polysaccharide; the gastric ulcer inhibition was 69.7%, at the dose of 100mg/Kg. Copyright © 2017 Elsevier B.V. All rights reserved.

Guidelines to reach high-quality purified recombinant proteins.

PubMed

Oliveira, Carla; Domingues, Lucília

2018-01-01

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

Inflated Sporopollenin Exine Capsules Obtained from Thin-Walled Pollen

NASA Astrophysics Data System (ADS)

Park, Jae Hyeon; Seo, Jeongeun; Jackman, Joshua A.; Cho, Nam-Joon

2016-06-01

Sporopollenin is a physically robust and chemically resilient biopolymer that comprises the outermost layer of pollen walls and is the first line of defense against harsh environmental conditions. The unique physicochemical properties of sporopollenin increasingly motivate the extraction of sporopollenin exine capsules (SECs) from pollen walls as a renewable source of organic microcapsules for encapsulation applications. Despite the wide range of different pollen species with varying sizes and wall thicknesses, faithful extraction of pollen-mimetic SECs has been limited to thick-walled pollen capsules with rigid mechanical properties. There is an unmet need to develop methods for producing SECs from thin-walled pollen capsules which constitute a large fraction of all pollen species and have attractive materials properties such as greater aerosol dispersion. Herein, we report the first successful extraction of inflated SEC microcapsules from a thin-walled pollen species (Zea mays), thereby overcoming traditional challenges with mechanical stability and loss of microstructure. Morphological and compositional characterization of the SECs obtained by the newly developed extraction protocol confirms successful protein removal along with preservation of nanoscale architectural features. Looking forward, there is excellent potential to apply similar strategies across a wide range of unexplored thin-walled pollen species.

Inflated Sporopollenin Exine Capsules Obtained from Thin-Walled Pollen

PubMed Central

Park, Jae Hyeon; Seo, Jeongeun; Jackman, Joshua A.; Cho, Nam-Joon

2016-01-01

Sporopollenin is a physically robust and chemically resilient biopolymer that comprises the outermost layer of pollen walls and is the first line of defense against harsh environmental conditions. The unique physicochemical properties of sporopollenin increasingly motivate the extraction of sporopollenin exine capsules (SECs) from pollen walls as a renewable source of organic microcapsules for encapsulation applications. Despite the wide range of different pollen species with varying sizes and wall thicknesses, faithful extraction of pollen-mimetic SECs has been limited to thick-walled pollen capsules with rigid mechanical properties. There is an unmet need to develop methods for producing SECs from thin-walled pollen capsules which constitute a large fraction of all pollen species and have attractive materials properties such as greater aerosol dispersion. Herein, we report the first successful extraction of inflated SEC microcapsules from a thin-walled pollen species (Zea mays), thereby overcoming traditional challenges with mechanical stability and loss of microstructure. Morphological and compositional characterization of the SECs obtained by the newly developed extraction protocol confirms successful protein removal along with preservation of nanoscale architectural features. Looking forward, there is excellent potential to apply similar strategies across a wide range of unexplored thin-walled pollen species. PMID:27302853

Determining the best method of Nellcor pulse oximeter sensor application in neonates.

PubMed

Saraswat, A; Simionato, L; Dawson, J A; Thio, M; Kamlin, C O F; Owen, L; Schmölzer, G; Davis, P G

2012-05-01

To identify the optimal sensor application method that gave the quickest display of accurate heart rate (HR) data using the Nellcor OxiMax N-600x pulse oximeter (PO). Stable infants who were monitored with an electrocardiograph were included. Three sensor application techniques were studied: (i) sensor connected to cable, then applied to infant; (ii) sensor connected to cable, applied to investigator's finger, and then to infant; (iii) sensor applied to infant, then connected to cable. The order of techniques tested was randomized for each infant. Time taken to apply the PO sensor, to display data and to display accurate data (HR(PO) = HR(ECG) ± 3 bpm) were recorded using a stopwatch. Forty infants were studied [mean (SD) birthweight, 1455 (872) g; gestational age, 31 (4) weeks; post-menstrual age, 34 (4) weeks]. Method 3 acquired any data significantly faster than methods 1 (p = 0.013; CI, -9.6 to -3.0 sec) and 2 (p = 0.004; CI, -5.9 to -1.2 sec). Method 3 acquired accurate data significantly faster than method 1 (p = 0.016; CI, -9.4 to -1.0 sec), but not method 2 (p = 0.28). Applying the sensor to the infant before connecting it to the cable yields the fastest acquisition of accurate HR data from the Nellcor PO. © 2011 The Author(s)/Acta Paediatrica © 2011 Foundation Acta Paediatrica.

Mathematical optimization techniques for managing selective catalytic reduction for a fleet of coal-fired power plants

NASA Astrophysics Data System (ADS)

Alanis Pena, Antonio Alejandro

Major commercial electricity generation is done by burning fossil fuels out of which coal-fired power plants produce a substantial quantity of electricity worldwide. The United States has large reserves of coal, and it is cheaply available, making it a good choice for the generation of electricity on a large scale. However, one major problem associated with using coal for combustion is that it produces a group of pollutants known as nitrogen oxides (NO x). NOx are strong oxidizers and contribute to ozone formation and respiratory illness. The Environmental Protection Agency (EPA) regulates the quantity of NOx emitted to the atmosphere in the United States. One technique coal-fired power plants use to reduce NOx emissions is Selective Catalytic Reduction (SCR). SCR uses layers of catalyst that need to be added or changed to maintain the required performance. Power plants do add or change catalyst layers during temporary shutdowns, but it is expensive. However, many companies do not have only one power plant, but instead they can have a fleet of coal-fired power plants. A fleet of power plants can use EPA cap and trade programs to have an outlet NOx emission below the allowances for the fleet. For that reason, the main aim of this research is to develop an SCR management mathematical optimization methods that, with a given set of scheduled outages for a fleet of power plants, minimizes the total cost of the entire fleet of power plants and also maintain outlet NO x below the desired target for the entire fleet. We use a multi commodity network flow problem (MCFP) that creates edges that represent all the SCR catalyst layers for each plant. This MCFP is relaxed because it does not consider average daily NOx constraint, and it is solved by a binary integer program. After that, we add the average daily NOx constraint to the model with a schedule elimination constraint (MCFPwSEC). The MCFPwSEC eliminates, one by one, the solutions that do not satisfy the average daily NOx constraint and the worst NH 3 slip until it finds the solution that satisfies that requirement. We introduce an algorithm called heuristic MCFPwSEC (HMCFPwSEC). When HMCFPwSEC algorithm starts, we calculate the cost of the edges estimating the average NH3 slip level, but after we have a schedule that satisfies the average daily NOx constraint and the worst NH3 slip, we update the cost of the edges with the average NH3 slip for this schedule. We repeat this process until we have the solution. Because HMCFPwSEC does not guarantee optimality, we compare its results with SGO, which is optimal, using computational experiments. The results between both models are very similar, the only important difference is the time to solve each model. Then, a fleet HMCFPwSEC (FHMCFPwSEC) uses HMCFPwSEC to create the SCR management plan for each plant of the fleet, with a discrete NOx emissions value for each plant. FHMCFPwSEC repeats this process with different discrete levels of NOx emissions, for each plant, in order to create a new problem with schedules with different cost and NO x emissions for each plant of the fleet. Finally, FHMCFPwSEC solves this new problem with a binary integer program, in order to satisfy a NO x emission value for the fleet that also minimizes the total cost for the fleet, and using each plant once. FHMCFPwSEC can work with single cut and also with multi-cut methods. Because FHMCFPwSEC does not guarantee optimality, we compare its results with fleet SGO (FSGO) using computational experiments. The results between both models are very similar, the only important difference is the time to solve each model. In the experiments, FHMCFPwSEC multi-cut targeting new layer always uses less time than FSGO.

[Markerless DNA deletion based on Red recombination and in vivo I-Sec I endonuclease cleavage in Escherichia coli chromosome].

PubMed

Zhu, Meiqin; Yu, Jian; Zhou, Changlin; Fang, Hongqing

2016-01-01

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.

Functional Description for the Department of the Army Movements Management System. Redesign Phase 1. (DAMMS-R1). Volume 3.

DTIC Science & Technology

1987-12-31

Maintan -.ountry-TbI e CARDINALiii 134; HL~.,N-L.". 1); bL t i 11L h I...MEMO: FCityCd-Memo SOURCE IS: ’TMAS DAlA MODEL’ ATTRIBUTE IS: MEDIA ’DISK’ SEC-CLASS ’UNCLASSIFIED’ volatility ’DYNAMIC’I UPDATE- METHOD ’INTERACTIVE...8217 ATTRIBUTE IS: MEDIA ’DISK’ SEC-CLASS ’UNCLASSIFIED’ volatility ’STATTO’ UPDATE- METHOD ’INTERACTIVE’ UPDATE-FREQUENCY ’AS PER CHANGE’ Tt.LLE-FORM ’COMMAND’

Unsteady aerodynamic characterization of a military aircraft in vertical gusts

NASA Technical Reports Server (NTRS)

Lebozec, A.; Cocquerez, J. L.

1985-01-01

The effects of 2.5-m/sec vertical gusts on the flight characteristics of a 1:8.6 scale model of a Mirage 2000 aircraft in free flight at 35 m/sec over a distance of 30 m are investigated. The wind-tunnel setup and instrumentation are described; the impulse-response and local-coefficient-identification analysis methods applied are discussed in detail; and the modification and calibration of the gust-detection probes are reviewed. The results are presented in graphs, and good general agreement is obtained between model calculations using the two analysis methods and the experimental measurements.

Precise measurement of instantaneous volume of eccrine sweat gland in mental sweating by optical coherence tomography

NASA Astrophysics Data System (ADS)

Sugawa, Yoshihiko; Fukuda, Akihiro; Ohmi, Masato

2015-03-01

We have demonstrated dynamic analysis of the physiological function of eccrine sweat glands underneath skin surface by optical coherence tomography (OCT). We propose a method for extraction of the target eccrine sweat gland by use of the connected component extraction process and the adaptive threshold method, where the en-face OCT images are constructed by the SS-OCT. Furthermore, we demonstrate precise measurement of instantaneous volume of the sweat gland in response to the external stimulus. The dynamic change of instantaneous volume of eccrine sweat gland in mental sweating is performed by this method during the period of 300 sec with the frame intervals of 3.23 sec.

Accelerated solvent extraction (ASE) for purification and extraction of silicone passive samplers used for the monitoring of organic pollutants.

PubMed

Brockmeyer, Berit; Kraus, Uta R; Theobald, Norbert

2015-12-01

Silicone passive samplers have gained an increasing attention as single-phased, practical and robust samplers for monitoring of organic contaminants in the aquatic environment in recent years. However, analytical challenges arise in routine application during the extraction of analytes as silicone oligomers are co-extracted and interfere severely during chemical analyses (e.g. gas chromatographic techniques). In this study, we present a fast, practical pre-cleaning method for silicone passive samplers applying accelerated solvent extraction (ASE) for the removal of silicone oligomers prior to the water deployment (hexane/dichloromethane, 100 °C, 70 min). ASE was also shown to be a very fast (10 min) and efficient extraction method for non-polar contaminants (non-exposed PRC recoveries 66-101 %) sampled by the silicone membrane. For both applications, temperature, extraction time and the solvent used for ASE have been optimized. Purification of the ASE extract was carried out by silica gel and high-pressure liquid size exclusion chromatography (HPLC-SEC). The silicone oligomer content was checked by total reflection X-ray fluorescence spectroscopy (TXRF) in order to confirm the absence of the silicone oligomers prior to analysis of passive sampler extracts. The established method was applied on real silicone samplers from the North- and Baltic Sea and showed no matrix effects during analysis of organic pollutants. Internal laboratory standard recoveries were in the same range for laboratory, transport and exposed samplers (85-126 %).

A statistical study of atypical wave modes in the Earth's foreshock region

NASA Astrophysics Data System (ADS)

Hsieh, W.; Shue, J.; Lee, B.

2010-12-01

The Earth's foreshock, the region upstream the Earth’s bow shock, is filled with back-streaming particles and ultra-low frequency waves. Three different wave modes have been identified in the region, including 30-sec waves, 3-sec waves, and shocklets. Time History of Events and Macroscale Interactions during Substorms (THEMIS), a satellite mission that consists of five probes, provides multiple measuements of the Earth’s foreshock region. The method of Hilbert-Huang transform (HHT) includes the procedures of empirical mode decomposition and instantaneous frequency calculation. In this study, we use HHT to decompose intrinsic wave modes and perform a wave analysis of chaotic magnetic fields in the Earth's foreshock region. We find that some individual atypical wave modes other than 30-sec and 3-sec appear in the region. In this presentation, we will show the statistical characteristics, such as wave frequency, wave amplitude, and wave polarization of the atypical intrinsic wave modes, with respect to different locations in the foreshock region and to different solar wind conditions.

MINS2: revisiting the molecular code for transmembrane-helix recognition by the Sec61 translocon.

PubMed

Park, Yungki; Helms, Volkhard

2008-08-15

To be fully functional, membrane proteins should not only fold, but also get inserted into the membrane, which is mediated by the Sec61 translocon. Recent experimental studies have attempted to elucidate how the Sec61 translocon accomplishes this delicate task by measuring the translocon-mediated membrane insertion free energies of 357 systematically designed peptides. On the basis of this data set, we have developed MINS2, a novel sequence-based computational method for predicting the membrane insertion free energies of protein sequences. A benchmark analysis of MINS2 shows that MINS2 signi.cantly outperforms previously proposed methods. Importantly, the application of MINS2 to known membrane protein structures shows that a better prediction of membrane insertion free energies does not lead to a better prediction of transmembrane segments of polytopic membrane proteins. A web server for MINS2 is publicly available at http://service.bioinformatik.uni-saarland.de/mins. Supplementary data are available at Bioinformatics online.

Charge Exchange of Ne^9+ for X-ray Emission

NASA Astrophysics Data System (ADS)

Lyons, David

2016-01-01

Using the molecular-orbital close-coupling (MOCC) method, single electron capture (SEC) cross sections were computed for Ne^9+ colliding with H.Potential energies and nonadiabatic couplings were calculated and used to obtain the MOCC cross sections which are final-quantum-state-resolved including a separation of singlet and triplet states. Atomic-orbital close-coupling, classical trajectory Monte Carlo, and multichannel Landau-Zener (MCLZ) calculations are also performed. Cross sections for more complicated targets including He, H2, N2, H2O, CO, and CO2, were obtained with the MCLZ method. The SEC results are compared with experimental and other theoretical data, where available. The SEC cross sections are being used in cascade models to predict X-ray emission spectra relevant to solar systemand astrophysical environments.D. Lyons, R. S. Cumbee, P. D. Mullen, P. C. Stancil (UGA), D. R. Schultz (UNT), P. Liebermann (Wuppertal Univ.),R. Buenker (NCSU).This work was partially supported by NASA grant NNX09AC46G.

H.R. 3627: This Act may be cited as the Brownfield Community Empowerment Act, introduced in the House of Representatives, One Hundred Fifth Congress, Second Session, April 1, 1998

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

NONE

1998-12-31

The purpose of this bill is to assist local governments and local citizens` organizations in the assessment and remediation of brownfield sites, and for other purposes. Attention is focused on the following: Sec. 101. Inventory, assessment, and training and grant program; Sec. 112. Grants for revolving loan programs; Sec. 113. Economic redevelopment grants; Sec. 114. Reports; Sec 115. Limitations on use of funds; Sec. 116 Siting of TSD facilities; Sec. 117. Effect on other laws; Sec. 118. Regulations; Sec. 119. Authorizations of appropriations; Sec 120. Research, development, and demonstration; Sec. 121. Assistance for workforce training; Sec. 122. Worker training andmore » education grants; and Sec. 201. Economic development grants in connection with community development loan guarantees.« less

Modeling the light-travel-time effect on the far-infrared size of IRC +10216

NASA Technical Reports Server (NTRS)

Wright, Edward L.; Baganoff, Frederick K.

1995-01-01

Models of the far-infrared emission from the large circumstellar dust envelope surrounding the carbon star IRC +10216 are used to assess the importance of the light-travel-time effect (LTTE) on the observed size of the source. The central star is a long-period variable with an average period of 644 +/- 17 days and a peak-to-peak amplitude of two magnituds, so a large light-travel-time effect is seen at 1 min radius. An attempt is made to use the LTTE to reconcile the discrepancy between the observations of Fazio et al. and Lester et al. regarding the far-infrared source size. This discrepancy is reviewed in light of recent, high-spatial-resolution observations at 11 microns by Danchi et al. We conclude that IRC +10216 has been resolved on the arcminute scale by Fazio et al. Convolution of the model intensity profile at 61 microns with the 60 sec x 90 sec Gaussian beam of Fazio et al. yields an observed source size full width at half maximum (FWHM) that ranges from approximately 67 sec to 75 sec depending on the phase of the star and the assumed distance to the source. Using a simple r(exp -2) dust distribution and the 106 deg phase of the Fazio et al. observations, the LTTE model reaches a peak size of 74.3 sec at a distance of 300 pc. This agrees favorably with the 78 sec x 6 sec size measured by Fazio et al. Finally, a method is outlined for using the LTTE as a distance indicator to IRC +10216 and other stars with extended mass outflows.

Inhibition of endogenous human dentin MMPs by Gluma

PubMed Central

Sabatini, Camila; Scheffel, Débora L.S.; Scheffel, Régis H.; Agee, Kelli A.; Rouch, Katelyn; Takahashi, Masahiro; Breschi, Lorenzo; Mazzoni, Annalisa; Tjäderhane, Leo; Tay, Franklin R.; Pashley, David H.

2014-01-01

Objective The objective of this study was to determine if Gluma dentin desensitizer (5.0% glutaraldehyde and 35% HEMA in water) can inhibit the endogenous MMPs of dentin matrices in 60 sec. and to evaluate its effect on dentin matrix stiffness and dry mass weight. Methods Dentin beams of 2×1×6 mm were obtained from extracted human third molars coronal dentin. To measure the influence of Gluma treatment time on total MMP activity of dentin, beams were dipped in 37% phosphoric acid (PA) for 15 sec. and rinsed in water. The acid-etched beams were then dipped in Gluma for 5, 15, 30 or 60 sec., rinsed in water and incubated into SensoLyte generic MMP substrate (AnaSpec, Inc.) for 60 min. Controls were dipped in water for 60 sec. Additional beams of 1×1×6 mm were completely demineralized in 37% PA for 18 h, rinsed and used to evaluate changes on the dry weight and modulus of elasticity (E) after 60 sec. of Gluma treatment followed by incubation in simulated body fluid buffer for zero, one or four weeks. E was measured by 3-pt flexure. Results Gluma treatment inhibited total MMP activity of acid-etched dentin by 44, 50, 84, 86 % after 5, 15, 30 or 60 sec. of exposure, respectively. All completely demineralized dentin beams lost stiffness after one and four weeks, with no significant differences between the control and Gluma-treated dentin. Gluma treatment for 60 sec. yielded significantly less dry mass loss than the control after four weeks. Significance The use of Gluma may contribute to the preservation of adhesive interfaces by its cross-linking and inhibitory properties of endogenous dentin MMPs. PMID:24846803

Counter-current motion in counter-current chromatography.

PubMed

Ito, Yoichiro

2014-12-12

After the CCC2012 meeting, I have received an e-mail regarding the terminology of "Countercurrent Chromatography". It stated that the term "Countercurrent" is a misnomer, because its stationary phase is motionless in the column and that the method should be renamed as liquid-liquid separations or centrifugal separations. However, it was found that these names are already used for various other techniques as found via Google search. The term "Countercurrent Chromatography" was originally made after two preparative methods of Countercurrent distribution and liquid Chromatography, both having no countercurrent motion in the column. However, it is surprising to find that this F1 hybrid method "Countercurrent Chromatography" can clearly exhibit countercurrent motion within the separation column in both hydrodynamic and hydrostatic equilibrium systems. This justifies that "Countercurrent Chromatography" is a proper term for this chromatographic method. Published by Elsevier B.V.

77 FR 14417 - Notice of Temporary Closure on Public Lands in Gooding and Elmore Counties, ID

Federal Register 2010, 2011, 2012, 2013, 2014

2012-03-09

... affected public lands is: Boise Meridian, Idaho T. 3 S., R. 10 E., Sec. 35. T. 4 S., R. 10 E., Secs. 1 and 2; Secs. 12 to 15, inclusive; Sec. 17; Secs. 20 to 28, inclusive; Secs. 33 to 35, inclusive. T. 5 S., R. 10 E., Secs. 3 and 4. T. 3 S., R. 11 E., Secs. 31 and 33. T. 4 S., R. 11 E., Secs. 3 to 15...

Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

PubMed Central

Feltcher, Meghan E.; Gibbons, Henry S.; Ligon, Lauren S.

2013-01-01

At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm. PMID:23204463

Direct identification of the site of binding on the chaperone SecB for the amino terminus of the translocon motor SecA.

PubMed

Randall, Linda L; Henzl, Michael T

2010-06-01

Protein export mediated by the general secretory Sec system in Escherichia coli proceeds by a dynamic transfer of a precursor polypeptide from the chaperone SecB to the SecA ATPase motor of the translocon and subsequently into and through the channel of the membrane-embedded SecYEG heterotrimer. The complex between SecA and SecB is stabilized by several separate sites of contact. Here we have demonstrated directly an interaction between the N-terminal residues 2 through 11 of SecA and the C-terminal 13 residues of SecB by isothermal titration calorimetry and analytical sedimentation velocity centrifugation. We discuss the unusual binding properties of SecA and SecB in context of a model for transfer of the precursor along the pathway of export.

Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development.

PubMed

Townley, Anna K; Feng, Yi; Schmidt, Katy; Carter, Deborah A; Porter, Robert; Verkade, Paul; Stephens, David J

2008-09-15

The COPII coat assembles on endoplasmic reticulum membranes to coordinate the collection of secretory cargo with the formation of transport vesicles. During COPII assembly, Sar1 deforms the membrane and recruits the Sec23-Sec24 complex (Sec23/24), which is the primary cargo-binding adaptor for the system, and Sec13-Sec31 (Sec13/31), which provides a structural outer layer for vesicle formation. Here we show that Sec13 depletion results in concomitant loss of Sec31 and juxtanuclear clustering of pre-budding complexes containing Sec23/24 and cargo. Electron microscopy reveals the presence of curved coated profiles on distended endoplasmic reticulum, indicating that Sec13/31 is not required for the generation or maintenance of the curvature. Surprisingly, export of tsO45-G-YFP, a marker of secretory cargo, is unaffected by Sec13/31 depletion; by contrast, secretion of collagen from primary fibroblasts is strongly inhibited. Suppression of Sec13 expression in zebrafish causes defects in proteoglycan deposition and skeletal abnormalities that are grossly similar to the craniofacial abnormalities of crusher mutant zebrafish and patients with cranio-lenticulo-sutural dysplasia. We conclude that efficient coupling of the inner (Sec23/24) and outer (Sec13/31) layers of the COPII coat is required to drive the export of collagen from the endoplasmic reticulum, and that highly efficient COPII assembly is essential for normal craniofacial development during embryogenesis.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice

PubMed Central

Khoriaty, Rami; Everett, Lesley; Chase, Jennifer; Zhu, Guojing; Hoenerhoff, Mark; McKnight, Brooke; Vasievich, Matthew P.; Zhang, Bin; Tomberg, Kärt; Williams, John; Maillard, Ivan; Ginsburg, David

2016-01-01

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes. PMID:27297878

78 FR 58554 - Notice of Application for Withdrawal Extension, and Notification of a Public Meeting, Desert...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-24

...., partially surveyed, Sec. 7, NW\\1/4\\ and S\\1/2\\. T. 16 S., R. 58 E., unsurveyed, Secs. 11 thru 14, inclusive; Secs. 23 thru 26, inclusive; Secs. 35 and 36. T. 15 S., R. 59 E., unsurveyed, Secs. 2 thru 11, inclusive; Secs. 14 thru 23, inclusive; Secs. 26 thru 35, inclusive. T. 16 S., R. 59 E., unsurveyed, Secs. 2...

Technical basis for inner container leak detection sensitivity goals in 3013 DE surveillance

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

Berg, John M.

Helium leak checking of 3013 inner container lids is under consideration for addition to DE Surveillance tasks as an improved means to detect any through-wall flaws that may have formed during storage. This white paper evaluates whether leak checking at DE could replace and improve upon the current method of comparing gas compositions and pressures within the inner and outer containers. We have used viscous and molecular flow equations in ANSI N14.5 to calculate what the measured standard helium leak rate would be for hypothetical leaks of three different sizes. For comparison, we have also calculated the effects on gasmore » composition and pressure differences as a function of pre-DE storage time for the same three leak sizes, using molecular and viscous flow equations as well as diffusion equations to predict the relevant gas transport. For a hypothetical leak that would be measured at 1x10 -7 std cc/sec, likely an achievable sensitivity using helium leak checking at DE, the calculations predict no measurable effect on pressure difference or gas composition as measured by DE gas analysis. We also calculate that it would take over 200 years for water vapor to diffuse through a 10 -7 std cc/sec leak enough to raise the RH outer container to half the RH value in the inner container. A leak 100 times larger, which would be measured at 1x10 -5 std cc/sec, the same water vapor diffusion would take at least 14 years. Our conclusion is that helium leak checking will be useful even at a sensitivity of 1x10 -5 std cc/sec, and a significant improvement over current DE methods at a sensitivity of 1x10 -7 std cc/sec.« less

Computation of Anisotropic Bi-Material Interfacial Fracture Parameters and Delamination Creteria

NASA Technical Reports Server (NTRS)

Chow, W-T.; Wang, L.; Atluri, S. N.

1998-01-01

This report documents the recent developments in methodologies for the evaluation of the integrity and durability of composite structures, including i) the establishment of a stress-intensity-factor based fracture criterion for bimaterial interfacial cracks in anisotropic materials (see Sec. 2); ii) the development of a virtual crack closure integral method for the evaluation of the mixed-mode stress intensity factors for a bimaterial interfacial crack (see Sec. 3). Analytical and numerical results show that the proposed fracture criterion is a better fracture criterion than the total energy release rate criterion in the characterization of the bimaterial interfacial cracks. The proposed virtual crack closure integral method is an efficient and accurate numerical method for the evaluation of mixed-mode stress intensity factors.

Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase.

PubMed

Kontinen, V P; Yamanaka, M; Nishiyama, K; Tokuda, H

1996-06-01

SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.

Spectroscopic planetary detection

NASA Technical Reports Server (NTRS)

Deming, Drake

1988-01-01

One of the most promising methods for the detection of extra-solar planets is the spectroscopic method, where a small Doppler shift (approximately 10 meters/sec) in the spectrum of the parent star reveals the presence of planetary companions. However, solar-type stars may show spurious Doppler shifts due to surface activity. If these effects are periodic, as is the solar activity cycle, then they may masquerade as planetary companions. The goal of this investigation is to determine whether the solar cycle affects the Doppler stability of integrated sunlight. Observations of integrated sunlight are made in the near infrared (approximately 2 micrometer), using the Kitt Peak McMath Fourier transform spectrometer, with an N2O gas absorption cell for calibration. Researchers currently achieve an accuracy of approximately 5 meters/sec. Solar rotation velocities vary by plus or minus 2000 meters/sec across the solar disk, and imperfect optical integration of these velocities is the principal source of error. We have been monitoring the apparent velocity of integrated sunlight since 1983. They initially saw a decrease of approximately 30 meters/sec in the integrated light velocity from 1983 through 1985, but in 1987 to 1988 the integrated light velocity returned to its 1983 level. It is too early to say whether these changes are solar-cycle related. Although the FTS, unlike a slit spectrograph, has a large field of view, researchers are always looking for ways to improve the optical integration of the solar disk. They recently made an improvement in the method used to optically collimate the FTS, and this has reduced the error level, eliminating some systematic effects seen earlier.

Synthesis of multiwalled carbon nanotube from different grades of carbon black using arc discharge method

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

Arora, Neha, E-mail: n4neha31@gmail.com; Sharma, N. N.; Director, School of Automobile, Mechanical & Mechatronics, Manipal University,Jaipur,India

2016-04-13

This paper describes the synthesis of nanotube from different grades (Tread * A(non-ASTM), N134,N121,N660 and N330)of carbon black using DC arc discharge method at 40A current for 60sec. Carbon black samples of different grades were procured from industry (Aditya Birla Science and Technology Limited, India). Scanning Electron Micrographs (SEM) of the deposited carbon nanostructures suggests that MWCNTs are formed at 40A and for a minimal exposure time of 60sec.The result formed indicates the N330 grade of carbon black gets converted to MWCNTs (Multiwall Carbon nanotube) as compared to other grades.

Max CAPR: high-resolution 3D contrast-enhanced MR angiography with acquisition times under 5 seconds.

PubMed

Haider, Clifton R; Borisch, Eric A; Glockner, James F; Mostardi, Petrice M; Rossman, Phillip J; Young, Phillip M; Riederer, Stephen J

2010-10-01

High temporal and spatial resolution is desired in imaging of vascular abnormalities having short arterial-to-venous transit times. Methods that exploit temporal correlation to reduce the observed frame time demonstrate temporal blurring, obfuscating bolus dynamics. Previously, a Cartesian acquisition with projection reconstruction-like (CAPR) sampling method has been demonstrated for three-dimensional contrast-enhanced angiographic imaging of the lower legs using two-dimensional sensitivity-encoding acceleration and partial Fourier acceleration, providing 1mm isotropic resolution of the calves, with 4.9-sec frame time and 17.6-sec temporal footprint. In this work, the CAPR acquisition is further undersampled to provide a net acceleration approaching 40 by eliminating all view sharing. The tradeoff of frame time and temporal footprint in view sharing is presented and characterized in phantom experiments. It is shown that the resultant 4.9-sec acquisition time, three-dimensional images sets have sufficient spatial and temporal resolution to clearly portray arterial and venous phases of contrast passage. It is further hypothesized that these short temporal footprint sequences provide diagnostic quality images. This is tested and shown in a series of nine contrast-enhanced MR angiography patient studies performed with the new method.

Selenium Levels in Serum, Red Blood Cells, and Cerebrospinal Fluid of Alzheimer's Disease Patients: A Report from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL).

PubMed

Cardoso, Bárbara R; Hare, Dominic J; Bush, Ashley I; Li, Qiao-Xin; Fowler, Christopher J; Masters, Colin L; Martins, Ralph N; Ganio, Katherine; Lothian, Amber; Mukherjee, Soumya; Kapp, Eugene A; Roberts, Blaine R

2017-01-01

Selenium (Se) protects cells against oxidative stress damage through a range of bioactive selenoproteins. Increased oxidative stress is a prominent feature of Alzheimer's disease (AD), and previous studies have shown that Se deficiency is associated with age-related cognitive decline. In this study, we assessed Se status in different biofluids from a subgroup of participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing. As Se in humans can either be an active component of selenoproteins or inactive via non-specific incorporation into other proteins, we used both size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and tandem mass spectrometry to characterize selenoproteins in serum. We observed no differences in total Se concentration in serum or cerebrospinal fluid of AD subjects compared to mildly cognitively impairment patients and healthy controls. However, Se levels in erythrocytes were decreased in AD compared to controls. SEC-ICP-MS analysis revealed a dominant Se-containing fraction. This fraction was subjected to standard protein purification and a bottom-up proteomics approach to confirm that the abundant Se in the fraction was due, in part, to selenoprotein P. The lack of change in the Se level is at odds with our previous observations in a Brazilian population deficient in Se, and we attribute this to the Australian cohort being Se-replete.

Characterization of Bioderived Polyhydroxyalkanoates by Size Exclusion Chromatography

NASA Astrophysics Data System (ADS)

Negulescu, Ioan; Cueto, Rafael; Rusch, Kelly; Gutierrez-Wing, Teresa; Stevens, Benjamin

2008-03-01

The plant derived polyesters, better known as polyhydroxyalkanoates, PHAs, are renewable and sustainable: [-O-CH(CH3)-(CH2)x-CO-]n. If x = 0 PHA is Poly(lactic acid), PLA; if x = 1 or 2 it is Poly(hydroxy butyrate), PHB, or Poly(hydroxy valerate), PHV. SEC and light scattering have been used before for determination of the absolute molecular mass of PLA dissolved in CHCl3 (Malmgren et al., J. Thermal Anal. Calorim., 2006, 83, 35-40). To our best knowledge there is no publication on the determination of the absolute MW of other PHAs. The bioderived polymers analyzed in this work were four catalog PHA samples: PHB Fluka 81329, PHB Natural Aldrich 363502, 95PHB/5PHV Aldrich 403105, and 92PHB/8PHV Aldrich 403113. SEC/LS instrumentation used: three Phenogel (1K-10000K) columns + a guard column, an Agilent pump and Wyatt Heleos MALS, QUELS (DLS), ViscoStar and rEX DRI detectors, all in series. The experimental dn/dc of PHB in CHCl3 (0.0336 ml/g at 658nm) allowed the determination of absolute MW of all PHA samples: PHB Fluka Mw 345,100 Mn 218,400; PHB Aldrich Mw 335,700 Mn 185,000; 92PHB/8PHV Mw 144,700 Mn 91,970; 95PHB/5PHV Mw 253,000 Mn 193,800.

Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs.

PubMed

Kawsar, S M A; Matsumoto, R; Fujii, Y; Yasumitsu, H; Dogasaki, C; Hosono, M; Nitta, K; Hamako, J; Matsui, T; Kojima, N; Ozeki, Y

2009-07-01

A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80 degrees C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, beta-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-alpha- and methyl-beta-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k(ass)) and dissociation rate constant (k(diss)) were determined for the lectin to be 4.3 x 10(5) M(-1) x sec(-1) and 2.2 x 10(-3) sec(-1), respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

Development of a gas chromatography method for the determination of isotretinoin and its degradation products in pharmaceuticals.

PubMed

Lima, Eliana Martins; Diniz, Danielle G Almeida; Antoniosi-Filho, Nelson R

2005-07-15

This paper describes the development of a gas chromatography (GC) method used for the assay of isotretinoin in its isolated form and in pharmaceutical formulations. Isotretinoin soft and hard gelatin capsules were prepared with various excipients. The performance of the proposed gas chromatography method was compared to that of traditional high performance liquid chromatography (HPLC) systems for this substance, and the GC parameters were established based on several preliminary tests, including thermal analysis of isotretinoin. Results showed that gas chromatography-flame ionization detector (GC-FID) exhibited a separation efficiency superior to that of HPLC, particularly for separating isotretinoin degradation products. This method was proven to be effectively applicable to stability evaluation assays of isotretinoin and isotretinoin based pharmaceuticals.

77 FR 36611 - Core Principles and Other Requirements for Designated Contract Markets

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-19

.... 38.154--Regulatory Services Provided by a Third Party vi. Sec. 38.155--Compliance Staff and Resources... vi. Sec. 38.256--Trade Reconstruction vii. Sec. 38.257--Regulatory Service Provider viii. Sec. 38.258... Integrity iii. Sec. 38.603--Protection of Customer Funds iv. Sec. 38.604--Financial Surveillance v. Sec. 38...

Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; determination of chlorinated pesticides in aquatic tissue by capillary-column gas chromatography with electron-capture detection

USGS Publications Warehouse

Leiker, Thomas J.; Madsen, J.E.; Deacon, J.R.; Foreman, W.T.

1995-01-01

A method for the determination of chlorinated organic compounds in aquatic tissue by dual capillary-column gas chromatography with electron-capture detection is described. Whole-body-fish or corbicula tissue is homogenized, Soxhlet extracted, lipid removed by gel permeation chromatography, and fractionated using alumina/silica adsorption chromatography. The extracts are analyzed by dissimilar capillary-column gas chromatography with electron-capture detection. The method reporting limits are 5 micrograms per kilogram (μg/kg) for chlorinated compounds, 50 μg/kg for polychlorinated biphenyls, and 200 μg/kg for toxaphene.

Analysis of lignans in Magnoliae Flos by turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry.

PubMed

Zhou, Xuan; Chen, Cen; Ye, Xiaolan; Song, Fenyun; Fan, Guorong; Wu, Fuhai

2016-04-01

In this study, a method coupling turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry was developed for analyzing the lignans in Magnoliae Flos. By the online pretreatment of turbulent flow chromatography solid-phase extraction, the impurities removal and analytes concentration were automatically processed, and the lignans were separated rapidly and well. Seven lignans of Magnoliae Flos including epieudesmin, magnolin, 1-irioresinol-B-dimethyl ether, epi-magnolin, fargesin aschantin, and demethoxyaschantin were identified by comparing their retention behavior, UV spectra, and mass spectra with those of reference substances or literature data. The developed method was validated, and the good results showed that the method was not only automatic and rapid, but also accurate and reliable. The turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry method holds a high potential to become an effective method for the quality control of lignans in Magnoliae Flos and a useful tool for the analysis of other complex mixtures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Agonal sequences in four filmed hangings: analysis of respiratory and movement responses to asphyxia by hanging.

PubMed

Sauvageau, Anny

2009-01-01

The human pathophysiology of asphyxia by hanging is still poorly understood, despite great advances in forensic science. In that context, filmed hangings may hold the key to answer questions regarding the sequence of events leading to death in human asphyxia. Four filmed hangings were analyzed. Rapid loss of consciousness was observed between 13 sec and 18 sec after onset of hanging, closely followed by convulsions (at 14-19 sec). A complex pattern of decerebration rigidity (19-21 sec in most cases), followed by a quick phase of decortication rigidity (1 min 00 sec-1 min 08 sec in most cases), an extended phase of decortication rigidity (1 min 04 sec-1 min 32 sec) and loss of muscle tone (1 min 38 sec-2 min 47 sec) was revealed. Very deep respiratory attempts started between 20 and 22 sec, the last respiratory attempt being detected between 2 min 00 sec and 2 min 04 sec. Despite differences in the types of hanging, this unique study reveals similarities that are further discussed.

A novel multidimensional protein identification technology approach combining protein size exclusion prefractionation, peptide zwitterion-ion hydrophilic interaction chromatography, and nano-ultraperformance RP chromatography/nESI-MS2 for the in-depth analysis of the serum proteome and phosphoproteome: application to clinical sera derived from humans with benign prostate hyperplasia.

PubMed

Garbis, Spiros D; Roumeliotis, Theodoros I; Tyritzis, Stavros I; Zorpas, Kostas M; Pavlakis, Kitty; Constantinides, Constantinos A

2011-02-01

The current proof-of-principle study was aimed toward development of a novel multidimensional protein identification technology (MudPIT) approach for the in-depth proteome analysis of human serum derived from patients with benign prostate hyperplasia (BPH) using rational chromatographic design principles. This study constituted an extension of our published work relating to the identification and relative quantification of potential clinical biomarkers in BPH and prostate cancer (PCa) tissue specimens. The proposed MudPIT approach encompassed the use of three distinct yet complementary liquid chromatographic chemistries. High-pressure size-exclusion chromatography (SEC) was used for the prefractionation of serum proteins followed by their dialysis exchange and solution phase trypsin proteolysis. The tryptic peptides were then subjected to offline zwitterion-ion hydrophilic interaction chromatography (ZIC-HILIC) fractionation followed by their online analysis with reversed-phase nano-ultraperformance chromatography (RP-nUPLC) hyphenated to nanoelectrospray ionization-tandem mass spectrometry using an ion trap mass analyzer. For the spectral processing, the sequential use of the SpectrumMill, Scaffold, and InsPecT software tools was applied for the tryptic peptide product ion MS(2) spectral processing, false discovery rate (FDR) assessment, validation, and protein identification. This milestone serum analysis study allowed the confident identification of over 1955 proteins (p ≤ 0.05; FDR ≤ 5%) with a broad spectrum of biological and physicochemical properties including secreted, tissue-specific proteins spanning approximately 12 orders of magnitude as they occur in their native abundance levels in the serum matrix. Also encompassed in this proteome was the confident identification of 375 phosphoproteins (p ≤ 0.05; FDR ≤ 5%) with potential importance to cancer biology. To demonstrate the performance characteristics of this novel MudPIT approach, a comparison was made with the proteomes resulting from the immunodepletion of the high abundant albumin and IgG proteins with offline first dimensional tryptic peptide separation with both ZIC-HILIC and strong cation exchange (SCX) chromatography and their subsequent online RP-nUPLC-nESI-MS(2) analysis.

Predicting Protein Aggregation during Storage in Lyophilized Solids Using Solid State Amide Hydrogen/Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS)

PubMed Central

2015-01-01

Solid state amide hydrogen/deuterium exchange with mass spectrometric analysis (ssHDX-MS) was used to assess the conformation of myoglobin (Mb) in lyophilized formulations, and the results correlated with the extent of aggregation during storage. Mb was colyophilized with sucrose (1:1 or 1:8 w/w), mannitol (1:1 w/w), or NaCl (1:1 w/w) or in the absence of excipients. Immediately after lyophilization, samples of each formulation were analyzed by ssHDX-MS and Fourier transform infrared spectroscopy (FTIR) to assess Mb conformation, and by dynamic light scattering (DLS) and size exclusion chromatography (SEC) to determine the extent of aggregation. The remaining samples were then placed on stability at 25 °C and 60% RH or 40 °C and 75% RH for up to 1 year, withdrawn at intervals, and analyzed for aggregate content by SEC and DLS. In ssHDX-MS of samples immediately after lyophilization (t = 0), Mb was less deuterated in solids containing sucrose (1:1 and 1:8 w/w) than in those containing mannitol (1:1 w/w), NaCl (1:1 w/w), or Mb alone. Deuterium uptake kinetics and peptide mass envelopes also indicated greater Mb structural perturbation in mannitol, NaCl, or Mb-alone samples at t = 0. The extent of deuterium incorporation and kinetic parameters related to rapidly and slowly exchanging amide pools (Nfast, Nslow), measured at t = 0, were highly correlated with the extent of aggregation on storage as measured by SEC. In contrast, the extent of aggregation was weakly correlated with FTIR band intensity and peak position measured at t = 0. The results support the use of ssHDX-MS as a formulation screening tool in developing lyophilized protein drug products. PMID:24816133

Secretome Analysis Defines the Major Role of SecDF in Staphylococcus aureus Virulence

PubMed Central

Quiblier, Chantal; Seidl, Kati; Roschitzki, Bernd; Zinkernagel, Annelies S.; Berger-Bächi, Brigitte; Senn, Maria M.

2013-01-01

The Sec pathway plays a prominent role in protein export and membrane insertion, including the secretion of major bacterial virulence determinants. The accessory Sec constituent SecDF has been proposed to contribute to protein export. Deletion of Staphylococcus aureus secDF has previously been shown to reduce resistance, to alter cell separation, and to change the expression of certain virulence factors. To analyse the impact of the secDF deletion in S. aureus on protein secretion, a quantitative secretome analysis was performed. Numerous Sec signal containing proteins involved in virulence were found to be decreased in the supernatant of the secDF mutant. However, two Sec-dependent hydrolases were increased in comparison to the wild type, suggesting additional indirect, regulatory effects to occur upon deletion of secDF. Adhesion, invasion, and cytotoxicity of the secDF mutant were reduced in human umbilical vein endothelial cells. Virulence was significantly reduced using a Galleria mellonella insect model. Altogether, SecDF is a promising therapeutic target for controlling S. aureus infections. PMID:23658837

The Immediate Effect of Neuromuscular Joint Facilitation (NJF) Treatment on Hip Muscle Strength.

PubMed

Wang, Hongdan; Huo, Ming; Huang, Qiuchen; Li, Desheng; Maruyama, Hitoshi

2013-11-01

[Purpose] This study investigated the change in hip muscle strength of younger persons after neuromuscular joint facilitation (NJF) treatment. [Subjects] The subjects were 45 healthy young people, who were divided into two groups: a NJF group and a proprioceptive neuromuscular facilitation (PNF) group. The NJF group consisted of 21 subjects (11 males, 10 females), and the PNF group consisted of 24 subjects (11 males, 13 females). [Methods] Participants in the NJF group received NJF treatment. We measured the maximal flexor strength and the maximal extensor strength during isokinetic movement of the hip joint before and after intervention in both groups. The angular velocities used were 60°/sec and 180°/sec. [Results] The NJF group showed significant increases in the maximal flexor strength and the maximal extensor strength after the intervention at each angular velocity. In the PNF group, the maximal flexor strength of 60°/sec and the maximal extensor strength of 180°/sec were significant increases. [Conclusion] These results suggest that there is an immediate effect of NJF intervention on hip muscle strength.

In Candida albicans hyphae, Sec2p is physically associated with SEC2 mRNA on secretory vesicles.

PubMed

Caballero-Lima, David; Hautbergue, Guillaume M; Wilson, Stuart A; Sudbery, Peter E

2014-11-01

Candida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

PubMed Central

Fang, H; Green, N

1994-01-01

The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane. Images PMID:7841522

The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

PubMed Central

Stalder, Danièle; Novick, Peter J.

2016-01-01

Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.

PubMed

Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid; Rapoport, Tom A

2017-05-12

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans

PubMed Central

Chayakulkeeree, Methee; Johnston, Simon Andrew; Oei, Johanes Bijosono; Lev, Sophie; Williamson, Peter Richard; Wilson, Christabel Frewen; Zuo, Xiaoming; Leal, Ana Lusia; Vainstein, Marilene Henning; Meyer, Wieland; Sorrell, Tania Christine; May, Robin Charles; Djordjevic, Julianne Teresa

2011-01-01

Summary Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein (PITP) ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1 (Lac1)-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnΔsec14-1 and CnΔsec14-1CnΔsfh5). Similar to the CnPLB1 deletion mutant (CnΔplb1), CnΔsec14-1 was hypo-virulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnΔsec14-1 and CnΔplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely-related CnSEC14-1 homologue, and CnSFH5, a distantly-related SEC fourteen-like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnΔsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis. PMID:21453402

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

PubMed Central

Ishii, Eiji; Chiba, Shinobu; Hashimoto, Narimasa; Kojima, Seiji; Homma, Michio; Ito, Koreaki; Akiyama, Yoshinori; Mori, Hiroyuki

2015-01-01

SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine–Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles. PMID:26392525

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

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

Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, wemore » report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.« less

Polysaccharides and Oligosaccharides Produced on Malvar Wines Elaborated with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 Native Yeasts from D.O. "Vinos de Madrid".

PubMed

García, Margarita; Apolinar-Valiente, Rafael; Williams, Pascale; Esteve-Zarzoso, Braulio; Arroyo, Teresa; Crespo, Julia; Doco, Thierry

2017-08-09

Polysaccharides and oligosaccharides released into Malvar white wines elaborated through pure, mixed, and sequential cultures with Torulaspora delbrueckii CLI 918 and Saccharomyces cerevisiae CLI 889 native yeasts from D.O. "Vinos de Madrid" were studied. Both fractions from different white wines were separated by high-resolution size-exclusion chromatography. Glycosyl composition and wine polysaccharide linkages were determined by GC-EI-MS chromatography. Molar-mass distributions were determined by SEC-MALLS, and intrinsic viscosity was determined by differential viscometer. Yeast species and type of inoculation have a significant impact on wine carbohydrate composition and structure. Mannose residues from mannoproteins were significantly predominant in those cultures where T. delbrueckii was present in the fermentation process in comparison with when pure cultures of S. cerevisiae were present in the fermenation process. Galactose residues from polysaccharides rich in arabinose and galactose presented greater values in pure cultures of S. cerevisiae, indicating that S. cerevisiae released fewer mannoproteins than T. delbrueckii. Moreover, we reported structural differences between mannoproteins released by T. delbrueckii CLI 918 and those released by S. cerevisiae CLI 889. These findings help to provide important information about the polysaccharides and oligosaccharides released from the cell walls of Malvar grapes and the carbohydrates released from each yeast species.

Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps.

PubMed

Lawoko, Martin; Henriksson, Gunnar; Gellerstedt, Göran

2005-01-01

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.

US-SOMO HPLC-SAXS module: dealing with capillary fouling and extraction of pure component patterns from poorly resolved SEC-SAXS data

PubMed Central

Brookes, Emre; Vachette, Patrice; Rocco, Mattia; Pérez, Javier

2016-01-01

Size-exclusion chromatography coupled with SAXS (small-angle X-ray scattering), often performed using a flow-through capillary, should allow direct collection of monodisperse sample data. However, capillary fouling issues and non-baseline-resolved peaks can hamper its efficacy. The UltraScan solution modeler (US-SOMO) HPLC-SAXS (high-performance liquid chromatography coupled with SAXS) module provides a comprehensive framework to analyze such data, starting with a simple linear baseline correction and symmetrical Gaussian decomposition tools [Brookes, Pérez, Cardinali, Profumo, Vachette & Rocco (2013 ▸). J. Appl. Cryst. 46, 1823–1833]. In addition to several new features, substantial improvements to both routines have now been implemented, comprising the evaluation of outcomes by advanced statistical tools. The novel integral baseline-correction procedure is based on the more sound assumption that the effect of capillary fouling on scattering increases monotonically with the intensity scattered by the material within the X-ray beam. Overlapping peaks, often skewed because of sample interaction with the column matrix, can now be accurately decomposed using non-symmetrical modified Gaussian functions. As an example, the case of a polydisperse solution of aldolase is analyzed: from heavily convoluted peaks, individual SAXS profiles of tetramers, octamers and dodecamers are extracted and reliably modeled. PMID:27738419

A "push and slide" mechanism allows sequence-insensitive translocation of secretory proteins by the SecA ATPase.

PubMed

Bauer, Benedikt W; Shemesh, Tom; Chen, Yu; Rapoport, Tom A

2014-06-05

In bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here we show that SecA moves polypeptides through the SecY channel by a "push and slide" mechanism. In its ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state or when SecA encounters a poorly interacting amino acid in its ATP-bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed push and slide mechanism is supported by a mathematical model and explains how SecA allows translocation of a wide range of polypeptides. This mechanism may also apply to hexameric polypeptide-translocating ATPases. Copyright © 2014 Elsevier Inc. All rights reserved.

Demonstration of a Specific Escherichia coli SecY–Signal Peptide Interaction†

PubMed Central

Wang, Ligong; Miller, Alexander; Rusch, Sharyn L.; Kendall, Debra A.

2011-01-01

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide–SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. PMID:15476412

SU-E-J-273: Skin Temperature Recovery Rate as a Potential Predictor for Radiation-Induced Skin Reactions

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

Biswal, N C; Wu, Z; Chu, J

Purpose: To assess the potential of dynamic infrared imaging to evaluate early skin reactions during radiation therapy in cancer patients. Methods: Thermal images were captured by our home-built system consisting of two flash lamps and an infrared (IR) camera. The surface temperature of the skin was first raised by ∼ 6 °C from ∼1 ms short flashes; the camera then captured a series of IR images for 10 seconds. For each image series, a basal temperature was recorded for 0.5 seconds before flash was triggered. The temperature gradients (ε) were calculated between a reference point (immediately after the flash) andmore » at a time point of 2sec, 4sec and 9sec after that. A 1.0 cm region of interest (ROI) on the skin was drawn; the mean and standard deviations of the ROIs were calculated. The standard ε values for normal human skins were evaluated by imaging 3 healthy subjects with different skin colors. All of them were imaged on 3 separate days for consistency checks. Results: The temperature gradient, which is the temperature recovery rate, depends on the thermal properties of underlying tissue, i.e. thermal conductivity. The average ε for three volunteers averaged over 3 measurements were 0.64±0.1, 0.72±0.2 and 0.80±0.3 at 2sec, 4sec and 9sec respectively. The standard deviations were within 1.5%–3.2%. One of the volunteers had a prior small skin burn on the left wrist and the ε values for the burned site were around 9% (at 4sec) and 13% (at 9sec) lower than that from the nearby normal skin. Conclusion: The temperature gradients from the healthy subjects were reproducible within 1.5%–3.2 % and that from a burned skin showed a significant difference (9%–13%) from the normal skin. We have an IRB approved protocol to image head and neck patients scheduled for radiation therapy.« less

Analysis of a waterborne disease model with socioeconomic classes.

PubMed

Collins, O C; Robertson, Suzanne L; Govinder, K S

2015-11-01

Waterborne diseases such as cholera continue to pose serious public health problems in the world today. Transmission parameters can vary greatly with socioeconomic class (SEC) and the availability of clean water. We formulate a multi-patch waterborne disease model such that each patch represents a particular SEC with its own water source, allowing individuals to move between SECs. For a 2-SEC model, we investigate the conditions under which each SEC is responsible for driving a cholera outbreak. We determine the effect of SECs on disease transmission dynamics by comparing the basic reproduction number of the 2-SEC model to that of a homogeneous model that does not take SECs into account. We conclude by extending several results of the 2-SEC model to an n-SEC model. Copyright © 2015 Elsevier Inc. All rights reserved.

Performance optimization of marine propellers

NASA Astrophysics Data System (ADS)

Lee, Chang-Sup; Choi, Young-Dal; Ahn, Byoung-Kwon; Shin, Myoung-Sup; Jang, Hyun-Gil

2010-12-01

Recently a Wide Chord Tip (WCT) propeller has been developed and applied to a commercial ship by STX Offshore & Shipbuilding. It is reported that the WCT propeller significantly reduces pressure fluctuations and also ship's noise and vibration. On the sea trial, vibration magnitude in the accommodations at NCR was measured at 0.9mm/sec which is only 10% of international allowable magnitude of vibration (9mm/sec). In this paper, a design method for increasing performance of the marine propellers including the WCT propeller is suggested. It is described to maximize the performance of the propeller by adjusting expanded areas of the propeller blade. Results show that efficiency can be increased up to over 2% through the suggested design method.

77 FR 2456 - Incorporation of Revised ASTM Standards That Provide Flexibility in the Use of Alternatives to...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-18

... (General Bomb Method), IBR approved for appendix A to subpart D. (4) ASTM D287-92, Standard Test Method for... Method for Sulfur in Petroleum Products (General Bomb Method), IBR approved for Sec. 94.108. (4) ASTM...

An improved method for predicting the effects of flight on jet mixing noise

NASA Technical Reports Server (NTRS)

Stone, J. R.

1979-01-01

The NASA method (1976) for predicting the effects of flight on jet mixing noise was improved. The earlier method agreed reasonably well with experimental flight data for jet velocities up to about 520 m/sec (approximately 1700 ft/sec). The poorer agreement at high jet velocities appeared to be due primarily to the manner in which supersonic convection effects were formulated. The purely empirical supersonic convection formulation of the earlier method was replaced by one based on theoretical considerations. Other improvements of an empirical nature included were based on model-jet/free-jet simulated flight tests. The revised prediction method is presented and compared with experimental data obtained from the Bertin Aerotrain with a J85 engine, the DC-10 airplane with JT9D engines, and the DC-9 airplane with refanned JT8D engines. It is shown that the new method agrees better with the data base than a recently proposed SAE method.

Comparison of isokinetic muscle strength and muscle power by types of warm-up.

PubMed

Sim, Young-Je; Byun, Yong-Hyun; Yoo, Jaehyun

2015-05-01

[Purpose] The purpose of this study was to clarify the influence of static stretching at warm-up on the isokinetic muscle torque (at 60°/sec) and muscle power (at 180°/sec) of the flexor muscle and extensor muscle of the knee joint. [Subjects and Methods] The subjects of this study were 10 healthy students with no medically specific findings. The warm-up group and warm-up with stretching group performed their respective warm-up prior to the isokinetic muscle torque evaluation of the knee joint. One-way ANOVA was performed by randomized block design for each variable. [Results] The results were as follows: First, the flexor peak torque and extensor peak torque of the knee joint tended to decrease at 60°/sec in the warm-up with stretching group compared with the control group and warm-up group, but without statistical significance. Second, extensor power at 180°/sec was also not statistically significant. However, it was found that flexor power increased significantly in the warm-up with stretching group at 180°/sec compared with the control group and warm-up group in which stretching was not performed. [Conclusion] Therefore, it is considered that in healthy adults, warm-up including two sets of stretching for 20 seconds per muscle group does not decrease muscle strength and muscle power.

Analytical linear energy transfer model including secondary particles: calculations along the central axis of the proton pencil beam

NASA Astrophysics Data System (ADS)

Marsolat, F.; De Marzi, L.; Pouzoulet, F.; Mazal, A.

2016-01-01

In proton therapy, the relative biological effectiveness (RBE) depends on various types of parameters such as linear energy transfer (LET). An analytical model for LET calculation exists (Wilkens’ model), but secondary particles are not included in this model. In the present study, we propose a correction factor, L sec, for Wilkens’ model in order to take into account the LET contributions of certain secondary particles. This study includes secondary protons and deuterons, since the effects of these two types of particles can be described by the same RBE-LET relationship. L sec was evaluated by Monte Carlo (MC) simulations using the GATE/GEANT4 platform and was defined by the ratio of the LET d distributions of all protons and deuterons and only primary protons. This method was applied to the innovative Pencil Beam Scanning (PBS) delivery systems and L sec was evaluated along the beam axis. This correction factor indicates the high contribution of secondary particles in the entrance region, with L sec values higher than 1.6 for a 220 MeV clinical pencil beam. MC simulations showed the impact of pencil beam parameters, such as mean initial energy, spot size, and depth in water, on L sec. The variation of L sec with these different parameters was integrated in a polynomial function of the L sec factor in order to obtain a model universally applicable to all PBS delivery systems. The validity of this correction factor applied to Wilkens’ model was verified along the beam axis of various pencil beams in comparison with MC simulations. A good agreement was obtained between the corrected analytical model and the MC calculations, with mean-LET deviations along the beam axis less than 0.05 keV μm-1. These results demonstrate the efficacy of our new correction of the existing LET model in order to take into account secondary protons and deuterons along the pencil beam axis.

Identification of O-linked β-d-N-acetylglucosamine-Modified Proteins from Arabidopsis

PubMed Central

Xu, Shou-Ling; Chalkley, Robert J.; Wang, Zhi-Yong; Burlingame, Alma L.

2013-01-01

The posttranslational modification of proteins with O-linked β-d-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues occurs in all animals and plants. This modification is dynamic and ubiquitous, and regulates many cellular processes, including transcription, signaling and cytokinesis and is associated with several diseases. Cycling of O-GlcNAc is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Plants have two OGTs, SPINDLY (SPY) and SECRET AGENT (SEC); disruption of both causes embryo lethality. Despite O-GlcNAc modification of proteins being discovered more than 20-years ago, identification and mapping of protein GlcNAcylation is still a challenging task. Here we describe the use of lectin affinity chromatography combined with electron transfer dissociation mass spectrometry to enrich and to detect O-GlcNAc modified peptides from Arabidopsis. PMID:22576084

Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul.

PubMed

Praznik, Werner; Löppert, Renate; Cruz Rubio, Josè M; Zangger, Klaus; Huber, Anton

2013-11-15

Fructo-oligosaccharides (FOSs) of a six year old agave plant variety, Agave tequilana, were isolated and fractionated by 2D preparative chromatography (SEC and rpHPLC). Structural analyses of different FOS-fractions were performed by reductive methylation analysis connected to GC/FID identification and NMR-analysis. FOSs from leaves (d.p. 3-8) contain single α-d-Glcp residues as well in terminal as internal position, however (2→1)-linked β-d-Fruf residues only. FOSs from stem, however, contain as well (2→1)- and (2→6)-linked β-d-Fruf residues with branched oligomeric repeating units. These characteristics indicate an enzymatically catalyzed metabolic regulation for the biosynthesis and transformation of fructans in A. tequilana which strongly depends on location and transport activities. Copyright © 2013 Elsevier Ltd. All rights reserved.

A novel high-throughput method for supported liquid extraction of retinol and alpha-tocopherol from human serum and simultaneous quantitation by liquid chromatography tandem mass spectrometry.

PubMed

Hinchliffe, Edward; Rudge, James; Reed, Paul

2016-07-01

Measurement of vitamin A (retinol) and E (alpha-tocopherol) in UK clinical laboratories is currently performed exclusively by high-performance liquid chromatography with ultraviolet detection. We investigated whether retinol and alpha-tocopherol could be measured simultaneously by liquid chromatography tandem mass spectrometry. Serum samples (100 μL) were extracted using Isolute + Supported Liquid Extraction plates. Chromatography was performed on a Phenomenex Kinetex Biphenyl 2.6 μm, 50 × 2.1 mm column, and liquid chromatography tandem mass spectrometry on a Waters Acquity TQD. Injection-to-injection time was 4.3 min. The assay was validated according to published guidelines. Patient samples were used to compare liquid chromatography tandem mass spectrometry and high-performance liquid chromatography with ultraviolet detection methods. For retinol and alpha-tocopherol, respectively, the assay was linear up to 6.0 and 80.0 μmol/L, and lower limit of quantification was 0.07 and 0.26 μmol/L. Intra and interassay imprecision were within desirable analytical specifications. Analysis of quality control material aligned to NIST SRM 968e, and relative spiked recovery from human serum, both yielded results within 15% of target values. Method comparison with high-performance liquid chromatography with ultraviolet detection methodology demonstrated a negative bias for retinol and alpha-tocopherol by the liquid chromatography tandem mass spectrometry method. Analysis of United Kingdom National External Quality Assurance Scheme samples yielded mean bias from the target value of +3.0% for retinol and -11.2% for alpha-tocopherol. We have developed a novel, high-throughput method for extraction of retinol and alpha-tocopherol from human serum followed by simultaneous quantitation by liquid chromatography tandem mass spectrometry. The method offers a rapid, sensitive, specific and cost-effective alternative to high-performance liquid chromatography with ultraviolet detection methodology, and is suitable for routine clinical monitoring of patients predisposed to fat-soluble vitamin malabsorption. © The Author(s) 2015.

[Analytical method and comparison for static and dynamic headspace gas chromatography of anisole in water].

PubMed

Zhang, Yan; Qian, Jie-feng; Liu, Lan-xia; Zhao, Hui-qin

2013-01-01

To establish and compare the method of static headspace gas chromatography hydrogen flame detector (static headspace method) and purge and trap gas chromatography-mass spectrometry (dynamic headspace method) of anisole in water. Nitrogen gas was used as carrier gas in the static headspace method, 5 g NaCl as matrix modifier was added into 10 ml water. The sample was balanced with high speed vibration at 75°C for 30 min, and anisole was detected by gas chromatography and quantified with external standard. Helium was used as carrier gas in dynamic headspace method, 5.0 ml water and 0.004 mg/L internal standard fluorobenzene was purged into the purge and trap apparatus. After purging, trapping and desorption, anisole was detected by the gas chromatography-mass spectrograph, confirmed by the retention time and comparison of mass-spectrogram in spectrum library and quantified with internal standard. The repeatability and sensitivity of assay were evaluated. A good linear range for anisole was observed in static headspace gas chromatography and dynamic headspace gas chromatography-mass spectrometry, within the range of 10 - 500 µg/L and 0.5 - 60.0 µg/L respectively. The linear regression equation was Y = 782.150X + 1.3446 and Y = 0.0358X - 0.0209 respectively, both the correlation coefficient ≥ 0.999. The detection limit (LOD) were 0.002 µg/L and 0.110 µg/L, the lower limit of quantitation (LOQ) were 0.006 µg/L and 0.350 µg/L, the relative standard deviation (RSD) were 1.8% - 2.3% and 2.0% - 3.4%, and the spiking recovery were 93% - 101% and 96% - 101% respectively. The methods of static headspace gas chromatography and dynamic headspace gas chromatography-mass spectrometry are simple and can measure anisole in water quickly, sensitively and accurately.

Efficacy of high-intensity pulsed light for the microbiological decontamination of chicken, associated packaging, and contact surfaces.

PubMed

Haughton, Pippa N; Lyng, James G; Morgan, Desmond J; Cronin, Denis A; Fanning, Seamus; Whyte, Paul

2011-01-01

The efficacy of high-intensity light pulse (HILP) technology (3 Hz, maximum of 505 J/pulse, and a pulse duration of 360 μs) for the decontamination of raw chicken and associated packaging and surface materials was investigated. Its ability to reduce microbial counts on raw chicken through plastic films was also examined. Complete inactivation of Campylobacter spp., Escherichia coli, and Salmonella Enteritidis in liquid was achieved after 30 sec HILP treatment. Reductions of 3.56, 4.69, and 4.60 log₁₀ cfu/cm²) were observed after 5 sec HILP treatment of Campylobacter jejuni, E. coli, and Salmonella Enteritidis inoculated onto packaging materials and contact surfaces, respectively. The greatest reductions on inoculated chicken skin were 1.22, 1.69, and 1.27 log₁₀ cfu/g for C. jejuni, E. coli, and Salmonella Enteritidis, respectively. Corresponding reductions on inoculated skinless breast meat were 0.96, 1.13, and 1.35 log₁₀ cfu/g. The effectiveness of HILP treatment for reducing microbial levels on chicken increased as the film thickness decreased. HILP treatments of 2 sec did not significantly affect the color of raw chicken although treatments of 30 sec impacted color. This study has shown HILP to be an effective method for the decontamination of packaging and surface materials. Additionally, it has demonstrated the potential of HILP to be used as a decontamination method for packaged chicken.

Diastolic blood pressure-estimated left ventricular dp/dt.

PubMed

Yilmaz, Hüseyin; Minareci, Kenan; Kabukçu, Mehmet; Sancaktar, Oktay

2002-02-01

Peak dp/dt is one of the best isovolumic phase indexes of the myocardial contractile state requiring invasive procedures or presence of mitral regurgitation severe enough to measure in clinical practice by Doppler echocardiography. In this study, we sought the correlation between two noninvasive methods of measurements for left ventricular dp/dt-diastolic blood pressure- (DBP) estimated and continuous-wave Doppler-derived dp/dt-min electrocardiographic/echocardiographic study to emphasize the clinical feasibility of the DBP-estimated method. Thirty-six randomized patients (27 male, 9 female; 58 +/- 8 years) with mild mitral regurgitation were enrolled in this study. DBP-estimated dp/dt was calculated from DBP minus the left ventricular end-diastolic pressure (LVEDP) over the isovolumetric contraction time (IVCT). LVEDP was assumed to be 10 mmHg for all patients. Doppler-determined left ventricular dp/dt was derived from the continuous-wave Doppler spectrum of mitral regurgitation jet by dividing the magnitude of the left ventricular atrial pressure gradient rise between 1 mm/sec-3 mm/sec of mitral regurgitant velocity signal by the time taken for this change. Left ventricular dp/dt by Doppler was 1122 +/- 303 mmHg/sec and blood pressure-estimated dp/dt was 1063 +/- 294 mmHg/sec. There was a high correlation (r = 0.97, P < 0.001) of dp/dt between the two techniques. DBP and IVCT can generate left ventricular dp/dt without invasive procedures, even in the absence of mitral regurgitation in clinical practice.

Flood Discharge Analysis with Nakayasu Method Using Combination of HEC-RAS Method on Deli River in Medan City

NASA Astrophysics Data System (ADS)

Harahap, Rumilla; Jeumpa, Kemala; Hadibroto, Bambang

2018-03-01

The problem in this research is how in the rainy season the water does not overflow, does not occur flood and during the dry season does not occur drought so it can adjust the condition or existence of Deli river which is around Medan city. Deli River floods often occur, either caused by a smaller capacity than the existing discharge, lack of maintenance and drainage and disposal systems that do not fit with the environment, resulting in flood subscriptions every year. The purpose of this research is to know flood discharge at Deli river as Flood control in Medan city. This research is analyzed on several methods such as log Pearson, Gumbel and hydrograph unit, while HEC-RAS method is modeling conducted in analyzing the water profile of the Deli River. Furthermore, the calculation of the periodic flood discharge using the Nakayasu Method. Calculation result at Deli River return period flood discharge 2 years with an area of 14.8 km2 annual flood hydrograph the total is 26.79 m3/sec on the hours at the 4th time. Return period flood discharge 5 years with an area of 14.8 km2 annual flood hydrograph the total is 73,44 m3/sec. While 25 annual return period total flood hydrograph is 146.50 m3/sec. With flood analysis can reduce and minimize the risk of losses and land can be mapped if in the area there is flooding.

Bactericidal active ingredient in cryopreserved plasma-treated water with the reduced-pH method for plasma disinfection

NASA Astrophysics Data System (ADS)

Kitano, Katsuhisa; Ikawa, Satoshi; Nakashima, Yoichi; Tani, Atsushi; Yokoyama, Takashi; Ohshima, Tomoko

2016-09-01

For the plasma disinfection of human body, plasma sterilization in liquid is crucial. We found that the plasma-treated water (PTW) has strong bactericidal activity under low pH condition. Physicochemical properties of PTW is discussed based on chemical kinetics. Lower temperature brings longer half-life and the bactericidal activity of PTW can be kept by cryopreservation. High performance PTW, corresponding to the disinfection power of 22 log reduction (B. subtilis spore), can be obtained by special plasma system equipped with cooling device. This is equivalent to 65% H2O2, 14% sodium hypochlorite and 0.33% peracetic acid, which are deadly poison for human. But, it is deactivated soon at higher temperature (4 sec. at body temperature), and toxicity to human body seems low. For dental application, PTW was effective on infected models of human extracted tooth. Although PTW has many chemical components, respective chemical components in PTW were isolated by ion chromatography. In addition to peaks of H2O2, NO2- and NO3-, a specific peak was detected. and only this fraction had bactericidal activity. Purified active ingredient of PTW is the precursor of HOO, and further details will be discussed in the presentation. MEXT (15H03583, 23340176, 25108505). NCCE (23-A-15).

Multi-fault clustering and diagnosis of gear system mined by spectrum entropy clustering based on higher order cumulants

NASA Astrophysics Data System (ADS)

Shao, Renping; Li, Jing; Hu, Wentao; Dong, Feifei

2013-02-01

Higher order cumulants (HOC) is a new kind of modern signal analysis of theory and technology. Spectrum entropy clustering (SEC) is a data mining method of statistics, extracting useful characteristics from a mass of nonlinear and non-stationary data. Following a discussion on the characteristics of HOC theory and SEC method in this paper, the study of signal processing techniques and the unique merits of nonlinear coupling characteristic analysis in processing random and non-stationary signals are introduced. Also, a new clustering analysis and diagnosis method is proposed for detecting multi-damage on gear by introducing the combination of HOC and SEC into the damage-detection and diagnosis of the gear system. The noise is restrained by HOC and by extracting coupling features and separating the characteristic signal at different speeds and frequency bands. Under such circumstances, the weak signal characteristics in the system are emphasized and the characteristic of multi-fault is extracted. Adopting a data-mining method of SEC conducts an analysis and diagnosis at various running states, such as the speed of 300 r/min, 900 r/min, 1200 r/min, and 1500 r/min of the following six signals: no-fault, short crack-fault in tooth root, long crack-fault in tooth root, short crack-fault in pitch circle, long crack-fault in pitch circle, and wear-fault on tooth. Research shows that this combined method of detection and diagnosis can also identify the degree of damage of some faults. On this basis, the virtual instrument of the gear system which detects damage and diagnoses faults is developed by combining with advantages of MATLAB and VC++, employing component object module technology, adopting mixed programming methods, and calling the program transformed from an *.m file under VC++. This software system possesses functions of collecting and introducing vibration signals of gear, analyzing and processing signals, extracting features, visualizing graphics, detecting and diagnosing faults, detecting and monitoring, etc. Finally, the results of testing and verifying show that the developed system can effectively be used to detect and diagnose faults in an actual operating gear transmission system.

Multi-fault clustering and diagnosis of gear system mined by spectrum entropy clustering based on higher order cumulants.

PubMed

Shao, Renping; Li, Jing; Hu, Wentao; Dong, Feifei

2013-02-01

Higher order cumulants (HOC) is a new kind of modern signal analysis of theory and technology. Spectrum entropy clustering (SEC) is a data mining method of statistics, extracting useful characteristics from a mass of nonlinear and non-stationary data. Following a discussion on the characteristics of HOC theory and SEC method in this paper, the study of signal processing techniques and the unique merits of nonlinear coupling characteristic analysis in processing random and non-stationary signals are introduced. Also, a new clustering analysis and diagnosis method is proposed for detecting multi-damage on gear by introducing the combination of HOC and SEC into the damage-detection and diagnosis of the gear system. The noise is restrained by HOC and by extracting coupling features and separating the characteristic signal at different speeds and frequency bands. Under such circumstances, the weak signal characteristics in the system are emphasized and the characteristic of multi-fault is extracted. Adopting a data-mining method of SEC conducts an analysis and diagnosis at various running states, such as the speed of 300 r/min, 900 r/min, 1200 r/min, and 1500 r/min of the following six signals: no-fault, short crack-fault in tooth root, long crack-fault in tooth root, short crack-fault in pitch circle, long crack-fault in pitch circle, and wear-fault on tooth. Research shows that this combined method of detection and diagnosis can also identify the degree of damage of some faults. On this basis, the virtual instrument of the gear system which detects damage and diagnoses faults is developed by combining with advantages of MATLAB and VC++, employing component object module technology, adopting mixed programming methods, and calling the program transformed from an *.m file under VC++. This software system possesses functions of collecting and introducing vibration signals of gear, analyzing and processing signals, extracting features, visualizing graphics, detecting and diagnosing faults, detecting and monitoring, etc. Finally, the results of testing and verifying show that the developed system can effectively be used to detect and diagnose faults in an actual operating gear transmission system.

Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

PubMed

Kanlaya, Rattiyaporn; Thongboonkerd, Visith

2016-08-01

Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

PubMed

Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

2016-07-01

An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The analysis of carbohydrates in milk powder by a new "heart-cutting" two-dimensional liquid chromatography method.

PubMed

Ma, Jing; Hou, Xiaofang; Zhang, Bing; Wang, Yunan; He, Langchong

2014-03-01

In this study, a new"heart-cutting" two-dimensional liquid chromatography method for the simultaneous determination of carbohydrate contents in milk powder was presented. In this two dimensional liquid chromatography system, a Venusil XBP-C4 analysis column was used in the first dimension ((1)D) as a pre-separation column, a ZORBAX carbohydrates analysis column was used in the second dimension ((2)D) as a final-analysis column. The whole process was completed in less than 35min without a particular sample preparation procedure. The capability of the new two dimensional HPLC method was demonstrated in the determination of carbohydrates in various brands of milk powder samples. A conventional one dimensional chromatography method was also proposed. The two proposed methods were both validated in terms of linearity, limits of detection, accuracy and precision. The comparison between the results obtained with the two methods showed that the new and completely automated two dimensional liquid chromatography method is more suitable for milk powder sample because of its online cleanup effect involved. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division.

PubMed

Tsui, Ho-Ching Tiffany; Keen, Susan K; Sham, Lok-To; Wayne, Kyle J; Winkler, Malcolm E

2011-01-01

The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed "ExPortal," in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥ 75%) of exponentially growing cells, S. pneumoniae SecA (SecA (Spn)) and SecY (Spn) located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecA(Spn) and SecY(Spn) remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells. In contrast, the HtrA (Spn) homologue localized to the equators and septa of most (> 90%) dividing cells, whereas the SrtA(Spn) sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrA(Spn)) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecA(Spn), SecY (Spn), and HtrA (Spn) to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrA(Spn) may play a general role in quality control of proteins exported by the Sec translocase.

Effects of Different Lifting Cadences on Ground Reaction Forces during the Squat Exercise

NASA Technical Reports Server (NTRS)

Bentley, Jason R.; Amonette, William E.; Hagan, R. Donald

2008-01-01

The purpose of this investigation was to determine the effect of different cadences on the ground reaction force (GRF(sub R)) during the squat exercise. It is known that squats performed with greater acceleration will produce greater inertial forces; however, it is not well understood how different squat cadences affect GRF(sub R). It was hypothesized that faster squat cadences will result in greater peak GRF(sub R). METHODS: Six male subjects (30.8+/-4.4 y, 179.5+/-8.9 cm, 88.8+/-13.3 kg) with previous squat experience performed three sets of three squats using three different cadences (FC = 1 sec descent/1 sec ascent; MC = 3 sec descent/1 sec ascent; SC = 4 sec descent/2 sec ascent) with barbell mass equal to body mass. Ground reaction force was used to calculate inertial force trajectories of the body plus barbell (FI(sub system)). Forces were normalized to body mass. RESULTS: Peak GRF(sub R) and peak FI(sub system) were significantly higher in FC squats compared to MC (p=0.0002) and SC (p=0.0002). Range of GRF(sub R) and FI(sub system) were also significantly higher in FC compared to MC (p<0.05), and MC were significantly higher than SC (p<0.05). DISCUSSION: Faster squat cadences result in significantly greater peak GRF(sub R) due to the inertia of the system. GRF(sub R) was more dependent upon decent cadence than on ascent cadence. PRACTICAL APPLICATION: This study demonstrates that faster squat cadences produce greater ground reaction forces. Therefore, the use of faster squat cadences might enhance strength and power adaptations to long-term resistance exercise training. Key Words: velocity, weight training, resistive exercise

76 FR 72245 - Implementation of Defense Trade Cooperation Treaties

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-22

... clarifying language to Sec. Sec. 123.9(c), (c)(1), and (c)(2); and adding the language of the current (c)(4) to (c)(3). New language pertaining to new Sec. Sec. 126.16 and 126.17 will comprise a new (c)(4). We... clarifying language to Sec. Sec. 123.9(e)(3) and (e)(4). We are adding Israel to the list of countries and...

40 CFR 172.3 - Scope of requirement.

Code of Federal Regulations, 2014 CFR

2014-07-01

....C. sec. 321(p) and sec. 355). (2) A “new animal drug” (21 U.S.C. sec. 321(w) and sec. 360(b)), or (3) An “animal feed” (21 U.S.C. sec. 321 (x)) containing a “new animal drug” (21 U.S.C. sec. 360(b)). (g... experimental use permit (EUP) is generally required for testing of any unregistered pesticide or any registered...

40 CFR 172.3 - Scope of requirement.

Code of Federal Regulations, 2013 CFR

2013-07-01

....C. sec. 321(p) and sec. 355). (2) A “new animal drug” (21 U.S.C. sec. 321(w) and sec. 360(b)), or (3) An “animal feed” (21 U.S.C. sec. 321 (x)) containing a “new animal drug” (21 U.S.C. sec. 360(b)). (g... experimental use permit (EUP) is generally required for testing of any unregistered pesticide or any registered...

40 CFR 172.3 - Scope of requirement.

Code of Federal Regulations, 2011 CFR

2011-07-01

....C. sec. 321(p) and sec. 355). (2) A “new animal drug” (21 U.S.C. sec. 321(w) and sec. 360(b)), or (3) An “animal feed” (21 U.S.C. sec. 321 (x)) containing a “new animal drug” (21 U.S.C. sec. 360(b)). (g... experimental use permit (EUP) is generally required for testing of any unregistered pesticide or any registered...

Determination of osthol and its metabolites in a phase I reaction system and the Caco-2 cell model by HPLC-UV and LC-MS/MS

PubMed Central

Yuan, Zhenting; Xu, Haiyan; Wang, Ke; Zhao, Zhonghua; Hu, Ming

2012-01-01

A straightforward and sensitive reversed-phase high-performance liquid chromatography (HPLC) assay was developed and validated for the analysis of osthol and its phase I metabolites (internal standard: umbelliferone). The method was validated for the determination of osthol with respect to selectivity, precision, linearity, limit of detection, recovery, and stability. The linear response range was 0.47 ~ 60 μM, and the average recoveries ranged from 98 to 101%. The inter-day and intra-day relative standard deviations were both less than 5%. Using this method, we showed that more than 80% of osthol was metabolized in 20 min in a phase I metabolic reaction system. Transport experiments in the Caco-2 cell culture model indicated that osthol was easily absorbed with high absorptive permeability (>10×10-6 cm/sec). The permeability did not display concentration-dependence or vectorial-dependence and is mildly temperature sensitive (activation energy less than 10 Kcal/mole), indicating passive mechanism of transport. When analyzed by LC-MS/MS, five metabolites were detected in a phase I reaction system and in the receiver side of a modified Caco-2 cell model, which was supplemented with the phase I reaction system. The major metabolites appeared to be desmethyl-osthol and multiple isomers of dehydro-osthol. In conclusion, a likely cause of poor osthol bioavailability is rapid phase I metabolism via the cytochrome P-450 pathways. PMID:19304430

Analysis of oversulfation in biglycan chondroitin/dermatan sulfate oligosaccharides by chip-based nanoelectrospray ionization multistage mass spectrometry.

PubMed

Flangea, Corina; Sisu, Eugen; Seidler, Daniela G; Zamfir, Alina D

2012-01-15

Biglycan (BGN) is a small proteoglycan that consists of a protein core containing leucine-rich repeat regions and two glycosaminoglycan (GAG) chains of either chondroitin sulfate (CS) or dermatan sulfate (DS) type. The development of novel, highly efficient analytical methods for structural identification of BGN-derived CS/DS motifs, possibly implicated in biological events, is currently the focus of research. In this work, an improved analytical method based on fully automated chip-nanoelectrospray ionization (nanoESI) in conjunction with high-capacity ion trap (HCT) multistage mass spectrometry (MS) by collision-induced dissociation (CID) was for the first time applied to BGN CS/DS oligosaccharide analysis. The CS/DS chains were released from transfected 293 BGN by β-elimination. The chain was digested with AC I lyase, and the resulting mixture was purified and subsequently separated by size exclusion chromatography (SEC). Di- and tetrasaccharide fractions were pooled and characterized in detail using the developed chip-nanoESI protocol. The chip-nanoESI MS profile in the negative ion mode revealed the presence of under-, regularly, and oversulfated species in both di- and tetrasaccharide fractions. CID MS(2)-MS(3) yielded sequence patterns consistent with unusual oversulfated 4,5-Δ-GlcA(2S)-GalNAc(4S) and 4,5-Δ-GlcA(2S)-GalNAc(6S)-IdoA(2S)-GalNAc(6S) motifs. Copyright © 2011 Elsevier Inc. All rights reserved.

Two-Dimensional Offline Chromatographic Fractionation for the Characterization of Humic-Like Substances in Atmospheric Aerosol Particles.

PubMed

Spranger, Tobias; van Pinxteren, Dominik; Herrmann, Hartmut

2017-05-02

Organic carbon in atmospheric particles comprises a large fraction of chromatographically unresolved compounds, often referred to as humic-like substances (HULIS), which influence particle properties and impact climate, human health, and ecosystems. To better understand its composition, a two-dimensional (2D) offline method combining size-exclusion (SEC) and reversed-phase liquid chromatography (RP-HPLC) using a new spiked gradient profile is presented. It separates HULIS into 55 fractions of different size and polarity, with estimated ranges of molecular weight and octanol/water partitioning coefficient (log P) from 160-900 g/mol and 0.2-3.3, respectively. The distribution of HULIS within the 2D size versus polarity space is illustrated with heat maps of ultraviolet absorption at 254 nm. It is found to strongly differ in a small example set of samples from a background site near Leipzig, Germany. In winter, the most intense signals were obtained for the largest molecules (>520 g/mol) with low polarity (log P ∼ 1.9), whereas in summer, smaller (225-330 g/mol) and more polar (log P ∼ 0.55) molecules dominate. The method reveals such differences in HULIS composition in a more detailed manner than previously possible and can therefore help to better elucidate the sources of HULIS in different seasons or at different sites. Analyzing Suwannee river fulvic acid as a common HULIS surrogate shows a similar polarity range, but the sizes are clearly larger than those of atmospheric HULIS.

Lst1p and Sec24p Cooperate in Sorting of the Plasma Membrane Atpase into Copii Vesicles in Saccharomyces cerevisiae

PubMed Central

Shimoni, Yuval; Kurihara, Tatsuo; Ravazzola, Mariella; Amherdt, Mylène; Orci, Lelio; Schekman, Randy

2000-01-01

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of α-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles. PMID:11086000

Simple and accurate method for determining dissolved inorganic carbon in environmental water by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2018-03-01

We investigate a simple and accurate method for quantitatively analyzing dissolved inorganic carbon in environmental water by reaction headspace gas chromatography. The neutralization reaction between the inorganic carbon species (i.e. bicarbonate ions and carbonate ions) in environmental water and hydrochloric acid is carried out in a sealed headspace vial, and the carbon dioxide formed from the neutralization reaction, the self-decomposition of carbonic acid, and dissolved carbon dioxide in environmental water is then analyzed by headspace gas chromatography. The data show that the headspace gas chromatography method has good precision (relative standard deviation ≤ 1.63%) and accuracy (relative differences ≤ 5.81% compared with the coulometric titration technique). The headspace gas chromatography method is simple, reliable, and can be well applied in the dissolved inorganic carbon detection in environmental water. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of proenkephalin products in brain tissue by high-performance liquid chromatography and a modified bioassay procedure.

PubMed

Bailey, C; Kitchen, I

1985-06-01

A method is described for the separation of proenkephalin products using gradient high-performance liquid chromatography preceded by Sep-Pak chromatography. Samples can be assayed simply by use of a modified mouse vas deferens bioassay which is sufficiently sensitive for most applications. The preliminary Sep-Pak chromatography method excludes alpha-neoendorphin and the dynorphins and thus provides a suitable procedure for separation of prodynorphin and proenkephalin products.

77 FR 53906 - Notice of Proposed Withdrawal and Opportunity for Public Meeting; California

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-04

...\\; Sec. 32, lots 4 and 5; Sec. 34, lot 4. T. 13 N., R. 10 E., Sec. 2, lot 1, and lots 3 to 15, inclusive; Sec. 9, lots 8, 12, and 13, and SW\\1/4\\NE\\1/4\\; Sec. 10, lots 1 to 10, inclusive, E\\1/2\\NE\\1/4\\, E\\1/2..., inclusive, S\\1/2\\ of lot 5, S\\1/2\\ of lot 8, lots 11 and 13; Sec. 19, lot 24; Sec. 20, lots 1, 2, 3, and 8...

Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses.

PubMed

Vincent, Delphine; Elkins, Aaron; Condina, Mark R; Ezernieks, Vilnis; Rochfort, Simone

2016-01-01

Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

Functional diversification of Arabidopsis SEC1-related SM proteins in cytokinetic and secretory membrane fusion.

PubMed

Karnahl, Matthias; Park, Misoon; Krause, Cornelia; Hiller, Ulrike; Mayer, Ulrike; Stierhof, York-Dieter; Jürgens, Gerd

2018-06-12

Sec1/Munc18 (SM) proteins contribute to membrane fusion by interacting with Qa-SNAREs or nascent trans -SNARE complexes. Gymnosperms and the basal angiosperm Amborella have only a single SEC1 gene related to the KEULE gene in Arabidopsis However, the genomes of most angiosperms including Arabidopsis encode three SEC1-related SM proteins of which only KEULE has been functionally characterized as interacting with the cytokinesis-specific Qa-SNARE KNOLLE during cell-plate formation. Here we analyze the closest paralog of KEULE named SEC1B. In contrast to the cytokinesis defects of keule mutants, sec1b mutants are homozygous viable. However, the keule sec1b double mutant was nearly gametophytically lethal, displaying collapsed pollen grains, which suggests substantial overlap between SEC1B and KEULE functions in secretion-dependent growth. SEC1B had a strong preference for interaction with the evolutionarily ancient Qa-SNARE SYP132 involved in secretion and cytokinesis, whereas KEULE interacted with both KNOLLE and SYP132. This differential interaction with Qa-SNAREs is likely conferred by domains 1 and 2a of the two SM proteins. Comparative analysis of all four possible combinations of the relevant SEC1 Qa-SNARE double mutants revealed that in cytokinesis, the interaction of SEC1B with KNOLLE plays no role, whereas the interaction of KEULE with KNOLLE is prevalent and functionally as important as the interactions of both SEC1B and KEU with SYP132 together. Our results suggest that functional diversification of the two SEC1-related SM proteins during angiosperm evolution resulted in enhanced interaction of SEC1B with Qa-SNARE SYP132, and thus a predominant role of SEC1B in secretion.

Intrinsic and chemically produced microheterogeneity of Staphylococcus aureus enterotoxin type C.

PubMed Central

Metzger, J F; Johnson, A D; Spero, L

1975-01-01

Staphylococcus aureus enterotoxins C1 (SEC1) and C2 (SEC2) produced from 50-liter quantities of crude culture supernatants were purified chromatographically in a neutral or acid milieu. Microheterogenity of SEC1 was markedly increased by treatment of the purified toxin with alkali, and new more acidic charged species appeared. SEC2 was more heterogenous than any of the other S. aureus enterotoxins and was affected only slightly by treatment with alkali. Prolonged incubation of the organism during production of the SEC2 produced changes in charged species that may be related to a bacterial deamidase, since similar changes were not seen with alkaline treatment of the purified toxin. Although SEC1 and SEC2 showed complete identity immunologically, they are separate, distinct toxins, and alkali treatment of SEC1 did not produce SEC2. Images PMID:237837

75 FR 2197 - Western Pacific Fisheries; Regulatory Restructuring

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-14

....16 Vessel identification. Sec. 665.17 Experimental fishing..... Sec. 665.17 Experimental fishing. Sec.... 0586, and -0589. Sec. 665.17 Experimental Sec. 665.17 0648-0214 and -0490. fishing. Experimental...) experimental fishing reports estimated at 4 hours (hr) per reporting action; (d) sales and transshipment...

The mechanism of protein export enhancement by the SecDF membrane component

PubMed Central

Tsukazaki, Tomoya; Nureki, Osamu

2011-01-01

Protein transport across membranes is a fundamental and essential cellular activity in all organisms. In bacteria, protein export across the cytoplasmic membrane, driven by dynamic interplays between the protein-conducting SecYEG channel (Sec translocon) and the SecA ATPase, is enhanced by the proton motive force (PMF) and a membrane-integrated Sec component, SecDF. However, the structure and function of SecDF have remained unclear. We solved the first crystal structure of SecDF, consisting of a pseudo-symmetrical 12-helix transmembrane domain and two protruding periplasmic domains. Based on the structural features, we proposed that SecDF functions as a membrane-integrated chaperone, which drives protein movement without using the major energetic currency, ATP, but with remarkable cycles of conformational changes, powered by the proton gradient across the membrane. By a series of biochemical and biophysical approaches, several functionally important residues in the transmembrane region have been identified and our model of the SecDF function has been verified. PMID:27857601

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

PubMed

Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Baldwin, Steve A; Radford, Sheena E; Tuma, Roman; Collinson, Ian

2016-05-16

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

Analysis of Explosives in Soil Using Solid Phase Microextraction and Gas Chromatography: Environmental Analysis

DTIC Science & Technology

2006-01-01

ENVIRONMENTAL ANALYSIS Analysis of Explosives in Soil Using Solid Phase Microextraction and Gas Chromatography Howard T. Mayfield Air Force Research...Abstract: Current methods for the analysis of explosives in soils utilize time consuming sample preparation workups and extractions. The method detection...chromatography/mass spectrometry to provide a con- venient and sensitive analysis method for explosives in soil. Keywords: Explosives, TNT, solid phase

Schinus terebinthifolius countercurrent chromatography (Part III): Method transfer from small countercurrent chromatography column to preparative centrifugal partition chromatography ones as a part of method development.

PubMed

das Neves Costa, Fernanda; Hubert, Jane; Borie, Nicolas; Kotland, Alexis; Hewitson, Peter; Ignatova, Svetlana; Renault, Jean-Hugues

2017-03-03

Countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are support free liquid-liquid chromatography techniques sharing the same basic principles and features. Method transfer has previously been demonstrated for both techniques but never from one to another. This study aimed to show such a feasibility using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. Heptane - ethyl acetate - methanol -water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3β-masticadienolic acids as target compounds. The optimized separation methodology previously described in Part I and II, was scaled up from an analytical hydrodynamic CCC column (17.4mL) to preparative hydrostatic CPC instruments (250mL and 303mL) as a part of method development. Flow-rate and sample loading were further optimized on CPC. Mobile phase linear velocity is suggested as a transfer invariant parameter if the CPC column contains sufficient number of partition cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification of lignans from Fructus Arctii using off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography.

PubMed

Yang, Bichao; Xin, Huaxia; Wang, Feier; Cai, Jianfeng; Liu, Yanfang; Fu, Qing; Jin, Yu; Liang, Xinmiao

2017-08-01

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high-purity compounds. In total, 12 high-purity compounds were isolated from Fructus Arctii. Their structures were identified by using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method for the purification of lignans from Fructus Arctii. The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preliminary design of a Primary Loop Pump Assembly (PLPA), using electromagnetic pumps

NASA Technical Reports Server (NTRS)

Moss, T. A.; Matlin, G.; Donelan, L.; Johnson, J. L.; Rowe, I.

1972-01-01

A preliminary design study of flight-type dc conduction-permanent magnetic, ac helical induction, and ac linear induction pumps for circulating 883 K (1130 F) NaK at 9.1 kg/sec (20 lb/sec) is described. Various electromagnetic pump geometrics are evaluated against hydraulic performance, and the effects of multiple windings and numbers of pumps per assembly on overall reliability were determined. The methods used in the electrical-hydraulic, stress, and thermal analysis are discussed, and the high temperature electrical materials selected for the application are listed.

First wall for polarized fusion reactors

DOEpatents

Greenside, H.S.; Budny, R.V.; Post, D.E. Jr.

1985-01-29

A first-wall or first-wall coating for use in a fusion reactor having polarized fuel may be formed of a low-Z non-metallic material having slow spin relaxation, i.e., a depolarization rate greater than 1 sec/sup -1/. Materials having these properties include hydrogenated and deuterated amorphous semiconductors. A method for preventing the rapid depolarization of a polarized plasma in a fusion device may comprise the step of providing a first-wall or first-wall coating formed of a low-Z, non-metallic material having a depolarization rate greater than 1 sec/sup -1/.

Methods of Evaluating Protective Clothing Relative to Heat and Cold Stress: Thermal Manikin, Biomedical Modeling, and Human Testing

DTIC Science & Technology

2011-01-01

in the climatic chamber housing the manikin. The most widely accepted test procedures for the operation of a TM are published by ASTM International...insulation value of a complete clothing ensemble. It requires a TM surface temperature of 35◦C and a climatic chamber controlled at 23◦C, 50% relative... climatic chamber controlled at 35◦C, 40% relative humidity, with a 0.4 m/sec air velocity. In addition to the tests conducted at 0.4 m/sec, USARIEM

Iris colour and the influence of local anaesthetics on pre-corneal tear film stability.

PubMed

Patel, S; Laidlaw, S; Mathewson, L; McCallum, L; Nicholson, C

1991-06-01

Using non-invasive methods we report here that the stability of the pre-corneal tear film is lower in the brown eye than in the blue eye. The average stability in the blue eye is 15.8 sec (SD +/- 5.8) and in the brown eye it is 12.3 sec (SD +/- 2.9). On average, instillation of topical anaesthetics, benoxinate hydrochloride (0.4%) or amethacaine hydrochloride (0.5%), depress the stability of the pre-corneal tear film in blue eyes but not in brown eyes.

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth1[OPEN

PubMed Central

Bloch, Daria; Pleskot, Roman; Vukašinović, Nemanja

2016-01-01

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. PMID:27516531

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.

PubMed

Bloch, Daria; Pleskot, Roman; Pejchar, Přemysl; Potocký, Martin; Trpkošová, Pavlína; Cwiklik, Lukasz; Vukašinović, Nemanja; Sternberg, Hasana; Yalovsky, Shaul; Žárský, Viktor

2016-10-01

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP 2 ) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP 2 However, the interaction with PIP 2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. © 2016 American Society of Plant Biologists. All Rights Reserved.

78 FR 73466 - Privacy Act

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-06

... Subpart A--General. Sec. 707.11 Scope and purpose. Sec. 707.12 Definitions. Sec. 707.13 Preservation of records. Subpart B--Requests for access to records; amendment of records, accounting of disclosures.... Sec. 707.23 Requests for amendment of records. Sec. 707.24 Requests for an accounting of record...

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

PubMed

Saito, Maiko; Kurosawa, Yae; Okuyama, Tsuneo

2012-02-01

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Complexation of Arsenite with Humic Acid in the Presence of Ferric Iron

PubMed Central

Liu, Guangliang; Fernandez, Aymara; Cai, Yong

2011-01-01

In the presence of iron (Fe), dissolved organic matter (DOM) may bind considerable amounts of arsenic (As), through formation of Fe-bridged As-Fe-DOM complexes and surface complexation of As on DOM-stabilized Fe-colloids (collectively referred to as As-Fe-DOM complexation). However, direct (e.g., chromatographic and spectroscopic) evidence and fundamental kinetic and stability constants have been rarely reported for this As-Fe-DOM complexation. Using a size exclusion chromatography (SEC)-UV-inductively coupled plasma mass spectrometry (ICP-MS) technique, arsenite (AsIII)-Fe-DOM complexation was investigated after adding AsIII into the priorly prepared Fe-DOM. A series of evidence, including coelution of As, Fe, and DOM from the SEC column and coretention of As, Fe, and DOM by 3 kDa MWCO centrifugal filtration membrane, demonstrated the occurrence of AsIII-Fe-DOM complexation. The kinetic data of AsIII-Fe-DOM complexation were well described by a pseudo-first order rate equation (R2 = 0.95), with the rate constant (k′) being 0.17±0.04 1/h. Stability of AsIII-Fe-DOM complexation was characterized by apparent stability constant (Ks) derived from two-site ligand binding model, with log Ks ranging from 4.4±0.2 to 5.6±0.4. Considering the kinetics (within hours) and stability (similar to typical metal-humates) of AsIII-Fe-DOM complexation, this complexation needs to be included when evaluating As mobility in Fe and DOM rich environments. PMID:21322632

Lipid cross-linking of nanolipoprotein particles substantially enhances serum stability and cellular uptake [Lipid crosslinking enhances the stability of nanolipoprotein particles in serum by multiple orders of magnitude

DOE PAGES

Gilmore, Sean F.; Blanchette, Craig D.; Scharadin, Tiffany M.; ...

2016-07-13

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100%more » serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ~10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. Lastly, these results collectively support the potential utility of X-NLPs for a variety of in vivo applications.« less

Effects of Lipid-Analog Detergent Solubilization on the Functionality and Lipidic Cubic Phase Mobility of the Torpedo californica Nicotinic Acetylcholine Receptor

PubMed Central

Padilla-Morales, Luis F.; Morales-Pérez, Claudio L.; De La Cruz-Rivera, Pamela C.; Asmar-Rovira, Guillermo; Báez-Pagán, Carlos A.

2011-01-01

Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β2-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility. PMID:21922299

Using GC-FID to Quantify the Removal of 4-sec-Butylphenol from NGS Solvent by NaOH

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

Sloop, Jr., Frederick V.; Moyer, Bruce A.

2014-12-01

A caustic wash of the solvent used in the Next-Generation Caustic-Side Solvent Extraction (NG-CSSX) process was found to remove the modifier breakdown product 4-sec-butylphenol (SBP) with varying efficiency depending on the aqueous NaOH concentration. Recent efforts at ORNL have aimed at characterizing the flowsheet chemistry and reducing the technical uncertainties of the NG-CSSX process. One technical uncertainty has been the efficacy of caustic washing of the solvent for the removal of lipophilic anions, in particular, the efficient removal of SBP, an important degradation product of the solvent modifier, Cs-7SB. In order to make this determination, it was necessary to developmore » a sensitive and reliable analytical technique for the detection and quantitation of SBP. This report recounts the development of a GC-FID-based (Gas Chromatography Flame Ionization Detection) technique for analyzing SBP and the utilization of the technique to subsequently confirm the ability of the caustic wash to efficiently remove SBP from the Next Generation Solvent (NGS) used in NG-CSSX. In particular, the developed technique was used to monitor the amount of SBP removed from a simple solvent and the full NGS by contact with sodium hydroxide wash solutions over a range of concentrations. The results show that caustic washing removes SBP with effectively the same efficiency as it did in the original Caustic-Side Solvent Extraction (CSSX) process.« less

The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase.

PubMed

Neira, José L; Medina-Carmona, Encarnación; Hernández-Cifre, José G; Montoliu-Gaya, Laia; Cámara-Artigás, Ana; Seffouh, Ilham; Gonnet, Florence; Daniel, Régis; Villegas, Sandra; de la Torre, José García; Pey, Angel L; Li, Fuchuan

2016-12-01

Sulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of β-mercaptoethanol (β-ME). EndoV secondary structure is formed by a mixture of α and β-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Lipid cross-linking of nanolipoprotein particles substantially enhances serum stability and cellular uptake [Lipid crosslinking enhances the stability of nanolipoprotein particles in serum by multiple orders of magnitude

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

Gilmore, Sean F.; Blanchette, Craig D.; Scharadin, Tiffany M.

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100%more » serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ~10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. Lastly, these results collectively support the potential utility of X-NLPs for a variety of in vivo applications.« less

Development and validation of an ultra-performance convergence chromatography method for the quality control of Angelica gigas Nakai.

PubMed

Kim, Hyo Seon; Chun, Jin Mi; Kwon, Bo-In; Lee, A-Reum; Kim, Ho Kyoung; Lee, A Yeong

2016-10-01

Ultra-performance convergence chromatography, which integrates the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies, is an environmentally friendly analytical method that uses dramatically reduced amounts of organic solvents. An ultra-performance convergence chromatography method was developed and validated for the quantification of decursinol angelate and decursin in Angelica gigas using a CSH Fluoro-Phenyl column (2.1 mm × 150 mm, 1.7 μm) with a run time of 4 min. The method had an improved resolution and a shorter analysis time in comparison to the conventional high-performance liquid chromatography method. This method was validated in terms of linearity, precision, and accuracy. The limits of detection were 0.005 and 0.004 μg/mL for decursinol angelate and decursin, respectively, while the limits of quantitation were 0.014 and 0.012 μg/mL, respectively. The two components showed good regression (correlation coefficient (r 2 ) > 0.999), excellent precision (RSD < 2.28%), and acceptable recoveries (99.75-102.62%). The proposed method can be used to efficiently separate, characterize, and quantify decursinol angelate and decursin in Angelica gigas and its related medicinal materials or preparations, with the advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electron capture in collisions of S4+ with helium

NASA Astrophysics Data System (ADS)

Wang, J. G.; Turner, A. R.; Cooper, D. L.; Schultz, D. R.; Rakovic, M. J.; Fritsch, W.; Stancil, P. C.; Zygelman, B.

2002-07-01

Charge transfer due to collisions of ground-state S4+(3s2 1S) ions with helium is investigated for energies between 0.1 meV u-1 and 10 MeV u-1. Total and state-selective single electron capture (SEC) cross sections and rate coefficients are obtained utilizing the quantum mechanical molecular-orbital close-coupling (MOCC), atomic-orbital close-coupling (AOCC), classical trajectory Monte Carlo (CTMC) and continuum distorted wave methods. The MOCC calculations utilize ab initio adiabatic potentials and nonadiabatic radial coupling matrix elements obtained with the spin-coupled valence-bond approach. Previous data are limited to a calculation of the total SEC rate coefficient using the Landau-Zener model that is, in comparison to the results presented here, three orders of magnitude smaller. The MOCC SEC cross sections at low energy reveal a multichannel interference effect. True double capture is also investigated with the AOCC and CTMC approaches while autoionizing double capture and transfer ionization (TI) is explored with CTMC. SEC is found to be the dominant process except for E>200 keV u-1 when TI becomes the primary capture channel. Astrophysical implications are briefly discussed.

Investigating the Degradation Behaviors of a Therapeutic Monoclonal Antibody Associated with pH and Buffer Species.

PubMed

Zheng, Songyan; Qiu, Difei; Adams, Monica; Li, Jinjiang; Mantri, Rao V; Gandhi, Rajesh

2017-01-01

This study aimed in understanding the degradation behaviors of an IgG 1 subtype therapeutic monoclonal antibody A (mAb-A) associated with pH and buffer species. The information obtained in this study can augment conventional, stability-based screening paradigms by providing the direction necessary for efficient experimental design. Differential scanning calorimetry (DSC) was used for studying conformational stability. Dynamic light scattering (DLS) was utilized to generate B 22 *, a modified second virial coefficient for the character of protein-protein interaction. Size-exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were employed to separate degradation products. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for determining the molecular size and liquid chromatography mass spectrometry (LC-MS) were used for identifying the sequence of the separated fragments. The results showed that both pH and buffer species played the roles in controlling the degradation behaviors of mAb-A, but the pH was more significant. In particular, pH 4.5 induced additional thermal transition peaks occurring at a low temperature compared with pH 6.5. A continual temperature-stress study illustrated that the additional thermal transition peaks related to the least stable structure and a greater fragmentation. Although mAb-A showed the comparable conformational structures and an identical amount of aggregates at time zero between the different types of buffer species at pH 6.5, the aggregation formation rate showed a buffer species-dependent discrepancy over a temperature-stress period. It was found that the levels of aggregations associated with the magnitudes of protein-protein interaction forces.

Spectroscopic planetary detection

NASA Technical Reports Server (NTRS)

Deming, Drake

1991-01-01

One of the most promising methods for the detection of extra-solar planets is the spectroscopic method, where a small Doppler shift (approx. 10 meter/sec) in the spectrum of the parent star reveals the presence of planetary companions. However, solar type stars may show spurious Doppler shifts due to surface activity. If these effects are periodic, as is the solar activity cycle, then they may masquerade as planetary companions. The goal of this study was to determine whether the solar cycle affects the Doppler stability of integrated sunlight. Observations of integrated sunlight were made in the near infrared (approx. 2 micron), using the Kitt Peak McMath Fourier transform spectrometer, with a N2O gas absorption cell for calibration. An accuracy of approx. 5 meter/sec was achieved.

75 FR 74743 - Notice of Proposed Withdrawal Extension, Corrections to Existing Withdrawal, and Opportunity for...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-01

.... 11 to 14, inclusive; Sec. 15, NE\\1/4\\, W\\1/2\\NW\\1/4\\, NW\\1/4\\SW\\1/4\\, and E\\1/2\\SE\\1/ 4\\; Sec. 19...\\; Secs. 23 to 34, inclusive; Sec. 35, NE\\1/4\\, NW\\1/4\\NW\\1/4\\, S\\1/2\\NW\\1/4\\, and S\\1/2\\; Sec. 36. T. 46 N., R. 22 E., Secs. 1 to 5, inclusive; Sec. 6, lots 1 and 2, S\\1/2\\NE\\1/4\\, E\\1/2\\SW\\1/4\\, and SE\\1...

Assessment of right liver graft perfusion effectiveness between one and two-catheter infusion methods.

PubMed

Jung, Bo-Hyun; Hwang, Shin; Ha, Tae-Yong; Song, Gi-Won; Jung, Dong-Hwan; Kim, Ki-Hun; Ahn, Chul-Soo; Moon, Deok-Bog; Park, Gil-Chun; Kang, Sung-Hwa; Yoon, Young-In; Lee, Sung-Gyu

2014-05-01

Conventional graft perfusion method using one small-caliber catheter takes a relatively long time for right liver graft perfusion, thus some modification is needed. In this study, we intended to assess the effectiveness of right liver graft perfusion methods through comparison of different infusion catheters. The study consisted of two parts including one bench experiment to obtain data of hydraulic infusion and one clinical trial of 40 cases on graft perfusion with one- versus two-catheter infusion methods. These two graft infusion methods were compared in terms of the perfusion time and washing-out efficiency. At bench experiment, the infusion flow rate and infusion pressure were 3.3 ml/sec and 1.9 cmH20 in one blood transfusion catheter group, and 11.7 ml/sec and 3.1 cmH20 in single transurethral resection of prostate irrigation catheter group, and 6.6 ml/sec and 2.0 cmH20 in two blood transfusion catheters group, respectively. In clinical trial with 40 right liver grafts, two-catheter group had a shorter graft portal perfusion time for the first 2 L of histidine-tryptophan-ketoglutarate (HTK) solution than the conventional one-catheter group (375±25 seconds vs. 662±34 seconds; p=0.001) and a lower rate of incomplete blood washing-out after the initial 2 L portal perfusion (40% vs. 85%; p=0.03). The two-catheter infusion method appears to be more effective than the conventional one-catheter infusion method for right liver graft perfusion at the back table. Large size of right liver grafts seems to be its good indication.

Comparison of DMSP and SECS region-1 and region-2 ionospheric current boundary

NASA Astrophysics Data System (ADS)

Weygand, J. M.; Wing, S.

2016-06-01

The region-1 and region-2 boundary has traditionally been identified using data from a single spacecraft crossing the auroral region and measuring the large scale changes in the cross track magnetic field. With data from the AUTUMN, CANMOS, CARISMA, GIMA, DTU MGS, MACCS, McMAC, STEP, THEMIS, and USGS ground magnetometer arrays we applied a state-of-art technique based on spherical elementary current system (SECS) method developed by Amm and Viljanen (1999) in order to calculate maps of region-1 and region-2 current system over the North American and Greenland auroral region. Spherical elementary current (SEC) amplitude (proxy for vertical currents) maps can be inferred at 10 s temporal resolution, ~1.5° geographic latitude (Glat), and 3.5° geographic longitude (Glon) spatial resolution. We compare the location of the region-1 and region-2 boundary obtained by the DMSP spacecraft with the region-1 and region-2 boundary observed in the SEC current amplitudes. We find that the boundaries typically agree within 0.2°±1.3°. These results indicate that the location of the region-1 and region-2 boundary can reasonably be determined from ground magnetometer data. The SECS maps represent a value-added product from the magnetometer database and can be used for contextual interpretation in conjunction with other missions as well as help with our understanding of magnetosphere-ionosphere coupling mechanisms using the ground arrays and the magnetospheric spacecraft data.

Comparison of DMSP and SECS region-1 and region-2 ionospheric current boundary✩

PubMed Central

Weygand, J.M.; Wing, S.

2017-01-01

The region-1 and region-2 boundary has traditionally been identified using data from a single spacecraft crossing the auroral region and measuring the large scale changes in the cross track magnetic field. With data from the AUTUMN, CANMOS, CARISMA, GIMA, DTU MGS, MACCS, McMAC, STEP, THEMIS, and USGS ground magnetometer arrays we applied a state-of-art technique based on spherical elementary current system (SECS) method developed by Amm and Viljanen (1999) in order to calculate maps of region-1 and region-2 current system over the North American and Greenland auroral region. Spherical elementary current (SEC) amplitude (proxy for vertical currents) maps can be inferred at 10 s temporal resolution, ~1.5° geographic latitude (Glat), and 3.5° geographic longitude (Glon) spatial resolution. We compare the location of the region-1 and region-2 boundary obtained by the DMSP spacecraft with the region-1 and region-2 boundary observed in the SEC current amplitudes. We find that the boundaries typically agree within 0.2° ± 1.3°. These results indicate that the location of the region-1 and region-2 boundary can reasonably be determined from ground magnetometer data. The SECS maps represent a value-added product from the magnetometer database and can be used for contextual interpretation in conjunction with other missions as well as help with our understanding of magnetosphere-ionosphere coupling mechanisms using the ground arrays and the magnetospheric spacecraft data. PMID:29056861

Comparison of DMSP and SECS region-1 and region-2 ionospheric current boundary.

PubMed

Weygand, J M; Wing, S

2016-06-01

The region-1 and region-2 boundary has traditionally been identified using data from a single spacecraft crossing the auroral region and measuring the large scale changes in the cross track magnetic field. With data from the AUTUMN, CANMOS, CARISMA, GIMA, DTU MGS, MACCS, McMAC, STEP, THEMIS, and USGS ground magnetometer arrays we applied a state-of-art technique based on spherical elementary current system (SECS) method developed by Amm and Viljanen (1999) in order to calculate maps of region-1 and region-2 current system over the North American and Greenland auroral region. Spherical elementary current (SEC) amplitude (proxy for vertical currents) maps can be inferred at 10 s temporal resolution, ~1.5° geographic latitude (Glat), and 3.5° geographic longitude (Glon) spatial resolution. We compare the location of the region-1 and region-2 boundary obtained by the DMSP spacecraft with the region-1 and region-2 boundary observed in the SEC current amplitudes. We find that the boundaries typically agree within 0.2° ± 1.3°. These results indicate that the location of the region-1 and region-2 boundary can reasonably be determined from ground magnetometer data. The SECS maps represent a value-added product from the magnetometer database and can be used for contextual interpretation in conjunction with other missions as well as help with our understanding of magnetosphere-ionosphere coupling mechanisms using the ground arrays and the magnetospheric spacecraft data.

Aortic PWV in Chronic Kidney Disease: A CRIC Ancillary Study

PubMed Central

Townsend, Raymond R.; Wimmer, Neil J.; Chirinos, Julio A.; Parsa, Afshin; Weir, Matthew; Perumal, Kalyani; Lash, James P.; Chen, Jing; Steigerwalt, Susan P.; Flack, John; Go, Alan S.; Rafey, Mohammed; Rahman, Mahboob; Sheridan, Angela; Gadegbeku, Crystal A.; Robinson, Nancy A.; Joffe, Marshall

2009-01-01

Background Aortic PWV is a measure of arterial stiffness and has proved useful in predicting cardiovascular morbidity and mortality in several populations of patients, including the healthy elderly, hypertensives and those with end stage renal disease receiving hemodialysis. Little data exist characterizing aortic stiffness in patients with chronic kidney disease who are not receiving dialysis, and in particular the effect of reduced kidney function on aortic PWV. Methods We performed measurements of aortic PWV in a cross-sectional cohort of participants enrolled in the Chronic Renal Insufficiency Cohort (CRIC) study to determine factors which predict increased aortic PWV in chronic kidney disease. Results PWV measurements were obtained in 2564 participants. The tertiles of aortic PWV (adjusted for waist circumference) were < 7.7 m/sec, 7.7–10.2 m/sec and > 10.2 m/sec with an overall mean (± S.D.) value of 9.48 ± 3.03 m/sec [95% CI = 9.35–9.61 m/sec]. Multivariable regression identified significant independent positive associations of age, blood glucose concentrations, race, waist circumference, mean arterial blood pressure, gender, and presence of diabetes with aortic PWV and a significant negative association with the level of kidney function. Conclusions The large size of this unique cohort, and the targeted enrollment of chronic kidney disease participants provides an ideal situation to study the role of reduced kidney function as a determinant of arterial stiffness. Arterial stiffness may be a significant component of the enhanced cardiovascular risk associated with kidney failure. PMID:20019670

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80.

PubMed

Login, G R; Aoki, M; Yamakawa, M; Lunardi, L O; Digenis, E C; Tanda, N; Schwartz, L B; Dvorak, A M

1997-10-01

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.

The Effect of Al2O3 Addition on the Thermal Diffusivity of Heat Activated Acrylic Resin

PubMed Central

Atla, Jyothi; Manne, Prakash; Gopinadh, A.; Sampath, Anche; Muvva, Suresh Babu; Kishore, Krishna; Sandeep, Chiramana; Chittamsetty, Harika

2013-01-01

Aim: This study aimed at investigating the effect of adding 5% to 20% by weight aluminium oxide powder (Al2O3) on thermal diffusivity of heat–polymerized acrylic resin. Material and Methods: Twenty five cylindrical test specimens with an embedded thermocouple were used to determine thermal diffusivity over a physiologic temperature range (0 to 70°C). The specimens were divided into five groups (5 specimens/group) which were coded A to E. Group A was the control group (unmodified acrylic resin specimens). The specimens of the remaining four groups were reinforced with 5%, 10%, 15%, and 20% Al2O3 by weight. Results were analysed by using one–way analysis of variance (ANOVA). Results: Test specimens which belonged to Group E showed the highest mean thermal diffusivity value of 10.7mm2/sec, followed by D (9.09mm2/sec), C (8.49mm2/sec), B(8.28mm2/sec) and A(6.48mm2/sec) groups respectively. Thermal diffusivities of the reinforced acrylic resins were found to be significantly higher than that of the unmodified acrylic resin. Thermal diffusivity was found to increase in proportion to the weight percentage of alumina filler. Conclusion: Al2O3 fillers have potential to provide increased thermal diffusivity. Increasing the heat transfer characteristics of the acrylic resin base material could lead to more patient satisfaction. PMID:24086917

Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

PubMed Central

Musial-Siwek, Monika; Rusch, Sharyn L.; Kendall, Debra A.

2007-01-01

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the Signal Peptide Binding Groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53-residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acids 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA. PMID:17084862

Simultaneous determination of niacin and pyridoxine at trace levels by using diode array high-performance liquid chromatography and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry.

PubMed

Sel, Sabriye; Öztürk Er, Elif; Bakırdere, Sezgin

2017-12-01

A highly sensitive and simple diode-array high-performance liquid chromatography and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry method was developed for the simultaneous determination of niacin and pyridoxine in pharmaceutical drugs, tap water, and wastewater samples. To determine the in vivo behavior of niacin and pyridoxine, analytes were subjected to simulated gastric conditions. The calibration plots of the diode-array high-performance liquid chromatography and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry method showed good linearity over a wide concentration range with close to 1.0 correlation coefficients for both analytes. The limit of detection/limit of quantitation values for liquid chromatography quadrupole time-of-flight tandem mass spectrometry analysis were 1.98/6.59 and 1.3/4.4 μg/L for niacin and pyridoxine, respectively, while limit of detection/limit of quantitation values for niacin and pyridoxine in high-performance liquid chromatography analysis were 3.7/12.3 and 5.7/18.9 μg/L, respectively. Recovery studies were also performed to show the applicability of the developed methods, and percentage recovery values were found to be 90-105% in tap water and 94-97% in wastewater for both analytes. The method was also successfully applied for the qualitative and quantitative determination of niacin and pyridoxine in drug samples. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphatidic Acid Sequesters Sec18p from cis-SNARE Complexes to Inhibit Priming.

PubMed

Starr, Matthew L; Hurst, Logan R; Fratti, Rutilio A

2016-10-01

Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/N-ethylmaleimide sensitive factor and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane-associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA-rich membrane microdomains before transferring to cis-SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA-binding domain blocked Sec18p binding to PA-liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis-SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis-SNAREs and complementation with plasmid-encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum

PubMed Central

1985-01-01

SEC53, a gene that is required for completion of assembly of proteins in the endoplasmic reticulum in yeast, has been cloned, sequenced, and the product localized by cell fractionation. Complementation of a sec53 mutation is achieved with unique plasmids from genomic or cDNA expression banks. These inserts contain the authentic gene, a cloned copy of which integrates at the sec53 locus. An open reading frame in the insert predicts a 29-kD protein with no significant hydrophobic character. This prediction is confirmed by detection of a 28-kD protein overproduced in cells that carry SEC53 on a multicopy plasmid. To follow Sec53p more directly, a LacZ-SEC53 gene fusion has been constructed which allows the isolation of a hybrid protein for use in production of antibody. With such an antibody, quantitative immune decoration has shown that the sec53-6 mutation decreases the level of Sec53p at 37 degrees C, while levels comparable to wild-type are seen at 24 degrees C. An eightfold overproduction of Sec53p accompanies transformation of cells with a multicopy plasmid containing SEC53. Cell fractionation, performed with conditions that preserve the lumenal content of the endoplasmic reticulum (ER), shows Sec53p highly enriched in the cytosol fraction. We suggest that Sec53p acts indirectly to facilitate assembly in the ER, possibly by interacting with a stable ER component, or by providing a small molecule, other than an oligosaccharide precursor, necessary for the assembly event. PMID:3905826

10 CFR Appendix A to Subpart A of... - Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec...

Code of Federal Regulations, 2013 CFR

2013-01-01

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Code of Federal Regulations, 2014 CFR

2014-01-01

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Code of Federal Regulations, 2012 CFR

2012-01-01

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Code of Federal Regulations, 2011 CFR

2011-01-01

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Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141 (42 U.S.C. 2161), Sec. 145 (42 U.S.C. 2165), Sec. 161 (42 U.S.C. 2201) A Appendix A to... to Subpart A of Part 710—Selected Provisions of the Atomic Energy Act of 1954, as Amended, Sec. 141...

Quality evaluation of moluodan concentrated pill using high-performance liquid chromatography fingerprinting coupled with chemometrics.

PubMed

Tao, Lingyan; Zhang, Qing; Wu, Yongjiang; Liu, Xuesong

2016-12-01

In this study, a fast and effective high-performance liquid chromatography method was developed to obtain a fingerprint chromatogram and quantitative analysis simultaneously of four indexes including gallic acid, chlorogenic acid, albiflorin and paeoniflorin of the traditional Chinese medicine Moluodan Concentrated Pill. The method was performed by using a Waters X-bridge C 18 reversed phase column on an Agilent 1200S high-performance liquid chromatography system coupled with diode array detection. The mobile phase of the high-performance liquid chromatography method was composed of 20 mmol/L phosphate solution and acetonitrile with a 1 mL/min eluent velocity, under a detection temperature of 30°C and a UV detection wavelength of 254 nm. After the methodology validation, 16 batches of Moluodan Concentrated Pill were analyzed by this high-performance liquid chromatography method and both qualitative and quantitative evaluation results were achieved by similarity analysis, principal component analysis and hierarchical cluster analysis. The results of these three chemometrics were in good agreement and all indicated that batch 10 and batch 16 showed significant differences with the other 14 batches. This suggested that the developed high-performance liquid chromatography method could be applied in the quality evaluation of Moluodan Concentrated Pill. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; determination of organochlorine pesticides and polychlorinated biphenyls in bottom sediment by dual capillary-column gas chromatography with electron-capture detection

USGS Publications Warehouse

Foreman, William T.; Connor, Brooke F.; Furlong, Edward T.; Vaught, Deborah G.; Merten, Leslie M.

1995-01-01

A method for the determination of 30 individual organochlorine pesticides, total toxaphene, and total polychlorinated biphenyls (PCBs) in bottom sediment is described. The method isolates the pesticides and PCBs by solvent extraction with dichlorobenzene, removes inorganic sulfur, large naturally occurring molecules, and other unwanted interferences by gel permeation chromatography, and further cleans up and class fractionates the extract using adsorption chromatography. The com- pounds then are instrumentally determined using dual capillary-column gas chromatography with electron-capture detection. Reporting limits range from 1 to 5 micrograms per kilogram for 30 individual pesticides, 50 micrograms per kilogram for total PCBs, and 200 micrograms per kilogram for total toxaphene. The method also is designed to allow the simultaneous isolation of 79 other semivolatile organic compounds from the sediment, which are separately quantified using gas chromatography with mass spectrometric detection. The method was developed in support of the U.S. Geological Survey's National Water-Quality Assessment program.

Determination of phytate in high molecular weight, charged organic matrices by two-dimensional size exclusion-ion chromatography

USDA-ARS?s Scientific Manuscript database

A two-dimensional chromatography method for analyzing anionic targets (specifically phytate) in complex matrices is described. Prior to quantification by anion exchange chromatography, the sample matrix was prepared by size exclusion chromatography, which removed the majority of matrix complexities....

Nature of alpha and beta particles in glycogen using molecular size distributions.

PubMed

Sullivan, Mitchell A; Vilaplana, Francisco; Cave, Richard A; Stapleton, David; Gray-Weale, Angus A; Gilbert, Robert G

2010-04-12

Glycogen is a randomly hyperbranched glucose polymer. Complex branched polymers have two structural levels: individual branches and the way these branches are linked. Liver glycogen has a third level: supramolecular clusters of beta particles which form larger clusters of alpha particles. Size distributions of native glycogen were characterized using size exclusion chromatography (SEC) to find the number and weight distributions and the size dependences of the number- and weight-average masses. These were fitted to two distinct randomly joined reference structures, constructed by random attachment of individual branches and as random aggregates of beta particles. The z-average size of the alpha particles in dimethylsulfoxide does not change significantly with high concentrations of LiBr, a solvent system that would disrupt hydrogen bonding. These data reveal that the beta particles are covalently bonded to form alpha particles through a hitherto unsuspected enzyme process, operative in the liver on particles above a certain size range.

Liquid chromatographic analysis of a formulated ester from a gas-turbine engine test

NASA Technical Reports Server (NTRS)

Jones, W. R., Jr.; Morales, W.

1983-01-01

Size exclusion chromatography (SEC) utilizing mu-Bondagel and mu-Styragel columns with a tetrahydrofuran mobile phase was used to determine the chemical degradation of lubricant samples from a gas-turbine engine test. A MIL-L-27502 candidate, ester-based lubricant was run in a J57-29 engine at a bulk oil temperature of 216 C. In general, the analyses indicated a progressive loss of primary ester, additive depletion, and formation of higher molecular weight material. An oil sample taken at the conclusion of the test showed a reversal of this trend because of large additions of new oil. The high-molecular-weight product from the degraded ester absorbed strongly in the ultraviolet region at 254 nanometers. This would indicate the presence of chromophoric groups. An analysis of a similar ester lubricant from a separate high-temperature bearing test yielded qualitatively similar results.

Balancing size exclusion and adsorption of polymers in nanopores

NASA Astrophysics Data System (ADS)

Kim, Won; Ryu, Chang Y.

2006-03-01

The liquid chromatography at critical condition (LCCC) presents the condition, at which the size exclusion and adsorption of polymer chains are balanced upon interactions with nanoporous substrates. In this study, we investigate how the polymer interactions with nanopores are affected by the solvent quality and nanopore size. Specifically, we measure the retention times of monodisperse polystyrenes in C18-bonded nanoporous silica column as a function of molecular weight, when a mixed solvent of methylene chloride and acetonitrile are used as elutent. C18-bonded silica particles with 70, 100, and 250 A pore size are used as a stationary phase to study how the transition from SEC-like to IC-like retention behavior depends on the condition of temperature and solvent composition. To locate the LCCC at various nanopore sizes, the temperature and solvent composition have been varied from 0 to 60 C and from 51 to 62 v/v% of methylene chloride, respectively.

Chromatographic resolution of a salt into its parent acid and base constituents.

PubMed

Davankov, Vadim; Tsyurupa, Maria

2006-12-08

Based on the results of the earlier proposed process of separation of mixtures of mineral electrolytes by size-exclusion chromatography (SEC), it has been suggested that a mineral salt must spontaneously resolve, at least partially, into its parent acid and base constituents, provided that the separating media discriminates the anion and cation of the salt according to their size. Indeed, migration of a zone of an aqueous salt solution through a bed of neutral nanoporous hypercrosslinked polystyrene-type packing was shown to result in the generation of acidic and alkaline effluent fractions. The principle of spontaneous salt resolution has been extended to other types of discriminating interactions between the stationary phase and the two ions of the salt. The idea was exemplified by the resolution of ammonium acetate, due to hydrophobic retention of the acetate, into fractions enriched in ammoniac and then acetic acid.

Moringa oleifera Lam.: Protease activity against blood coagulation cascade

PubMed Central

Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

2012-01-01

Background: The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Materials and Methods: Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Results: Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. Conclusions: These findings support the traditional usage of M. oleifera extracts for wound healing. PMID:22224061

Lipidomic analysis of biological samples: Comparison of liquid chromatography, supercritical fluid chromatography and direct infusion mass spectrometry methods.

PubMed

Lísa, Miroslav; Cífková, Eva; Khalikova, Maria; Ovčačíková, Magdaléna; Holčapek, Michal

2017-11-24

Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography - mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemical Synthesis Coxiella Burnetti Lipopolysaccharides: Structural Studies of Coxiella Burnetti Lipopolysaccharides.

DTIC Science & Technology

1987-11-30

currently evaluating two instrumental techniques which seem highly appropriate to this LPS project, supercritical fluid chromatography (SFC) and...NEW INSTRUMENTAL TECHNIQUES AND METHODS OF APPROACH 1. Supercritical Fluid Chromatography (SFC) ............... 6. 2. SFC and Mass Spectrometry...details are discussed below in the appropriate sections. B. NEW INSTRUMENTAL TECHNIQUES AND METHODS OF APPROACH 1. Supercritical Fluid Chromatography (SFC

Homestead Valley, California, aftershocks (March 17-18, 1979) recorded on portable seismographs

USGS Publications Warehouse

Perry-Huston, Sue; Eberhart-Phillips, Donna

1994-01-01

A method for the determination of chlorinated organic compounds in aquatic tissue by dual capillary-column gas chromatography with electron- capture detection is described. Whole-body-fish or corbicula tissue is homogenized, Soxhlet extracted, lipid removed by gel permeation chromatography, and fractionated using alumina/silica adsorption chromatography. The extracts are analyzed by dissimilar capillary-column gas chromatography with electron-capture detection. The method reporting limits are 5 micrograms per kilogram (ug/kg) for chlorinated compounds, 50 ug/kg for polychlorinated biphenyls, and 200 ug/kg for toxaphene.

Using chromatography – desorption method of manufacturing gas mixtures for analytical instruments calibration

NASA Astrophysics Data System (ADS)

Platonov, I. A.; Kolesnichenko, I. N.; Lange, P. K.

2018-05-01

In this paper, the chromatography desorption method of obtaining gas mixtures of known compositions stable for a time sufficient to calibrate analytical instruments is considered. The comparative analysis results of the preparation accuracy of gas mixtures with volatile organic compounds using diffusion, polyabarbotage and chromatography desorption methods are presented. It is shown that the application of chromatography desorption devices allows one to obtain gas mixtures that are stable for 10...60 hours in a dynamic condition. These gas mixtures contain volatile aliphatic and aromatic hydrocarbons with a concentration error of no more than 7%. It is shown that it is expedient to use such gas mixtures for analytical instruments calibration (chromatographs, spectrophotometers, etc.)

Characterization of experimentally induced, nonaflatoxigenic variant strains of Aspergillus parasiticus.

PubMed Central

Kale, S P; Cary, J W; Bhatnagar, D; Bennett, J W

1996-01-01

Six previously isolated, nonaflatoxigenic variants of Aspergillus parasiticus, designated sec mutants, were characterized morphologically by electron microscopy, biochemically by biotransformation studies with an aflatoxin precursor, and genetically by Northern (RNA) hybridization analysis of aflatoxin biosynthetic gene transcripts. Scanning electron micrographs clearly demonstrated that compared with the parental sec+ forms, the variant sec forms had an abundance of vegetative mycelia, orders of magnitude reduced number of conidiophores and conidia, and abnormal metulae. Conidiospores were detected in sec cultures only at higher magnifications (x 500), in contrast to the sec+ (wild-type) strain, in which abundant conidiospores (masking the vegetative mycelia) were observed at even lower magnifications (x 300). All sec+ forms, but none of the sec forms, showed bioconversion of sterigmatocystin to aflatoxins. Northern blots probed with pathway genes demonstrated lack of expression of both the aflatoxin biosynthetic pathway structural (nor-1 and omtA) and regulatory (aflR) genes in the sec forms; PCR and Southern hybridization analysis confirmed the presence of the genes in the sec genomes. Thus, the loss of aflatoxigenic capabilities in the sec form is correlated with alterations in the conidial morphology of the fungus, suggesting that the regulation of aflatoxin synthesis and conidiogenesis may be interlinked. PMID:8795232

Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp.

PubMed

Loh, Eva; Hong, Wanjin

2002-06-14

Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus.

Selenocysteine in proteins-properties and biotechnological use.

PubMed

Johansson, Linda; Gafvelin, Guro; Arnér, Elias S J

2005-10-30

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

One year quality of life measured with SEC-QoL in levonorgestrel 52 mg IUS users.

PubMed

Cristobal, Ignacio; Lete, Luis Ignacio; Viuda, Esther de la; Perulero, Nuria; Arbat, Agnes; Canals, Ignasi

2016-04-01

The present study aims to prospectively evaluate quality of life (QoL) of women using 52-mg levonorgestrel intrauterine system (LNG-IUS) for contraception determined through the Sociedad Española de Contracepción (Spanish contraception Society) (SEC)-QoL, a questionnaire specifically designed to assess the impact of contraceptive methods on QoL of fertile women. We conducted a prospective observational multicenter study of 201 reproductive age women who initiated the LNG-IUS for contraception. Sociodemographic and clinical data were collected at baseline and 12 months afterwards. Participants filled in the SEC-QoL questionnaire at both visits. SEQ-QoL scores range from 0 (worst QoL) to 100 (best QoL). Participants claimed an increased QoL 12 months after insertion in all five dimensions of SEC-QoL due to its high contraceptive efficacy and its capability to reduce other menstrual symptoms (e.g., heavy menstrual bleeding or dysmenorrhoea), overall exerting a positive impact on user's satisfaction. SEC-QoL general overall score went from a mean (S.D.) score of 46.3 (17.3) at baseline to 72.2 (14.8) 12 months afterwards (p<.001). Overall, 94.6% of women claimed having found additional benefits other than contraception. No pregnancies were reported during the 12 months of study duration, and only 14 women discontinued use of LNG-IUS (only two of them due to an adverse event), representing a continuation rate of 93%. Women using LNG-IUS for contraception have an increased QoL after 12 months of use, demonstrated by the increased score in all dimensions of the SEC-QoL questionnaire. The present study prospectively evaluated QoL of women using LNG-IUS for contraception through the SEC-QoL questionnaire. Participants claimed increased QoL 12 months afterwards, implying that women using LNG-IUS for contraception in usual clinical practise also benefit from the reduction of period-related symptoms, overall leading to very low discontinuation rates. Copyright © 2016 Elsevier Inc. All rights reserved.

A screen for over-secretion of proteins by yeast based on a dual component cellular phosphatase and immuno-chromogenic stain for exported bacterial alkaline phosphatase reporter

PubMed Central

2013-01-01

Background To isolate over-secretors, we subjected to saturation mutagenesis, a strain of P.pastoris exporting E. coli alkaline phosphatase (EAP) fused to the secretory domain of the yeast α factor pheromone through cellular PHO1/KEX2 secretory processing signals as the α-sec-EAP reporter protein. Direct chromogenic staining for α-sec-EAP activity is non-specific as its NBT/BCIP substrate cross-reacts with cellular phosphatases which can be inhibited with Levulinic acid. However, the parental E(P) strain only exports detectable levels of α-sec-EAP at 69 hours and not within the 36 hour period post-seeding required for effective screening with the consequent absence of a reference for secretion. We substituted the endogenous cellular phosphatase activity as a comparative reference for secretion rate and levels as well as for colony alignment while elevating specificity and sensitivity of detection of the exported protein with other innovative modifications of the immuno-chromogenic staining application for screening protein export mutants. Results Raising the specificity and utility of staining for α-sec-EAP activity required 5 modifications including some to published methods. These included, exploitation of endogenous phosphatase activity, reduction of the cell/protein burden, establishment of the direct relation between concentrations of transcriptional inducer and exported membrane immobilized protein and concentrations of protein exported into growth media, amplification of immuno-specificity and sensitivity of detection of α-sec-EAP reporter enzyme signal and restriction of staining to optimal concentrations of antisera and time periods. The resultant immuno-chromogenic screen allows for the detection of early secretion and as little as 1.3 fold over-secretion of α-sec-EAP reporter protein by E(M) mutants in the presence of 10 fold -216 fold higher concentrations of HSA. Conclusions The modified immuno-chromogenic screen is sensitive, specific and has led to the isolation of mutants E(M) over-secreting the α-sec-EAP reporter protein by a minimum of 50 fold higher levels than that exported by non-mutagenized E(P) parental strains. Unselected proteins were also over-secreted. PMID:23602005

77 FR 8143 - Section 482; Methods To Determine Taxable Income in Connection With a Cost Sharing Arrangement...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-14

... 482; Methods To Determine Taxable Income in Connection With a Cost Sharing Arrangement; Correction... on Thursday, December 22, 2011 (76 FR 80082), relating to section 482 and methods to determine... revisions read as follows: Sec. 1.482-7 Methods to determine taxable income in connection with a cost...

Improvements To Micro Contact Performance And Reliability

DTIC Science & Technology

2016-12-22

BAKE : □ 1 min 110°C hot plate bake OPTIONAL - For Grayscale Lithography: 1818 COAT: □ Flood wafer with 1818 □ 4 sec spread at 500 rpm...30 sec spin at 4,000 rpm, ramp=200 □ 75 sec 110°C hot plate bake GRAYSCALE PATTERNING: □ Follow grayscale patterning process for patterns...8217 □ 2 min 200°C hot plate bake 1818 COAT: □ Flood wafer with 1818 □ 4 sec spread at 500 rpm □ 30 sec spin at 4,000 rpm, ramp=200 □ 75 sec 110

Application of Solid Phase Microextraction Coupled with Gas Chromatography/Mass Spectrometry as a Rapid Method for Field Sampling and Analysis of Chemical Warfare Agents and Toxic Industrial Chemicals

DTIC Science & Technology

2003-01-01

PHASE MICROEXTRACTION COUPLED WITH GAS CHROMATOGRAPHY/MASS SPECTROMETRY AS A RAPID METHOD FOR FIELD SAMPLING AND ANALYSIS OF CHEMICAL WARFARE AGENTS...SAMPLING AND ANALYSIS OF CHEMICAL WARFARE AGENTS AND TOXIC INDUSTRIAL CHEMICALS 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6...GAS CHROMATOGRAPHY/MASS SPECTROMETRY AS A RAPID METHOD FOR FIELD SAMPLING AND ANALYSIS OF CHEMICAL WARFARE AGENTS AND TOXIC INDUSTRIAL CHEMICALS

In Vitro and in Vivo Evaluation of Native Glucagon and Glucagon Analog (MAR-D28) during Aging: Lack of Cytotoxicity and Preservation of Hyperglycemic Effect

PubMed Central

Ward, W Kenneth; Massoud, Ryan G; Szybala, Cory J; Engle, Julia M; El Youssef, Joseph; Carroll, Julie M; Roberts, Charles T; DiMarchi, Richard D

2010-01-01

Background For automated prevention of hypoglycemia, there is a need for glucagon (or an analog) to be sufficiently stable so that it can be indwelled in a portable pump for at least 3 days. However, under some conditions, solutions of glucagon can form amyloid fibrils. Currently, the usage instructions for commercially available glucagon allow only for its immediate use. Methods In NIH 3T3 fibroblasts, we tested amyloid formation and cytotoxicity of solutions of native glucagon and the glucagon analog MAR-D28 after aging under different conditions for 5 days. In addition, aged native glucagon was subjected to size-exclusion chromatography (SEC). We also studied whether subcutaneous aged Novo Nordisk GlucaGen® would have normal bioactivity in octreotide-treated, anesthetized, nondiabetic pigs. Results We found no evidence of cytotoxicity from native glucagon or MAR-D28 (up to 2.5 mg/ml) at a pH of 10 in a glycine solvent. We found a mild cytotoxicity for both compounds in Tris buffer at pH 8.5. A high concentration of the commercial glucagon preparation (GlucaGen) caused marked cytotoxicity, but low pH and/or a high osmolarity probably accounted primarily for this effect. With SEC, the decline in monomeric glucagon over time was much lower when aged in glycine (pH 10) than when aged in Tris (pH 8.5) or in citrate (pH 3). Congo red staining for amyloid was very low with the glycine preparation (pH 10). In the pig studies, the hyperglycemic effect of commercially available glucagon was preserved despite aging conditions associated with marked amyloid formation. Conclusions Under certain conditions, aqueous solutions of glucagon and MAR-D28 are stable for at least 5 days and are thus very likely to be safe in mammals. Glycine buffer at a pH of 10 appears to be optimal for avoiding cytotoxicity and amyloid fibril formation. PMID:21129325

Potential air contamination during CO2 angiography using a hand-held syringe: theoretical considerations and gas chromatography.

PubMed

Cho, David R; Cho, Kyung J; Hawkins, Irvin F

2006-01-01

To assess air contamination in the hand-held syringes currently used for CO2 delivery and to determine whether there is an association between their position and the rate of air contamination. Assessment of air contamination in the syringe (20 ml) included theoretical modeling, mathematical calculation, and gas chromatography (GC). The model was used with Fick's first law to calculate the diffusion of CO2 and the amount of air contamination. For GC studies, the syringes were placed in the upright, horizontal, and inverted positions and gas samples were obtained after 5, 10, 20, 30, and 60 min. All trials with each position for each sampling time were performed five times. The amounts of air contamination with time calculated mathematically were 5-10% less than those of GC. With the diffusivity of air-CO2 at 0.1599 cm2/sec (9.594 cm2/min), air contamination was calculated to be 60% at 60 min. With GC air contamination was 13% at 5 min, 31% at 20 min, 43% at 30 min, and 68% at 60 min. There was no difference in air contamination between the different syringe positions. Air contamination occurs in hand-held syringes filled with CO2 when they are open to the ambient air. The amounts of air contamination over time are similar among syringes placed in the upright, horizontal, and inverted positions.

Human Growth Hormone Delivery with a Microneedle Transdermal System: Preclinical Formulation, Stability, Delivery and PK of Therapeutically Relevant Doses

PubMed Central

Ameri, Mahmoud; Kadkhodayan, Miryam; Nguyen, Joe; Bravo, Joseph A.; Su, Rebeca; Chan, Kenneth; Samiee, Ahmad; Daddona, Peter E.

2014-01-01

This study evaluated the feasibility of coating formulated recombinant human growth hormone (rhGH) on a titanium microneedle transdermal delivery system, Zosano Pharma (ZP)-hGH, and assessed preclinical patch delivery performance. Formulation rheology and surface activity were assessed by viscometry and contact angle measurement. rhGH liquid formulation was coated onto titanium microneedles by dip-coating and drying. The stability of coated rhGH was determined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Preclinical delivery and pharmacokinetic studies were conducted in female hairless guinea pigs (HGP) using rhGH coated microneedle patches at 0.5 and 1 mg doses and compared to Norditropin® a commercially approved rhGH subcutaneous injection. Studies demonstrated successful rhGH formulation development and coating on microneedle arrays. The ZP-hGH patches remained stable at 40 °C for six months with no significant change in % aggregates. Pharmacokinetic studies showed that the rhGH-coated microneedle patches, delivered with high efficiency and the doses delivered indicated linearity with average Tmax of 30 min. The absolute bioavailability of the microneedle rhGH patches was similar to subcutaneous Norditropin® injections. These results suggest that ZP-transdermal microneedle patch delivery of rhGH is feasible and may offer an effective and patient-friendly alternative to currently marketed rhGH injectables. PMID:24838219

29 CFR 5.1 - Purpose and scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

..., Health Statistics, and Medical Libraries Act of 1974 (sec. 107, see sec. 308(h)(2) thereof, 88 Stat. 370... 1965 (sec. 712; 79 Stat. 575 as amended; 42 U.S.C. 3222). 42. Juvenile Delinquency Prevention Act (sec...

Agonal sequences in a filmed suicidal hanging: analysis of respiratory and movement responses to asphyxia by hanging.

PubMed

Sauvageau, Anny; Racette, Stéphanie

2007-07-01

The forensic literature on the pathophysiology of human hanging is still limited. Therefore, forensic pathologists often feel uncomfortable when confronted with related questions. Here presented is the filmed suicidal hanging of a 37-year-old man. This recording allows a unique analysis of agonal movement sequences: loss of consciousness (13 sec), convulsions (15 sec), decortication rigidity (21 sec), decerebration rigidity (46 sec), second decortication rigidity (1 min 11 sec), loss of muscle tone, (1 min 38 sec) and last isolated muscle movement (4 min 10 sec). As for respiratory responses, very deep respiratory attempts started at 20 sec. Respiratory movements progressively decreased and completely stopped at 2 min. Despite the fact that extending the presented data on all cases of hanging asphyxia would be a mistake, this case gives a very interesting insight into movement and respiratory response to asphyxia by hanging.

Revisiting the human seminiferous epithelium cycle.

PubMed

Nihi, F; Gomes, M L M; Carvalho, F A R; Reis, A B; Martello, R; Melo, R C N; Almeida, F R C L; Chiarini-Garcia, H

2017-06-01

Can all types of testicular germ cells be accurately identified by microscopy techniques and unambiguously distributed in stages of the human seminiferous epithelium cycle (SEC)? By using a high-resolution light microscopy (HRLM) method, which enables an improved visualization of germ cell morphological features, we identified all testicular germ cells in the seminiferous epithelium and precisely grouped them in six well-delimitated SEC stages, thus providing a reliable reference source for staging in man. Morphological characterization of germ cells in human has been done decades ago with the use of conventional histological methods (formaldehyde-based fixative -Zenker-formal- and paraffin embedding). These early studies proposed a classification of the SEC in six stages. However, the use of stages as baseline for morphofunctional evaluations of testicular parenchyma has been difficult because of incomplete morphological identification of germ cells and their random distribution in the human SEC. Testicular tissue from adult and elderly donors with normal spermatogenesis according to Levin's, Johnsen's and Bergmann's scores were used to evaluate germ cell morphology and validate their distribution and frequency in stages throughout human spermatogenesis. Testicular tissue from patients diagnosed with congenital bilateral agenesis of vas deferens (n = 3 adults) or prostate cancer (n = 3 elderly) were fixed in glutaraldehyde and embedded in araldite epoxy resin. Morphological analyses were performed by both light and transmission electron microscopy. HRLM method enabled a reliable morphological identification of all germ cells (spermatogonia, spermatocytes and spermatids) based on high-resolution aspects of euchromatin, heterochromatin and nucleolus. Moreover, acrosomal development of spermatids was clearly revealed. Altogether, our data redefined the limits of each stage leading to a more reliable determination of the SEC in man. Occasionally, germ cells can be absent in some tubular sections. In this situation, it has to be taken into account the germ cell association proposed in the present study to classify the stages. Our findings bring a new focus on the morphology and development of germ cells during the SEC in human. Application of HRLM may be a valuable tool for research studies and clinical andrology helping to understand some testicular diseases and infertility conditions which remain unsolved. Experiments were partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors declare that there are no conflicts of interest. Not applicable. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Collaborative trial validation study of two methods, one based on high performance liquid chromatography-tandem mass spectrometry and on gas chromatography-mass spectrometry for the determination of acrylamide in bakery and potato products.

PubMed

Wenzl, Thomas; Karasek, Lubomir; Rosen, Johan; Hellenaes, Karl-Erik; Crews, Colin; Castle, Laurence; Anklam, Elke

2006-11-03

A European inter-laboratory study was conducted to validate two analytical procedures for the determination of acrylamide in bakery ware (crispbreads, biscuits) and potato products (chips), within a concentration range from about 20 microg/kg to about 9000 microgg/kg. The methods are based on gas chromatography-mass spectrometry (GC-MS) of the derivatised analyte and on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) of native acrylamide. Isotope dilution with isotopically labelled acrylamide was an integral part of both methods. The study was evaluated according to internationally accepted guidelines. The performance of the HPLC-MS/MS method was found to be superior to that of the GC-MS method and to be fit-for-the-purpose.

Enantioselective supercritical fluid chromatography-tandem mass spectrometry method for simultaneous estimation of risperidone and its 9-hydroxyl metabolites in rat plasma.

PubMed

Prasad, Thatipamula R; Joseph, Siji; Kole, Prashant; Kumar, Anoop; Subramanian, Murali; Rajagopalan, Sudha; Kr, Prabhakar

2017-11-01

Objective of the current work was to develop a 'green chemistry' compliant selective and sensitive supercritical fluid chromatography-tandem mass spectrometry method for simultaneous estimation of risperidone (RIS) and its chiral metabolites in rat plasma. Methodology & results: Agilent 1260 Infinity analytical supercritical fluid chromatography system resolved RIS and its chiral metabolites within runtime of 6 min using a gradient chromatography method. Using a simple protein precipitation sample preparation followed by mass spectrometric detection achieved a sensitivity of 0.92 nM (lower limit of quantification). With linearity over four log units (0.91-7500 nM), the method was found to be selective, accurate, precise and robust. The method was validated and was successfully applied for simultaneous estimation of RIS and 9-hydroxyrisperidone metabolites (R & S individually) after intravenous and per oral administration to rats.

An Analysis of Alternatives to Verbal FM Radio Tactical Command and Control Communications

DTIC Science & Technology

1975-06-06

specialist’s ,, , nwarted by these means even more reliablv than by conventional weapons.16 renaoiy an enemy attack can be th1 Marshal Vasili ...Mort Pit Squad(4) FDC F0(3) LDR FAC Bn LN0(2) Conmel Cff Survl Sec Sgt Commo Pit Gnd Survl Sec(6) Spt Pit Co Maint Sec AVLB Sec Med Sec Bn

76 FR 2609 - Proposed Amendment of Class E Airspace; Pueblo, CO

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-14

...]21[sec] N., long. 104[deg]29[min]47[sec] W.) That airspace extending upward from 700 feet above the... Memorial Airport clockwise between the 070[deg] and 133[deg] bearing from the airport; that airspace...., long. 105[deg]00[min]02[sec] W.; to lat. 38[deg]09[min]25[sec] N., long. 105[deg]08[min]06[sec] W.; to...

40 CFR Appendix I to Part 600 - Highway Fuel Economy Driving Schedule (Applicable to 1978 and Later Model Year Automobiles)

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 30 2011-07-01 2011-07-01 false Highway Fuel Economy Driving Schedule (Applicable to 1978 and Later Model Year Automobiles) I Appendix I to Part 600 Protection of Environment... to 1978 and Later Model Year Automobiles) [ speed (mph) vs time (sec) ] SEC MPH SEC MPH SEC MPH SEC...

FINCAP Analysis: A Method for Financial Capability Analysis of Air Force Contractors

DTIC Science & Technology

1979-03-01

obtained directly from the companies or through the SEC Pu’lications contractor (in hard copy or microfiche): Disclosure, Inc., 4827 Rugby Avenue...contracts- labor union wage agreements - material prices - escalation clauses in contracts - contractor accounting methods - level of capacity utilization

DETERMINATION OF CARBENDAZIM IN WATER BY HIGH-PERFORMANCE IMMUNOAFFINITY CHROMATOGRAPHY ON-LINE WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE-ARRAY OR MASS SPECTROMETRIC DETECTION

EPA Science Inventory

An automated method for the determination of carbendazim in water that combines high-performance immunoaffinity chromatography (HPIAC), high-performance liquid chromatography (HPLC) in the reversed-phase mode, and detection by either UV-Vis diode array detector (DAD) spectroscopy...

Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography.

PubMed

Ko, K Y; Ahn, D U

2007-02-01

The objective of this study was to develop an economical, simple, and large-scale separation method for IgY from egg yolk. Egg yolk diluted with 9 volumes of cold water was centrifuged after adjusting the pH to 5.0. The supernatant was added with 0.01% charcoal or 0.01% carrageenan and centrifuged at 2,800 x g for 30 min. The supernatant was filtered through a Whatman no. 1 filter paper and then the filtrate was concentrated to 20% original volume using ultrafiltration. The concentrated solution was further purified using either cation exchange chromatography or ammonium sulfate precipitation. For the cation exchange chromatography method, the concentrated sample was loaded onto a column equilibrated with 20 mM citrate-phosphate buffer at pH 4.8 and eluted with 200 mM citrate-phosphate buffer at pH 6.4. For the ammonium sulfate precipitation method, the concentrated sample was twice precipitated with 40% ammonium sulfate solution at pH 9.0. The yield and purity of IgY were determined by ELISA and electrophoresis. The yield of IgY from the cation exchange chromatography method was 30 to 40%, whereas that of the ammonium sulfate precipitation was 70 to 80%. The purity of IgY from the ammonium sulfate method was higher than that of the cation exchange chromatography. The cation exchange chromatography could handle only a small amount of samples, whereas the ammonium sulfate precipitation could handle a large volume of samples. This suggests that ammonium sulfate precipitation was a more efficient and useful purification method than cation exchange chromatography for the large-scale preparation of IgY from egg yolk.

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Repeated sprint ability related to recovery time in young soccer players.

PubMed

Padulo, J; Tabben, M; Ardigò, L P; Ionel, M; Popa, C; Gevat, C; Zagatto, A M; Dello Iacono, A

2015-01-01

This study aimed to describe the influence of recovery duration during a repeated sprint ability (RSA) test (6 × 40 m) by investigating a number of variables, such as general performance, metabolic demand, and muscular stretch-shortening performance. Seventeen male soccer outfield players (16 ± 0 years, 66 ± 10 kg) performed three field shuttle-running tests with 15, 20, and 25-sec recoveries. In addition to specific shuttle test's variables, blood lactate concentration and vertical jump height were assessed. Resulting measures were highly reliable (intra-class correlation coefficient up to 0.86). 25-sec recovery improved test performance (-3% total time from 15-sec to 25-sec recovery), vertical jump height (+7% post-test height from 15-sec to 25-sec recovery), and decreased blood lactate accumulation (-33% post-test from 15-sec to 25-sec recovery). Study findings suggest that metabolic acidosis plays a role in worsening performance and fatigue development during the shuttle test. A 25-sec recovery duration maximized performance, containing metabolic-anaerobic power involvement and muscular stretch-shortening performance deterioration during a RSA test.

Recent applications of gas chromatography with high-resolution mass spectrometry.

PubMed

Špánik, Ivan; Machyňáková, Andrea

2018-01-01

Gas chromatography coupled to high-resolution mass spectrometry is a powerful analytical method that combines excellent separation power of gas chromatography with improved identification based on an accurate mass measurement. These features designate gas chromatography with high-resolution mass spectrometry as the first choice for identification and structure elucidation of unknown volatile and semi-volatile organic compounds. Gas chromatography with high-resolution mass spectrometry quantitative analyses was previously focused on the determination of dioxins and related compounds using magnetic sector type analyzers, a standing requirement of many international standards. The introduction of a quadrupole high-resolution time-of-flight mass analyzer broadened interest in this method and novel applications were developed, especially for multi-target screening purposes. This review is focused on the development and the most interesting applications of gas chromatography coupled to high-resolution mass spectrometry towards analysis of environmental matrices, biological fluids, and food safety since 2010. The main attention is paid to various approaches and applications of gas chromatography coupled to high-resolution mass spectrometry for non-target screening to identify contaminants and to characterize the chemical composition of environmental, food, and biological samples. The most interesting quantitative applications, where a significant contribution of gas chromatography with high-resolution mass spectrometry over the currently used methods is expected, will be discussed as well. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Harassment and Violence Among Men Who Have Sex with Men (MSM) and Hijras After Reinstatement of India's "Sodomy Law".

PubMed

Li, Dennis H; Rawat, Shruta; Rhoton, Jayson; Patankar, Pallav; Ekstrand, Maria L; Simon Rosser, B R; Wilkerson, J Michael

2017-09-01

On December 11, 2013, the Indian Supreme Court recriminalized non-peno-vaginal sex under Sec. 377 of the Indian Penal Code (IPC), overturning a 2009 ruling that deemed IPC Sec. 377 unconstitutional. Similar "sodomy laws" in other countries have been associated with increased violence, harassment, and other discrimination against men who have sex with men (MSM) and transgender women. However, few studies have looked at the effects of such a law in an Indian context. This study examined experiences of victimization among MSM and hijra/transgender women (MSM-H) in the State of Maharashtra using a mixed-method approach. Data came from a quantitative survey and qualitative focus groups and interviews from an HIV prevention study as well as qualitative media and case reports from a local MSM-H-serving community-based organization. MSM-H in Maharashtra reported experiencing a high frequency of harassment, violence, and extortion, particularly from male sex partners met online and police. IPC Sec. 377 was implicated across qualitative sources as creating a culture of protection for harassment against MSM-H by being used directly as a tool for harassment, hindering victims of harassment from seeking legal recourse, and adversely impacting HIV and healthcare services. The reinstated IPC Sec. 377 may directly and indirectly facilitate negative health outcomes among MSM-H. Health agencies and advocates should continue to monitor the impact of IPC Sec. 377, incorporate rights-based approaches to protect MSM-H identities while addressing their health and well-being, and explore avenues to initiate discussions with the government to work toward repealing the law.

Head impacts in a junior rugby league team measured with a wireless head impact sensor: an exploratory analysis.

PubMed

King, Doug; Hume, Patria; Gissane, Conor; Clark, Trevor

2017-01-01

OBJECTIVE The aim of this study was to investigate the frequency, magnitude, and distribution of head impacts sustained by players in a junior rugby league over a season of matches. METHODS The authors performed a prospective cohort analysis of impact magnitude, frequency, and distribution on data collected with instrumented XPatches worn behind the ear of players in an "under-11" junior rugby league team (players under 11 years old). RESULTS A total of 1977 impacts were recorded. Over the course of the study, players sustained an average of 116 impacts (average of 13 impacts per player per match). The measured linear acceleration ranged from 10g to 123g (mean 22g, median 16g, and 95th percentile 57g). The rotational acceleration ranged from 89 rad/sec 2 to 22,928 rad/sec 2 (mean 4041 rad/sec 2 , median 2773 rad/sec 2 , and 95th percentile 11,384 rad/sec 2 ). CONCLUSIONS The level of impact severity based on the magnitude of impacts for linear and rotational accelerations recorded was similar to the impacts reported in studies of American junior and high school football, collegiate football, and youth ice hockey players, but the players in the rugby league cohort were younger, had less body mass, and played at a slower speed than the American players. Junior rugby league players are required to tackle the player to the ground and use a different tackle technique than that used in American football, likely increasing the rotational accelerations recorded at the head.

Kinetics of cycle length dependence of ventricular repolarization: effect of autonomic blockade

NASA Technical Reports Server (NTRS)

Raeder, E. A.; Albrecht, P.; Perrott, M.; Cohen, R. J.

1995-01-01

INTRODUCTION: Beat-to-beat adaptation of ventricular repolarization duration to cardiac cycle length and autonomic activity has not been previously characterized in the spontaneously beating human heart. METHODS AND RESULTS: The ECG of 14 healthy subjects was recorded from the supine and upright positions. Autonomic blockade was accomplished by atropine and propranolol. RR and RT intervals were measured by a computer algorithm, and the impulse response (h) from RR to RT computed. In the supine position the maximal adjustment of the RT interval occurred in the first beat following a change in cycle length (hpeak = 17.8 +/- 1.6 msec/sec), but continued to be detectable for 3.8 seconds (2.9-4.7 sec). Propranolol attenuated the peak impulse response to 15.8 +/- 4.0 msec/sec (P = NS). In the standing position the peak impulse response was increased to 25.2 +/- 5.0 msec/sec (P = 0.004 vs supine), and the impulse response duration (hdur) shortened to 1.4 seconds (1.3-1.6). This was reversed by beta blockade (hpeak = 10.7 +/- 3.6 [P = 0.005 vs standing]; hdur = 5.5 sec [4.8-6.1]). Parasympathetic and combined autonomic blockade resulted in too little residual heart rate variability to estimate the impulse response accurately. The slope of the regression of delta RT and delta RR in the supine position was 0.0177 +/- 0.0016, which was closely correlated with the peak impulse response (r = 0.91). CONCLUSIONS: System identification techniques can assist in characterizing the cycle dependence of ventricular repolarization and may provide new insights into conditions associated with abnormal repolarization.

Preparation of Yellow-Green-Emissive Carbon Dots and Their Application in Constructing a Fluorescent Turn-On Nanoprobe for Imaging of Selenol in Living Cells.

PubMed

Wang, Qin; Zhang, Shengrui; Zhong, Yaogang; Yang, Xiao-Feng; Li, Zheng; Li, Hua

2017-02-07

Selenocysteine (Sec) carries out the majority of the functions of the various Se-containing species in vivo. Thus, it is of great importance to develop sensitive and selective assays to detect Sec. Herein, a carbon-dot-based fluorescent turn-on probe for highly selective detection of selenol in living cells is presented. The highly photoluminescent carbon dots that emit yellow-green fluorescence (Y-G-CDs; λ max = 520 nm in water) were prepared by using m-aminophenol as carbon precursor through a facile solvothermal method. The surface of Y-G-CDs was then covalently functionalized with 2,4-dinitrobenzenesulfonyl chloride (DNS-Cl) to afford the 2,4-dinitrobenzene-functionalized CDs (CD-DNS) as a nanoprobe for selenol. CD-DNS is almost nonfluorescent. However, upon treating with Sec, the DNS moiety of CD-DNS can be readily cleaved by selenolate through a nucleophilic substitution process, resulting in the formation of highly fluorescent Y-G-CDs and hence leads to a dramatic increase in fluorescence intensity. The proposed nanoprobe exhibits high sensitivity and selectivity toward Sec over biothiols and other biological species. A preliminary study shows that CD-DNS can function as a useful tool for fluorescence imaging of exogenous and endogenous selenol in living cells.

Natural flow and water consumption in the Milk River basin, Montana and Alberta, Canada

USGS Publications Warehouse

Thompson, R.E.

1986-01-01

A study was conducted to determine the differences between natural and nonnatural Milk River streamflow, to delineate and quantify the types and effects of water consumption on streamflow, and to refine the current computation procedure into one which computes and apportions natural flow. Water consumption consists principally of irrigated agriculture, municipal use, and evapotranspiration. Mean daily water consumption by irrigation ranged from 10 cu ft/sec to 26 cu ft/sec in the Canada part and from 6 cu ft/sec to 41 cu ft/sec in the US part. Two Canadian municipalities consume about 320 acre-ft and one US municipality consumes about 20 acre-ft yearly. Evaporation from the water surface comprises 80% 0 90% of the flow reduction in the Milk River attributed to total evapotranspiration. The current water-budget approach for computing natural flow of the Milk River where it reenters the US was refined into an interim procedure which includes allowances for man-induced consumption and a method for apportioning computed natural flow between the US and Canada. The refined procedure is considered interim because further study of flow routing, tributary inflow, and man-induced consumption is needed before a more accurate procedure for computing natural flow can be developed. (Author 's abstract)

Comparison of Diagnostic Accuracy between Octopus 900 and Goldmann Kinetic Visual Fields

PubMed Central

Rowe, Fiona J.; Rowlands, Alison

2014-01-01

Purpose. To determine diagnostic accuracy of kinetic visual field assessment by Octopus 900 perimetry compared with Goldmann perimetry. Methods. Prospective cross section evaluation of 40 control subjects with full visual fields and 50 patients with known visual field loss. Comparison of test duration and area measurement of isopters for Octopus 3, 5, and 10°/sec stimulus speeds. Comparison of test duration and type of visual field classification for Octopus versus Goldmann perimetry. Results were independently graded for presence/absence of field defect and for type and location of defect. Statistical evaluation comprised of ANOVA and paired t test for evaluation of parametric data with Bonferroni adjustment. Bland Altman and Kappa tests were used for measurement of agreement between data. Results. Octopus 5°/sec perimetry had comparable test duration to Goldmann perimetry. Octopus perimetry reliably detected type and location of visual field loss with visual fields matched to Goldmann results in 88.8% of results (K = 0.775). Conclusions. Kinetic perimetry requires individual tailoring to ensure accuracy. Octopus perimetry was reproducible for presence/absence of visual field defect. Our screening protocol when using Octopus perimetry is 5°/sec for determining boundaries of peripheral isopters and 3°/sec for blind spot mapping with further evaluation of area of field loss for defect depth and size. PMID:24587983

40 CFR 162.156 - General requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... thereunder: (i) Sec. 12(a)(1) (A) through (E), in accordance with: (A) Sec. 2(q)(1) (A) through (G). (B) Sec... registered by the Administrator in accordance with sec. 7 of FIFRA and 40 CFR part 167. (c) Books and records...

7 CFR 235.4 - Allocation of funds to States.

Code of Federal Regulations, 2010 CFR

2010-01-01

.... (Sec. 14, Pub. L. 95-166, 91 Stat. 1338 (42 U.S.C. 1776); sec. 7, Pub. L. 95-627, 92 Stat. 3621 (42 U.S.C. 1776); sec. 7(a), Pub. L. 95-627, 92 Stat. 3622 (42 U.S.C. 1751); Pub. L. 96-499, secs. 201 and..., 1759a, 1774 and 1776); E.O. 12372 (July 14, 1982, 47 FR 30959); sec. 401(b) Intergovernmental...

7 CFR 235.4 - Allocation of funds to States.

Code of Federal Regulations, 2012 CFR

2012-01-01

.... (Sec. 14, Pub. L. 95-166, 91 Stat. 1338 (42 U.S.C. 1776); sec. 7, Pub. L. 95-627, 92 Stat. 3621 (42 U.S.C. 1776); sec. 7(a), Pub. L. 95-627, 92 Stat. 3622 (42 U.S.C. 1751); Pub. L. 96-499, secs. 201 and..., 1759a, 1774 and 1776); E.O. 12372 (July 14, 1982, 47 FR 30959); sec. 401(b) Intergovernmental...

7 CFR 235.4 - Allocation of funds to States.

Code of Federal Regulations, 2013 CFR

2013-01-01

.... (Sec. 14, Pub. L. 95-166, 91 Stat. 1338 (42 U.S.C. 1776); sec. 7, Pub. L. 95-627, 92 Stat. 3621 (42 U.S.C. 1776); sec. 7(a), Pub. L. 95-627, 92 Stat. 3622 (42 U.S.C. 1751); Pub. L. 96-499, secs. 201 and..., 1759a, 1774 and 1776); E.O. 12372 (July 14, 1982, 47 FR 30959); sec. 401(b) Intergovernmental...

7 CFR 235.4 - Allocation of funds to States.

Code of Federal Regulations, 2014 CFR

2014-01-01

.... (Sec. 14, Pub. L. 95-166, 91 Stat. 1338 (42 U.S.C. 1776); sec. 7, Pub. L. 95-627, 92 Stat. 3621 (42 U.S.C. 1776); sec. 7(a), Pub. L. 95-627, 92 Stat. 3622 (42 U.S.C. 1751); Pub. L. 96-499, secs. 201 and..., 1759a, 1774 and 1776); E.O. 12372 (July 14, 1982, 47 FR 30959); sec. 401(b) Intergovernmental...

7 CFR 235.4 - Allocation of funds to States.

Code of Federal Regulations, 2011 CFR

2011-01-01

.... (Sec. 14, Pub. L. 95-166, 91 Stat. 1338 (42 U.S.C. 1776); sec. 7, Pub. L. 95-627, 92 Stat. 3621 (42 U.S.C. 1776); sec. 7(a), Pub. L. 95-627, 92 Stat. 3622 (42 U.S.C. 1751); Pub. L. 96-499, secs. 201 and..., 1759a, 1774 and 1776); E.O. 12372 (July 14, 1982, 47 FR 30959); sec. 401(b) Intergovernmental...

Rubber.

ERIC Educational Resources Information Center

Krishen, Anoop

1989-01-01

This review covers methods for identification, characterization, and determination of rubber and materials in rubber. Topics include: general information, nuclear magnetic resonance spectroscopy, infrared spectroscopy, thermal methods, gel permeation chromatography, size exclusion chromatography, analysis related to safety and health, and…

Chromatographic and electrophoretic approaches in ink analysis.

PubMed

Zlotnick, J A; Smith, F P

1999-10-15

Inks are manufactured from a wide variety of substances that exhibit very different chemical behaviors. Inks designed for use in different writing instruments or printing methods have quite dissimilar components. Since the 1950s chromatographic and electrophoretic methods have played important roles in the analysis of inks, where compositional information may have bearing on the investigation of counterfeiting, fraud, forgery, and other crimes. Techniques such as paper chromatography and electrophoresis, thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gel electrophoresis, and the relatively new technique of capillary electrophoresis have all been explored as possible avenues for the separation of components of inks. This paper reviews the components of different types of inks and applications of the above separation methods are reviewed.

Decreased proteinase A excretion by strengthening its vacuolar sorting and weakening its constitutive secretion in Saccharomyces cerevisiae.

PubMed

Chen, Yefu; Song, Lulu; Han, Yueran; Liu, Mingming; Gong, Rui; Luo, Weiwei; Guo, Xuewu; Xiao, Dongguang

2017-01-01

Proteinase A (PrA), encoded by PEP4 gene, is detrimental to beer foam stability. There are two transport pathways for the new synthesized PrA in yeast, sorting to the vacuole normally, or excreting out of the cells under stress conditions. They were designated as the Golgi-to-vacuole pathway and the constitutive secretory pathway, respectively. To reduce PrA excretion in some new way instead of its coding gene deletion, which had a negative effect on cell metabolism and beer fermentation, we modified the PrA transport based on these above two pathways. In the Golgi-to-vacuole pathway, after the verification that Vps10p is the dominant sorting receptor for PrA Golgi-to-vacuolar transportation by VPS10 deletion, VPS10 was then overexpressed. Furthermore, SEC5, encoding exocyst complexes' central subunit (Sec5p) in the constitutive secretory pathway, was deleted. The results show that PrA activity in the broth fermented with WGV10 (VPS10 overexpressing strain) and W∆SEC5 (SEC5 deletion strain) was lowered by 76.96 and 32.39%, compared with the parental strain W303-1A, at the end of main fermentation. There are negligible changes in fermentation performance between W∆SEC5 and W303-1A, whereas, surprisingly, WGV10 had a significantly improved fermentation performance compared with W303-1A. WGV10 has an increased growth rate, resulting in higher biomass and faster fermentation speed; finally, wort fermentation is performed thoroughly. The results show that the biomass production of WGV10 is always higher than that of W∆SEC5 and W303-1A at all stages of fermentation, and that ethanol production of WGV10 is 1.41-fold higher than that of W303-1A. Obviously, VPS10 overexpression is beneficial for yeast and is a more promising method for reduction of PrA excretion.

Understanding the Current State of Infection Prevention to Prevent Clostridium difficile Infection: A Human Factors and Systems Engineering Approach

PubMed Central

Yanke, Eric; Zellmer, Caroline; Van Hoof, Sarah; Moriarty, Helene; Carayon, Pascale; Safdar, Nasia

2015-01-01

Background Achieving and sustaining high levels of healthcare worker (HCW) compliance with contact isolation precautions is challenging. The aim of this study was to determine HCW work system barriers to, and facilitators of, adherence to contact isolation for patients with suspected or confirmed Clostridium difficile infection (CDI) using a human factors and systems engineering approach. Methods Prospective cohort study from September 2013 to November 2013 at a large academic medical center (hospital A) and an affiliated Veterans Administration (VA) hospital (hospital B). A human factors engineering (HFE) model for patient safety – the Systems Engineering Initiative for Patient Safety (SEIPS) model – was used to guide work system analysis and direct observation data collection. 288 observations were conducted. HCWs and visitors were assessed for compliance with all components of contact isolation precautions (hand hygiene, gowning, and gloving) before and after patient contact. Time required to complete contact isolation precautions was measured and adequacy of contact isolation supplies was assessed. Results Full compliance with contact isolation precautions was low at both hospitals: hospital A, 7%; hospital B, 22%. Lack of appropriate hand hygiene prior to room entry (Compliance: hospital A, 18%; hospital B, 29%) was the most common reason for lack of full compliance. More time was required for full compliance as compared to compliance with no components of contact isolation precautions before patient room entry, inside patient room, and after patient room exit (59.9 sec vs. 3.2 sec; P < .001; 507.3 sec vs. 149.7 sec; P = .006; 15.2 sec vs. 1.3 sec; P < .001). Compliance was lower when contact isolation supplies were inadequate (4% vs. 16%; P = .005). Conclusions Adherence to contact isolation precautions for CDI is a complex, time-consuming process. HFE analysis indicates multiple work system components serve as barriers and facilitators to full compliance with contact isolation precautions and should be addressed further to prevent CDI. PMID:25728149

Apigenin potentiates the antitumor activity of 5-FU on solid Ehrlich carcinoma: Crosstalk between apoptotic and JNK-mediated autophagic cell death platforms

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

Gaballah, Hanaa H., E-mail: hanaahibishy@hotmail.c

Background: Although 5- Fluorouracil (5-FU) has exhibited effectiveness against cancer, novel therapeutic strategies are needed to enhance its antitumor efficiency and modulate its cytotoxity. Apigenin, a flavonoid present in fruits and vegetables, is a potent dietary phytochemical effective in cancer chemoprevention. Aim: This study was undertaken to investigate the potential synergistic antitumor activity of apigenin and 5-FU on Solid Ehrlich carcinoma (SEC). Methods: Eighty Swiss albino male mice were divided into four equal groups: vehicle treated control SEC, SEC + 5-FU, SEC + apigenin, SEC + 5-FU + apigenin. Beclin-1 and caspases 3, 9 and JNK activities were estimated bymore » ELISA; mRNA expression levels of the antiapoptotic gene Mcl-1 were estimated using quantitative real-time RT-PCR, while tissue malondialdehyde (MDA), glutathione peroxidase and total antioxidant capacity were evaluated spectrophotometrically. A part of the tumor was examined for histopathological and Ki-67 immunohistochemistry analysis. Results: 5-FU and/or apigenin caused significant increase in tissue levels of Beclin-1, caspases 3, 9 and JNK activities, MDA with significant decrease in tumor volume, Mcl-1expression, tissue glutathione peroxidase and total antioxidant capacity and alleviated the histopathological changes with significant decrease of Ki-67 proliferation index compared to vehicle treated SEC control group. In conclusion: The combination of 5-FU and apigenin had a greater effect than each of 5-FU or apigenin alone against solid Ehrlich carcinoma in mice. - Highlights: • Apigenin potentiated 5-FU cytotoxicity in EAC solid tumor models in vivo. • It acted via autophagy stimulation, downregulating MCL-1 and Ki-67 expression. • It caused JNK activation and ROS accumulation; resulted in tumor growth inhibition. • Apigenin can be used as a co-adjuvant agent in cancer therapy.« less

Geographical Variation in Diabetes Prevalence and Detection in China: Multilevel Spatial Analysis of 98,058 Adults

PubMed Central

Zhou, Maigeng; Astell-Burt, Thomas; Bi, Yufang; Feng, Xiaoqi; Jiang, Yong; Li, Yichong; Page, Andrew; Wang, Limin; Xu, Yu

2015-01-01

OBJECTIVE To investigate the geographic variation in diabetes prevalence and detection in China. RESEARCH DESIGN AND METHODS Self-report and biomedical data were collected from 98,058 adults aged ≥18 years (90.5% response) from 162 areas spanning mainland China. Diabetes status was assessed using American Diabetes Association criteria. Among those with diabetes, detection was defined by prior diagnosis. Choropleth maps were used to visually assess geographical variation in each outcome at the provincial level. The odds of each outcome were assessed using multilevel logistic regression, with adjustment for person- and area-level characteristics. RESULTS Geographic visualization at the provincial level indicated widespread variation in diabetes prevalence and detection across China. Regional prevalence adjusted for age, sex, and urban/rural socioeconomic circumstances (SECs) ranged from 8.3% (95% CI 7.2%, 9.7%) in the northeast to 12.7% (11.1%, 14.6%) in the north. A clear negative gradient in diabetes prevalence was observed from 13.1% (12.0%, 14.4%) in the urban high-SEC to 8.7% (7.8%, 9.6%) in rural low-SEC counties/districts. Adjusting for health literacy and other person-level characteristics only partially attenuated these geographic variations. Only one-third of participants living with diabetes had been previously diagnosed, but this also varied substantively by geography. Regional detection adjusted for age, sex, and urban/rural SEC, for example, spanned from 40.4% (34.9%, 46.3%) in the north to 15.6% (11.7%, 20.5%) in the southwest. Compared with detection of 40.8% (37.3%, 44.4%) in urban high-SEC counties, detection was poorest among rural low-SEC counties at just 20.5% (17.7%, 23.7%). Person-level characteristics did not fully account for these geographic variations in diabetes detection. CONCLUSIONS Strategies for addressing diabetes risk and improving detection require geographical targeting. PMID:25352654

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

PubMed

Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

2018-02-01

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

PubMed Central

Bruno, Joanne; Chaudhary, Natasha; Iaea, David

2016-01-01

RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10–SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4. PMID:27354378

Liver diffusion-weighted MR imaging: reproducibility comparison of ADC measurements obtained with multiple breath-hold, free-breathing, respiratory-triggered, and navigator-triggered techniques.

PubMed

Chen, Xin; Qin, Lei; Pan, Dan; Huang, Yanqi; Yan, Lifen; Wang, Guangyi; Liu, Yubao; Liang, Changhong; Liu, Zaiyi

2014-04-01

To prospectively compare the reproducibility of normal liver apparent diffusion coefficient (ADC) measurements by using different respiratory motion compensation techniques with multiple breath-hold (MBH), free-breathing (FB), respiratory-triggered (RT), and navigator-triggered (NT) diffusion-weighted (DW) imaging and to compare the ADCs at different liver anatomic locations. The study protocol was approved by the institutional review board, and written informed consent was obtained from each participant. Thirty-nine volunteers underwent liver DW imaging twice. Imaging was performed with a 1.5-T MR imager with MBH, FB, RT, and NT techniques (b = 0, 100, and 500 sec/mm(2)). Three representative sections--superior, central, and inferior--were selected on left and right liver lobes, respectively. On each selected section, three regions of interest were drawn, and ADCs were measured. Analysis of variance was used to assess ADCs among the four techniques and various anatomic locations. Reproducibility of ADCs was assessed with the Bland-Altman method. ADCs obtained with MBH (range: right lobe, [1.641-1.662] × 10(-3)mm(2)/sec; left lobe, [2.034-2.054] ×10(-3)mm(2)/sec) were higher than those obtained with FB (right, [1.349-1.391] ×10(-3)mm(2)/sec; left, [1.630-1.700] ×10(-3)mm(2)/sec), RT (right, [1.439-1.455] ×10(-3)mm(2)/sec; left, [1.720-1.755] ×10(-3)mm(2)/sec), or NT (right, [1.387-1.400] ×10(-3)mm(2)/sec; left, [1.661-1.736] ×10(-3)mm(2)/sec) techniques (P < .001); however, no significant difference was observed between ADCs obtained with FB, RT, and NT techniques (P = .130 to P >.99). ADCs showed a trend to decrease moving from left to right. Reproducibility in the left liver lobe was inferior to that in the right, and the central middle segment in the right lobe had the most reproducible ADC. Statistical differences in ADCs were observed in the left-right direction in the right lobe (P < .001), but they were not observed in the superior-inferior direction (P = .144-.450). However, in the left liver lobe, statistical differences existed in both directions (P = .001 to P = .016 in the left-right direction, P < .001 in the superior-inferior direction). Both anatomic location and DW imaging technique influence liver ADC measurements and their reproducibility. FB DW imaging is recommended for liver DW imaging because of its good reproducibility and shorter acquisition time compared with that of MBH, RT, and NT techniques. RSNA, 2014

27 CFR 19.456 - Adding denaturants.

Code of Federal Regulations, 2010 CFR

2010-04-01

... methods of mixing denaturants and spirits if he deems such denaturation will not hinder effective... Denaturation § 19.456 Adding denaturants. Denaturants and spirits shall be mixed in packages, tanks, or bulk... proprietor shall submit a flow diagram of the intended process or method of adding denaturants. (Sec. 201...

Oil composition and method of making the same

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

Herman, S.M.

1931-10-24

A method is described for production of a mineral oil having a Saybolt viscosity of approximately 200 to 350 sec at 100/sup 0/F. The oil contains partially or wholly saponified oxidized rape oil in an amount sufficient to render the mineral oil emulsifiable in water.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

PubMed

De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie

2014-01-01

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

PubMed

Pinto, Nuno D S; Uplekar, Shaunak D; Moreira, Antonio R; Rao, Govind; Frey, Douglas D

2017-01-01

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Development of an on-line mixed-mode gel liquid chromatography×reversed phase liquid chromatography method for separation of water extract from Flos Carthami.

PubMed

Wang, Yu-Qing; Tang, Xu; Li, Jia-Fu; Wu, Yun-Long; Sun, Yu-Ying; Fang, Mei-Juan; Wu, Zhen; Wang, Xiu-Min; Qiu, Ying-Kun

2017-10-13

A novel on-line comprehensive two-dimensional liquid chromatography (2D-LC) method by coupling mixed-mode gel liquid chromatography (MMG-LC) with reversed phase liquid chromatography (RPLC) was developed. A mixture of 17 reference compounds was used to study the separation mechanism. A crude water extract of Flos Carthami was applied to evaluate the performance of the novel 2D-LC system. In the first dimension, the extract was eluted with a gradient of water/methanol over a cross-linked dextran gel Sephadex LH-20 column. Meanwhile, the advantages of size exclusion, reversed phase partition and adsorption separation mechanism were exploited before further on-line reversed phase purification on the second dimension. This novel on-line mixed-mode Sephadex LH-20×RPLC method provided higher peak resolution, sample processing ability (2.5mg) and better orthogonality (72.9%) versus RPLC×RPLC and hydrophilic interaction liquid chromatography (HILIC)×RPLC. To the best of our knowledge, this is the first report of a mixed-mode Sephadex LH-20×RPLC separation method with successful applications in on-line mode, which might be beneficial for harvesting targets from complicated medicinal plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of mycotoxins produced by Fusarium isolates from banana fruits by capillary gas chromatography and high-performance liquid chromatography.

PubMed

Jiménez, M; Mateo, R

1997-08-22

A method of analysis for trichothecenes (nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, T-2 tetraol, T-2 and HT-2 toxins), zearalenone and zearalenols, and another method for determination of fumonisin B1 are described and applied to cultures of Fusarium isolated from bananas. Both methods were adapted from different techniques of extraction, clean-up and determination of these mycotoxins. The first method involves extraction with methanol-1% aqueous sodium chloride, clean-up of extracts by partition with hexane and dichloromethane, additional solid reversed-phase clean-up and analysis of two eluates by both high-performance liquid chromatography with ultraviolet detection and capillary gas chromatography. The method for fumonisin B1 implies extraction with aqueous methanol, concentration, clean-up with water and methanol on Amberlite XAD-2 column, formation of a fluorescent 4-fluoro-7-nitrobenzofurazan derivative and analysis by high-performance liquid chromatography with fluorescence detection. Both procedures give good limits of detection and recoveries, and are considered suitable for the detection and quantification of the studied toxins in corn and rice cultures of Fusarium spp. isolated from banana fruits.

Advantages of estimating rate corrections during dynamic propagation of spacecraft rates: Applications to real-time attitude determination of SAMPEX

NASA Technical Reports Server (NTRS)

Challa, M. S.; Natanson, G. A.; Baker, D. F.; Deutschmann, J. K.

1994-01-01

This paper describes real-time attitude determination results for the Solar, Anomalous, and Magnetospheric Particle Explorer (SAMPEX), a gyroless spacecraft, using a Kalman filter/Euler equation approach denoted the real-time sequential filter (RTSF). The RTSF is an extended Kalman filter whose state vector includes the attitude quaternion and corrections to the rates, which are modeled as Markov processes with small time constants. The rate corrections impart a significant robustness to the RTSF against errors in modeling the environmental and control torques, as well as errors in the initial attitude and rates, while maintaining a small state vector. SAMPLEX flight data from various mission phases are used to demonstrate the robustness of the RTSF against a priori attitude and rate errors of up to 90 deg and 0.5 deg/sec, respectively, as well as a sensitivity of 0.0003 deg/sec in estimating rate corrections in torque computations. In contrast, it is shown that the RTSF attitude estimates without the rate corrections can degrade rapidly. RTSF advantages over single-frame attitude determination algorithms are also demonstrated through (1) substantial improvements in attitude solutions during sun-magnetic field coalignment and (2) magnetic-field-only attitude and rate estimation during the spacecraft's sun-acquisition mode. A robust magnetometer-only attitude-and-rate determination method is also developed to provide for the contingency when both sun data as well as a priori knowledge of the spacecraft state are unavailable. This method includes a deterministic algorithm used to initialize the RTSF with coarse estimates of the spacecraft attitude and rates. The combined algorithm has been found effective, yielding accuracies of 1.5 deg in attitude and 0.01 deg/sec in the rates and convergence times as little as 400 sec.

Facile preparation of an alternating copolymer-based high molecular shape-selective organic phase for reversed-phase liquid chromatography.

PubMed

Mallik, Abul K; Noguchi, Hiroki; Rahman, Mohammed Mizanur; Takafuji, Makoto; Ihara, Hirotaka

2018-06-22

The synthesis of a new alternating copolymer-grafted silica phase is described for the separation of shape-constrained isomers of polycyclic aromatic hydrocarbons (PAHs) and tocopherols in reversed-phase high-performance liquid chromatography (RP-HPLC). Telomerization of the monomers (octadecyl acrylate and N-methylmaleimide) was carried out with a silane coupling agent; 3-mercaptopropyltrimethoxysilane (MPS), and the telomer (T) was grafted onto porous silica surface to prepare the alternating copolymer-grafted silica phase (Sil-alt-T). The new hybrid material was characterized by elemental analyses, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, and solid-state 13 C and 29 Si cross-polarization magic-angle spinning (CP/MAS) NMR spectroscopy. The results of 13 C CP/MAS NMR demonstrated that the alkyl chains of the grafted polymers in Sil-alt-T remained disordered, amorphous, and mobile represented by gauche conformational form. Separation abilities and molecular-shape selectivities of the prepared organic phase were evaluated by the separation of PAHs isomers and Standard Reference Material 869b, Column Selectivity Test Mixture for Liquid Chromatography, respectively and compared with commercially available octadecylsilylated silica (ODS) and C 30 columns as well as previously reported alternating copolymer-based column. The effectiveness of this phase is also demonstrated by the separation of tocopherol isomers. Oriented functional groups along the polymer main chains and cavity formations are investigated to be the driving force for better separation with multiple-interactions with the solutes. One of the advantages of the Sil-alt-T phase to that of the previously reported phase is the synthesis of the telomer first and then immobilized onto silica surface. In this case, the telomer was characterized easily with simple spectroscopic techniques and the molecular mass and polydispersity index of the telomer were determined by size exclusion chromatography (SEC) before grafting onto silica surface. Moreover, both of the monomers were commercially available. Therefore, the technique of preparation was very facile and better separation was achieved with the Sil-alt-T phase compared to the ODS, C 30 and other previously reported alternating copolymer-based columns. Copyright © 2018 Elsevier B.V. All rights reserved.

Near-Infrared Fluorescent Turn-on Probe with a Remarkable Large Stokes Shift for Imaging Selenocysteine in Living Cells and Animals.

PubMed

Feng, Weiyong; Li, Meixing; Sun, Yao; Feng, Guoqiang

2017-06-06

Selenocysteine (Sec) is the 21st naturally occurring amino acid and has emerged as an important sensing target in recent years. However, fluorescent detection of Sec in living systems is challenging. To date, very few fluorescent Sec probes have been reported and most of them respond fluorescence to Sec in the visible region. In this paper, a very promising near-infrared fluorescent probe for Sec was developed. This probe works in aqueous solution over a wide pH range under mild conditions and can be used for rapid, highly selective and sensitive detection of Sec with significant near-infrared fluorescent turn-on signal changes. In addition, it features a remarkable large Stokes shift (192 nm) and a low detection limit (60 nM) for Sec with a wide linear range (0-70 μM). Moreover, this probe can be conveniently used to detect Sec in serum samples, living cells, and animals, indicating it holds great promise for biological applications.

A Pseudo-Atomic Model of the COPII Cage Obtained from CryoEM and Mass Spectrometry Analyses

PubMed Central

Noble, Alex J.; Zhang, Qian; O’Donnell, Jason; Hariri, Hanaa; Bhattacharya, Nilakshee; Marshall, Alan G.

2012-01-01

COPII vesicles transport proteins from the ER to the Golgi apparatus. Previous cryoEM structures of the COPII cage lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12Å-resolution structure of the COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13-Sec31 complex to create a reliable pseudo-atomic model of the COPII cage. We combined this model with hydrogen/deuterium exchange mass spectrometry analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the 2-fold symmetry of the Sec31 dimeric region of Sec13-31 is broken on cage formation, and that the resulting hinge is essential to form the proper edge geometry in COPII cages. PMID:23262493

Agonal sequences in eight filmed hangings: analysis of respiratory and movement responses to asphyxia by hanging.

PubMed

Sauvageau, Anny; LaHarpe, Romano; Geberth, Vernon J

2010-09-01

It has been proposed that filmed hangings may hold the key to a better understanding of human asphyxia, and The Working Group on Human Asphyxia was formed to systematically review and compare these video recordings. This study analyzed eight filmed hangings. Considering time 0 to represent the onset of the final hanging, rapid loss of consciousness was observed (at 8-18 sec), closely followed by convulsions (at 10-19 sec). A complex pattern of decerebrate rigidity and decorticate rigidity then followed. Between 1 min 38 sec and 2 min 15 sec, muscle tone seemed to be lost, the body becoming progressively flaccid. From then on, isolated body movements were observed from time to time, the last one occurring between 1 min 2 sec and 7 min 31 sec. As for the respiratory responses, all cases presented deep rhythmic abdominal respiratory movements (last one between 1 min 2 sec and 2 min 5 sec). © 2010 American Academy of Forensic Sciences.

Regulation of the protein-conducting channel by a bound ribosome

PubMed Central

Gumbart, James; Trabuco, Leonardo G.; Schreiner, Eduard; Villa, Elizabeth; Schulten, Klaus

2009-01-01

Summary During protein synthesis, it is often necessary for the ribosome to form a complex with a membrane-bound channel, the SecY/Sec61 complex, in order to translocate nascent proteins across a cellular membrane. Structural data on the ribosome-channel complex are currently limited to low-resolution cryo-electron microscopy maps, including one showing a bacterial ribosome bound to a monomeric SecY complex. Using that map along with available atomic-level models of the ribosome and SecY, we have determined, through molecular dynamics flexible fitting (MDFF), an atomic-resolution model of the ribosome-channel complex. We characterized computationally the sites of ribosome-SecY interaction within the complex and determined the effect of ribosome binding on the SecY channel. We also constructed a model of a ribosome in complex with a SecY dimer by adding a second copy of SecY to the MDFF-derived model. The study involved 2.7-million-atom simulations over altogether nearly 50 ns. PMID:19913480

Simultaneous analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography.

PubMed

Taguchi, Kaori; Fukusaki, Eiichiro; Bamba, Takeshi

2014-10-03

Chromatography techniques usually use a single state in the mobile phase, such as liquid, gas, or supercritical fluid. Chromatographers manage one of these techniques for their purpose but are sometimes required to use multiple methods, or even worse, multiple techniques when the target compounds have a wide range of chemical properties. To overcome this challenge, we developed a single method covering a diverse compound range by means of a "unified" chromatography which completely bridges supercritical fluid chromatography and liquid chromatography. In our method, the phase state was continuously changed in the following order; supercritical, subcritical and liquid. Moreover, the gradient of the mobile phase starting at almost 100% CO2 was replaced with 100% methanol at the end completely. As a result, this approach achieved further extension of the polarity range of the mobile phase in a single run, and successfully enabled the simultaneous analysis of fat- and water-soluble vitamins with a wide logP range of -2.11 to 10.12. Furthermore, the 17 vitamins were exceptionally separated in 4min. Our results indicated that the use of dense CO2 and the replacement of CO2 by methanol are practical approaches in unified chromatography covering diverse compounds. Additionally, this is a first report to apply the novel approach to unified chromatography, and can open another door for diverse compound analysis in a single chromatographic technique with single injection, single column and single system. Copyright © 2014. Published by Elsevier B.V.

Comparative chromatography of chloroplast pigment

NASA Technical Reports Server (NTRS)

Grandolfo, M.; Sherma, J.; Strain, H. H.

1969-01-01

Methods for isolation of low concentration pigments of the cocklebur species are described. The methods entail two step chromatography so that the different sorption properties of the various pigments in varying column parameters can be utilized. Columnar and thin layer methods are compared. Many conditions influence separability of the chloroplasts.

Electrochemistry and Spectroelectrochemistry of Luminescent Europium Complexes

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

Lines, Amanda M.; Wang, Zheming; Clark, Sue B.

Fast, cost effective, and robust means of detecting and quantifying lanthanides are needed for supporting more efficient tracking within the nuclear, medicinal, and industrial fields. Spectroelectrochemistry (SEC) is a powerful technique combining electrochemistry and spectroscopy that can meet those needs. The primary limitation of SEC as a detection method for lanthanides is their low molar absorptivity in absorbance based measurements and low emission intensities in fluorescence based measurements; both lead to high limits of detection. These limitations can be circumvented by complexing the lanthanides with sensitizing ligands that enhance fluorescence, thereby dropping the limits of detection. Complexation may also stabilizemore » the metal ions in solution and improve the electrochemical reversibility, or Nernstian behavior, of the redox couples. To demonstrate this concept, studies were completed using europium in complexes with four different sensitizing ligands. Initial work indicates Eu in the four complexes studied does display the necessary characteristics for SEC analysis, which was successfully and reproducibly applied to all Eu complexes.« less

Why do Ladybugs Smell Bad? In-vivo Quantification of Odorous Insect Kairomones with SPME and Multidimensional GC-MS-Olfactometry

NASA Astrophysics Data System (ADS)

Cai, Lingshuang; Koziel, Jacek A.; O'Neal, Matthew E.

2009-05-01

Winemakers, small fruit growers, and homeowners are concerned with noxious compounds released by multicolored Asian ladybird beetles (Harmonia axyridis, Coleoptera: Coccinellidae). New method based on headspace solid phase microextraction (HS-SPME) coupled with multidimensional gas chromatography mass spectrometry—olfactometry (MDGC-MS-O) system was developed for extraction, isolation and simultaneous identification of compounds responsible for the characteristic odor of live H. axyridis. Four methoxypyrazines (MPs) were identified in headspace volatiles of live H. axyridis as those responsible for the characteristic odor: 2, 5-dimethy1-3-methoxypyrazine (DMMP), 2-isopropy1-3-methoxypyrazine (IPMP), 2-sec-buty1-3-methoxypyrazine (SBMP), and 2-isobuty1-3-methoxypyrazine (IBMP). To the best of our knowledge this is the first report of H. axyridis releasing DMMP and the first report of this compound being a component of the H. axyridis characteristic odor. Quantification of three MPs (IPMP, SBMP and IBMP) emitted from live H. axyridis were performed using external calibration with HS-SPME and direct injections. A linear relationship (R2>0.9958 for all 3 MPs) between MS response and concentration of standard was observed over a concentration range from 0.1 ng L-1 to 0.05 μg L-1 for HS-SPME-GC-MS. The method detection limits (MDL) based on multidimensional GC-MS approach for three MPs were estimated to be between 0.020 ng L-1. to 0.022 ng L-1. This methodology is applicable for in vivo determination of odor-causing chemicals associated with emissions of volatiles from insects.

Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reaction.

PubMed

Gullón, Beatriz; Montenegro, María I; Ruiz-Matute, Ana I; Cardelle-Cobas, Alejandra; Corzo, Nieves; Pintado, Manuela E

2016-02-10

Chitosan (Chit) was submitted to the Maillard reaction (MR) by co-heating a solution with glucose (Glc). Different reaction conditions as temperature (40, 60 and 80 °C), Glc concentration (0.5%, 1%, and 2%, w/v), and reaction time (72, 52 and 24h) were evaluated. Assessment of the reaction extent was monitored by measuring changes in UV absorbance, browning and fluorescence. Under the best conditions, 2% (w/v) of Chit, 2% (w/v) of Glc at 60°C and 32 h of reaction time, a chitosan-glucose (Chit-Glc) derivative was purified and submitted to structural characterization to confirm its formation. Analysis of its molecular weight (MW) and the degree of substitution (DS) was carried out by HPLC-Size Exclusion Chromatography (SEC) and a colloid titration method, respectively. FT-IR and (1)H NMR were also used to analyze the functional groups and evaluate the introduction of Glc into the Chit molecule. According to our objectives, the results obtained in this work allowed to better understand the key parameters influencing the MR with Chit as well as to confirm the successful introduction of Glc into the Chit molecule obtaining a Chit-Glc derivative with a DS of 64.76 ± 4.40% and a MW of 210.37 kDa. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polyvinylidene Fluoride Micropore Membranes as Solid-Phase Extraction Disk for Preconcentration of Nanoparticulate Silver in Environmental Waters.

PubMed

Zhou, Xiao-Xia; Lai, Yu-Jian; Liu, Rui; Li, Sha-Sha; Xu, Jing-Wen; Liu, Jing-Fu

2017-12-05

Efficient separation and preconcentration of trace nanoparticulate silver (NAg) from large-volume environmental waters is a prerequisite for reliable analysis and therefore understanding the environmental processes of silver nanoparticles (AgNPs). Herein, we report the novel use of polyvinylidene fluoride (PVDF) filter membrane for disk-based solid phase extraction (SPE) of NAg in 1 L of water samples with the disk-based SPE system, which consists of a syringe pump and a syringe filter holder to embed the filter membrane. While the PVDF membrane can selectively adsorb NAg in the presence of Ag + , aqueous solution of 2% (m/v) FL-70 is found to efficiently elute NAg. Analysis of NAg is performed following optimization of filter membrane and elution conditions with an enrichment factor of 1000. Additionally, transmission electron microscopy (TEM), UV-vis spectroscopy, and size-exclusion chromatography coupled with ICP-MS (SEC-ICP-MS) analysis showed that the extraction gives rise to no change in NAg size or shape, making this method attractive for practical applications. Furthermore, feasibility of the protocol is verified by applying it to extract NAg in four real waters with recoveries of 62.2-80.2% at 0.056-0.58 μg/L spiked levels. This work will facilitate robust studies of trace NAg transformation and their hazard assessments in the environment.

Application of radar chart array analysis to visualize effects of formulation variables on IgG1 particle formation as measured by multiple analytical techniques

PubMed Central

Kalonia, Cavan; Kumru, Ozan S.; Kim, Jae Hyun; Middaugh, C. Russell; Volkin, David B.

2013-01-01

This study presents a novel method to visualize protein aggregate and particle formation data to rapidly evaluate the effect of solution and stress conditions on the physical stability of an IgG1 monoclonal antibody (mAb). Radar chart arrays were designed so that hundreds of Microflow Digital Imaging (MFI) solution measurements, evaluating different mAb formulations under varying stresses, could be presented in a single figure with minimal loss of data resolution. These MFI radar charts show measured changes in subvisible particle number, size and morphology distribution as a change in the shape of polygons. Radar charts were also created to visualize mAb aggregate and particle formation across a wide size range by combining data sets from size exclusion chromatography (SEC), Archimedes resonant mass measurements, and MFI. We found that the environmental/mechanical stress condition (e.g., heat vs. agitation) was the most important factor in influencing the particle size and morphology distribution with this IgG1 mAb. Additionally, the presence of NaCl exhibited a pH and stress dependent behavior resulting in promotion or inhibition mAb particle formation. This data visualization technique provides a comprehensive analysis of the aggregation tendencies of this IgG1 mAb in different formulations with varying stresses as measured by different analytical techniques. PMID:24122556

Radiation-induced controlled polymerization of acrylic acid by RAFT and RAFT-MADIX methods in protic solvents

NASA Astrophysics Data System (ADS)

Sütekin, S. Duygu; Güven, Olgun

2018-01-01

The kinetic investigation of one-pot synthesis of poly(acrylic acid) (PAA) prepared via gamma radiation induced controlled polymerization was reported. PAA homopolymers were prepared by Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization in the presence of trithiocarbonate-based chain transfer agent (CTA) 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) and also by Reversible Addition-Fragmentation/Macromolecular Design by Inter-change of Xanthates (RAFT/MADIX) polymerization in the presence of a xanthate based CTA O-ethyl-S-(1-methoxycarbonyl) ethyl dithiocarbonate (RA1). The polymerizations were performed at room temperature by the virtue of ionizing radiation. Protic solvents were used for the RAFT polymerization of AA considering environmental profits. The linear first-order kinetic plot, close control of molecular weight by the monomer/CTA molar ratio supported that the polymerization proceeds in a living fashion. The linear increase in molecular weight with conversion monitored by Size Exclusion Chromatography (SEC) is another proof of controlling of polymerization. [Monomer]/[RAFT] ratio and conversion was controlled to obtain PAA in the molecular weight range of 6900-35,800 with narrow molecular weight distributions. Reaction kinetics and effect of the amount of RAFT agent were investigated in detail. Between two different types of CTA, trithiocarbonate based DDMAT was found to be more efficient in terms of low dispersity (Đ) and linear first-order kinetic behavior for the radiation induced controlled synthesis of PAA homopolymers.

Tuberculin purified protein derivative (PPD) immunoassay as an in vitro alternative assay for identity and confirmation of potency.

PubMed

Ho, Mei M; Kairo, Satnam K; Corbel, Michael J

2006-01-01

Tuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

Spray-Freeze Drying: a Suitable Method for Aerosol Delivery of Antibodies in the Presence of Trehalose and Cyclodextrins.

PubMed

Pouya, Maryam Amini; Daneshmand, Behnaz; Aghababaie, Shabnam; Faghihi, Homa; Vatanara, Alireza

2018-05-08

We aimed to prepare spray-freeze-dried powder of IgG considering physicochemical stability and aerodynamic aspects. Spray-freeze drying (SFD) exposes proteins to various stresses which should be compensated by suitable stabilizers. The competence of cyclodextrins (CDs), namely beta-cyclodextrin (βCD) and hydroxypropyl βCD (HPβCD), at very low concentrations, was investigated in the presence of separate mannitol- and trehalose-based formulations. Spray-freeze-dried preparations were quantified in terms of monomer recovery and conformation by size exclusion chromatography (SEC-HPLC) and Fourier transform infrared (FTIR) spectroscopy, respectively. Differential scanning calorimetry (DSC) and X-ray diffractometry (XRD) were employed to identify the thermal characteristics of powders. Particle morphology was visualized by scanning electron microscopy (SEM). Aerodynamic behavior of powders was checked through an Anderson cascade impactor (ACI). Although all formulations protected antibody from aggregation during the SFD process (aggregation < 1%), mannitol-containing ones failed upon the storage (19.54% in the worst case). Trehalose-HPβCD incomparably preserved the formulation with fine particle fraction (FPF) of 51.29%. Crystallization of mannitol resulted in IgG destabilization upon storage. Although employed concentration of CDs is too low (less than 50:1 molar ratio to protein), they successfully served as stabilizing agents in SFD with perfect improvement in aerosol functionality. Graphical Abstract ᅟ.

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

Lou, K; Rice University, Houston, TX; Sun, X

Purpose: To study the feasibility of clinical on-line proton beam range verification with PET imaging Methods: We simulated a 179.2-MeV proton beam with 5-mm diameter irradiating a PMMA phantom of human brain size, which was then imaged by a brain PET with 300*300*100-mm{sup 3} FOV and different system sensitivities and spatial resolutions. We calculated the mean and standard deviation of positron activity range (AR) from reconstructed PET images, with respect to different data acquisition times (from 5 sec to 300 sec with 5-sec step). We also developed a technique, “Smoothed Maximum Value (SMV)”, to improve AR measurement under a givenmore » dose. Furthermore, we simulated a human brain irradiated by a 110-MeV proton beam of 50-mm diameter with 0.3-Gy dose at Bragg peak and imaged by the above PET system with 40% system sensitivity at the center of FOV and 1.7-mm spatial resolution. Results: MC Simulations on the PMMA phantom showed that, regardless of PET system sensitivities and spatial resolutions, the accuracy and precision of AR were proportional to the reciprocal of the square root of image count if image smoothing was not applied. With image smoothing or SMV method, the accuracy and precision could be substantially improved. For a cylindrical PMMA phantom (200 mm diameter and 290 mm long), the accuracy and precision of AR measurement could reach 1.0 and 1.7 mm, with 100-sec data acquired by the brain PET. The study with a human brain showed it was feasible to achieve sub-millimeter accuracy and precision of AR measurement with acquisition time within 60 sec. Conclusion: This study established the relationship between count statistics and the accuracy and precision of activity-range verification. It showed the feasibility of clinical on-line BR verification with high-performance PET systems and improved AR measurement techniques. Cancer Prevention and Research Institute of Texas grant RP120326, NIH grant R21CA187717, The Cancer Center Support (Core) Grant CA016672 to MD Anderson Cancer Center.« less

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

Wainwright, W. W.

It is suggested that film speed is the most important single factor in reducing dental radiation exposure but has been given little attention. A necessary step in this direction is the application of quantitative film rating systems (of the type used in general radiography) to dental radiography and attention to exposure development factors. To this end, a sensitometric method is presented for measurement of undesired dental radiation overexposure resulting from underdevelopment. The method is based on a universal curve of density-log relative exposure for dental x-ray film. The curve is applicable to any given film and machine setting in intraoralmore » roentgenography. Correct exposure time can be predicted from the curve after exposure of only two dental films and use of a lead-aluminum penetrometer. This dental penetrometer and the universal sensitometric curve make it possible to conduct mass surveys of the amount of radiation overexposure from exposure-development factors in dental offices. An example of a typical determination of the effect of exposuredevelopment factors on radiation dose is given. The densities were measured with a densitometer in the range from 0 to 8. With an exposure of 1/2 sec and development for 1 1/2 min at 64 deg F, the hypothetical dentist obtained a density of 1.95 under aluminum. Full development gave a much greater density, 4.05, which was found by reference to the universal curve to represent a radiation exposure of 3.5 times normal. In other words, the underdevelopment (1 1/2 min at 64 deg F) was compensated by overexposure (1/2 sec), so that films of normal density could be obtained. The dentist was informed of the overexposure, and it was predicted that by dividing his time (1/2 sec) by the radiation exposure (3.5), with full development, he would be able to reduce exposure time from 0.5 to 0.143 sec. On the corrected film with an exposure time of 0.15 sec, the density is 1.72. By changing to full development, the dentist obtained normal density with 1/3 the amount of radiation.« less

78 FR 18234 - Section 482: Methods To Determine Taxable Income in Connection With a Cost Sharing Arrangement

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-26

... DEPARTMENT OF THE TREASURY Internal Revenue Service 26 CFR Part 1 Section 482: Methods To Determine Taxable Income in Connection With a Cost Sharing Arrangement CFR Correction 0 In Title 26 of the...) * * * (1) * * * See Sec. 1.482-8 for examples of the application of the best method rule...

75 FR 5852 - Proposed Collection; Comment Request for Regulation Project

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-04

... Method in De Minimis Cases (Sec. 1.460-6). DATES: Written comments should be received on or before April... INFORMATION: Title: Election Not to Apply Look-Back Method in De Minimis Cases. OMB Number: 1545-1572... may elect not to apply the look-back method to long-term contracts in de minimis cases. The taxpayer...

Behavior of Paramecium sp. in solutions containing Sr and Pb: Do Paramecium sp. alter chemical forms of those metals?

NASA Astrophysics Data System (ADS)

Kozai, Naofumi; Ohnuki, Toshihiko; Koka, Masahi; Satoh, Takahiro; Kamiya, Tomihiro

2011-10-01

The behavior of Paramecium sp. (Paramecium bursaria) in aqueous solutions containing Sr and Pb was investigated to determine the role of protozoa in the migration of radionuclides in the environment. Precultured living cells of P. bursaria were exposed to aqueous solutions containing 0.01 or 0.05 mM Sr or Pb at pH 7 for 24 h. For comparison, pre-killed cells were treated with the metal solutions in the same way. Two-dimensional elemental mappings of cells were obtained by micro-PIXE. Aquatic species of Sr and Pb were analyzed by size exclusion chromatography (SEC) coupled online to ultraviolet (UV) spectroscopy and inductivity coupled plasma mass spectroscopy (ICP-MS). The amounts of Sr adsorbed or taken up by the cells surviving for 24 h and adsorbed on pre-killed cells were below the detection limit. Cells of P. bursaria adsorbed or took up a fraction of Pb. The Pb adsorbed or taken up by the cells surviving for 24 h in the Pb solution was barely detectable, while the Pb adsorbed on pre-killed cells was clearly mappable. These findings suggest that living cells of P. bursaria have functions that reduce adsorption or uptake of Pb on the cells. Quantitative and SEC-UV-ICP-MS analyses of the Sr and Pb in aqueous phases showed no clear evidences that living cells of P. bursaria alter the chemical form of Sr or Pb remaining in the aqueous phases after the cell-solution contact.

Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia

PubMed Central

Paranjape, Geeta S.; Terrill, Shana E.; Gouwens, Lisa K.; Ruck, Benjamin M.; Nichols, Michael R.

2012-01-01

Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1–42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia. PMID:23242692

Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles

PubMed Central

Martin, Sophie G.

2012-01-01

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP2 and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk. PMID:22768263

Inhibition of selenocysteine tRNA[Ser]Sec aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA

PubMed Central

Kim, Jin Young; Carlson, Bradley A.; Xu, Xue-Ming; Zeng, Yu; Chen, Shawn; Gladyshev, Vadim N.; Lee, Byeong Jae; Hatfield, Dolph L.

2011-01-01

There are two isoforms of selenocysteine (Sec) tRNA[Ser]Sec that differ by a single methyl group, Um34. The non-Um34 isoform supports the synthesis of a subclass of selenoproteins, designated housekeeping, while the Um34 isoform supports the expression of another subclass, designated stress-related selenoproteins. Herein, we investigated the relationship between tRNA[Ser]Sec aminoacylation and Um34 synthesis which is the last step in the maturation of this tRNA. Mutation of the discriminator base at position 73 in tRNA[Ser]Sec dramatically reduced aminoacylation with serine, as did an inhibitor of seryl-tRNA synthetase, SB-217452. Although both the mutation and the inhibitor prevented Um34 synthesis, neither precluded the synthesis of any other of the known base modifications on tRNA[Ser]Sec following microinjection and incubation of the mutant tRNA[Ser]Sec transcript, or the wild type transcript along with inhibitor, in Xenopus oocytes. The data demonstrate that Sec tRNA[Ser]Sec must be aminoacylated for Um34 addition. The fact that selenium is required for Um34 methylation suggests that Sec must be attached to its tRNA for Um34 methylation. This would explain why selenium is essential for the function of Um34 methylase and provides further insights into the hierarchy of selenoprotein expression. PMID:21624347

[Analysis of microalbuminuria with immunonephelometry and high performance liquid chromatography. Evaluation of new criteria].

PubMed

Markó, Lajos; Molnár, Gergo Attila; Wagner, Zoltán; Koszegi, Tamás; Matus, Zoltán; Mohás, Márton; Kuzma, Mónika; Szijártó, István András; Wittmann, István

2008-01-13

Hypertension as well as type 2 diabetes mellitus is a major factor in population mortality. Both diseases damage the endothelium, the early sign of which is microalbuminuria, which can be screened by dipstick and can be diagnosed by using immuno-based and high performance liquid chromatography methods. Using high performance liquid chromatography, the non-immunoreactive albumin can be detected as well. The authors aimed at the examination of albuminuria in the case of immunonephelometrically negative patients with high performance liquid chromatography, in diabetic and hypertensive and non-diabetic hypertensive populations. The authors also wanted to compare the present (albumin-creatinine ratio: male: > or =2.5 mg/mmol, female: > or =3.5 mg/mmol) and a new criteria of the Heart Outcomes Prevention Evaluation study (patients without diabetes: immunological method, > or =0.7 mg/mmol; high performance liquid chromatography, > or =3.1 mg/mmol; individuals with diabetes: immunological method, > or =1.4 mg/mmol; high performance liquid chromatography, > or =5.2 mg/mmol) of microalbuminuria. Examination of fresh urines of 469 microalbuminuria negative patients by dipstick were performed by immunonephelometry. Patients, who were microalbuminuria negative by immunonephelometry as well, were further analyzed by high performance liquid chromatography using the Accumintrade mark Kit, based on size-exclusion chromatography. Three times higher albuminuria were found with high performance liquid chromatography than with immunonephelometry. The intraindividual coefficient of variation did not differ in the two methods (37 +/- 31% vs. 40 +/- 31%, p = 0.869; immunonephelometry vs. high performance liquid chromatography; mean +/- standard deviation). Using the present criteria for microalbuminuria, 43% of immunonephelometrically negative patients proved to be microalbuminuric by high performance liquid chromatography. Using the new criteria of the Heart Outcomes Prevention Evaluation study, the rate of microalbuminuria positivity among the immunonephelometrically negative patients decreased to 14.5% by high performance liquid chromatography and the decrease in the number of microalbuminuria positive cases by high performance liquid chromatography could be observed mainly in the diabetic and hypertensive group (49% vs. 7.5%), while slighter decrease could be observed in the non-diabetic hypertensive group (37% vs. 26.5%). Applying the traditional criteria, the strongest predictor was the male gender by the logistic regression analysis. In 28% of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established using high performance liquid chromatography. Almost in one-third of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established by high performance liquid chromatography for which diagnosis three constitutive urine examinations are still needed. New criteria determined by the Heart Outcomes Prevention Evaluation study can be used neither in case of diabetic and hypertensive patients, nor in the case of non-diabetic hypertensive patients. The gender as the most important predictor of microalbuminuria cannot be ignored.

Off-line exhaled nitric oxide measurements in children.

PubMed

Barreto, M; Villa, M P; Martella, S; Falasca, C; Guglielmi, F; Pagani, J; Darder, M T; Ronchetti, R

2001-08-01

The concentration of exhaled nitric oxide (eNO) is a useful marker of asthmatic bronchial inflammation. eNO can now be measured away from the laboratory (off-line), even in children. Short exhalation maneuvers (8 sec) and small samples (1 L) of exhaled gas are probably sufficient in children, but more information is needed about the effect of different measurement conditions. As a preliminary step before conducting epidemiological studies in schoolchildren, we investigated the effects of expiratory flow, dead space, and expiratory time on eNO concentrations collected in 1-L mylar collection bags. We studied 101 cooperative subjects (62 males) aged 5-18 years (30 healthy volunteers, 51 asthmatics, and 20 children with various other respiratory diseases) in our pulmonary function laboratory. On-line and off-line eNO were compared in a single session, and analyzed with a Sievers NOA 280 nitric oxide analyzer. For both methods of collecting expired gas, subjects did a single exhalation without breath-holding against an expiratory pressure 10 cm H(2)O. We investigated the effects of expiratory flow, dead space, and exhalation time on eNO; we also compared on-line and off-line eNO measurements, and the repeatability of both techniques at a given flow rate. Expiratory flows of 58 mL/sec provided more reproducible data than lower flows (coefficient of repeatability 1.1 ppb for 58 mL/sec vs. 2.8 for 27 mL/sec vs. 5.7 for 18 mL/sec). eNO concentrations were about 25% higher in off-line than in on-line recordings if the initial 250 mL of exhaled gas were not eliminated, and 37% higher if exhalation lasted longer (16 sec vs. 8 sec). Eliminating 250 mL of dead space and shortening the filling time to 8 sec yielded off-line eNO values close to those on-line (geometric mean off-line eNO 14.4 ppb, 95% confidence interval: 12.2-17.0) vs. on-line eNO 13.8 ppb (95% confidence interval: 11.6-16.5). On-line and off-line results were highly correlated (r = 0.996, P = 0.000) and had similar coefficients of variation (on-line eNO 2.6%, off-line 2.8%). Neither agreement nor repeatability of eNO measurements were affected by disease status or baseline FEV(1) (% predicted values). Once standardized, the off-line eNO technique using 1-L gas collection bags will provide results similar to those recorded on-line. Copyright 2001 Wiley-Liss, Inc.

Transposon-disruption of a maize nuclear gene, tha1, encoding a chloroplast SecA homologue: in vivo role of cp-SecA in thylakoid protein targeting.

PubMed

Voelker, R; Mendel-Hartvig, J; Barkan, A

1997-02-01

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced > or = 40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

Association of early- and adult-life socioeconomic circumstances with muscle strength in older age.

PubMed

Cheval, Boris; Boisgontier, Matthieu P; Orsholits, Dan; Sieber, Stefan; Guessous, Idris; Gabriel, Rainer; Stringhini, Silvia; Blane, David; van der Linden, Bernadette W A; Kliegel, Matthias; Burton-Jeangros, Claudine; Courvoisier, Delphine S; Cullati, Stéphane

2018-05-01

socioeconomic circumstances (SEC) during a person's lifespan influence a wide range of health outcomes. However, solid evidence of the association of early- and adult-life SEC with health trajectories in ageing is still lacking. This study assessed whether early-life SEC are associated with muscle strength in later life-a biomarker of health-and whether this relationship is caused by adult-life SEC and health behaviours. we used data from the Survey of Health Ageing and Retirement in Europe, a 12-year population-based cohort study with repeated measurement in six waves (2004-15) and retrospective collection of life-course data. Participants' grip strength was assessed by using a handheld dynamometer. Confounder-adjusted logistic mixed-effect models were used to examine the associations of early- and adult-life SEC with the risk of low muscle strength (LMS) in older age. a total of 24,179 participants (96,375 observations) aged 50-96 living in 14 European countries were included in the analyses. Risk of LMS was increased with disadvantaged relative to advantaged early-life SEC. The association between risk of LMS and disadvantaged early-life SEC gradually decreased when adjusting for adult-life SEC for both sexes and with unhealthy behaviours for women. After adjusting for these factors, all associations between risk of LMS and early-life SEC remained significant for women. early-life SEC are associated with muscle strength after adjusting for adult-life SEC and behavioural lifestyle factors, especially in women, which suggests that early life may represent a sensitive period for future health.

Development of Impurity Profiling Methods Using Modern Analytical Techniques.

PubMed

Ramachandra, Bondigalla

2017-01-02

This review gives a brief introduction about the process- and product-related impurities and emphasizes on the development of novel analytical methods for their determination. It describes the application of modern analytical techniques, particularly the ultra-performance liquid chromatography (UPLC), liquid chromatography-mass spectrometry (LC-MS), high-resolution mass spectrometry (HRMS), gas chromatography-mass spectrometry (GC-MS) and high-performance thin layer chromatography (HPTLC). In addition to that, the application of nuclear magnetic resonance (NMR) spectroscopy was also discussed for the characterization of impurities and degradation products. The significance of the quality, efficacy and safety of drug substances/products, including the source of impurities, kinds of impurities, adverse effects by the presence of impurities, quality control of impurities, necessity for the development of impurity profiling methods, identification of impurities and regulatory aspects has been discussed. Other important aspects that have been discussed are forced degradation studies and the development of stability indicating assay methods.

76 FR 41993 - Customs and Border Protection Officer Retirement

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-18

... Border Protection Officers Sec. 831.1601 Applicability and purpose. 831.1602 Definitions. 831.1603... position is a prerequisite. Sec. 831.1603 Conditions for coverage in primary positions. (a) An employee's... determination under Sec. Sec. 831.1603(a) and 831.1604(a) must be based solely on the official position...

75 FR 13555 - Compliance Policy Guide Sec. 540.375 Canned Salmon - Adulteration Involving Decomposition (CPG...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-22

...] (Formerly Docket No. 1998N-0046) Compliance Policy Guide Sec. 540.375 Canned Salmon -- Adulteration... of Compliance Policy Guide Sec. 540.375 Canned Salmon -- Adulteration Involving Decomposition (CPG... relating to decomposition in fish and fishery products, including canned salmon, is provided in CPG Sec...

Preconcentration for Improved Long-Term Monitoring of Contaminants in Groundwater: Sorbent Development

DTIC Science & Technology

2013-02-11

calibration curves was ±5%. Ion chromatography (IC) was used for analysis of perchlorate and other ionic targets. Analysis was carried out on a...The methods utilize liquid or gas chromatography , techniques that do not lend themselves well to portable devices and methods. Portable methods are...

Rapid characterization of the chemical constituents of Cortex Fraxini by homogenate extraction followed by UHPLC coupled with Fourier transform ion cyclotron resonance mass spectrometry and GC-MS.

PubMed

Wang, Yinan; Han, Fei; Song, Aihua; Wang, Miao; Zhao, Min; Zhao, Chunjie

2016-11-01

Cortex Fraxini is an important traditional Chinese medicine. In this work, a rapid and reliable homogenate extraction method was applied for the fast extraction for Cortex Fraxini, and the method was optimized by response surface methodology. Ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry and gas chromatography with mass spectrometry were established for the separation and characterization of the constituents of Cortex Fraxini. Liquid chromatography separation was conducted on a C 18 column (150 mm × 2.1 mm, 1.8 μm), and gas chromatography separation was performed on a capillary with a 5% phenyl-methylpolysiloxane stationary phase (30 m × 0.25 mm × 0.25 mm) by injection of silylated samples. According to the results, 33 chemical compounds were characterized by liquid chromatography with mass spectrometry, and 11 chemical compounds were characterized by gas chromatography with mass spectrometry, and coumarins were the major components characterized by both gas chromatography with mass spectrometry and liquid chromatography with mass spectrometry. The proposed homogenate extraction was an efficient and rapid method, and coumarins, phenylethanoid glycosides, iridoid glycosides, phenylpropanoids, and lignans were the main constituents of Cortex Fraxini. This work laid the foundation for further study of Cortex Fraxini and will be helpful for the rapid extraction and characterization of ingredients in other traditional Chinese medicines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

PubMed

Lu, Shaoying; Ouyang, Mingxing; Seong, Jihye; Zhang, Jin; Chien, Shu; Wang, Yingxiao

2008-07-25

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2)/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2)/sec and outside: 0.18+/-0.02 microm(2)/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF) stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

Noise Reduction of 1sec Geomagnetic Observatory Data without Information Loss

NASA Astrophysics Data System (ADS)

Brunke, Heinz-Peter; Korte, Monika; Rudolf, Widmer-Schnidrig

2017-04-01

Traditional fluxgate magnetometers used at geomagnetic observatories are optimized towards long-term stability. Typically, such instruments can only resolve background geomagnetic field variations up to a frequency of approximately 0.04 Hz and are limited by instrumental self-noise above this frequency. However, recently the demand for low noise 1 Hz observatory data has increased. IAGA has defined a standard for definitive 1sec data. Induction coils have low noise at these high frequencies, but lack long-term stability. We present a method to numerically combine the data from a three axis induction coil system with a typical low-drift observatory fluxgate magnetometer. The resulting data set has a reduced noise level above 0.04 Hz while maintaining the long term stability of the fluxgate magnetometer. Numerically we fit a spline to the fluxgate data. But in contrast to such a low pass filtering process, our method reduces the noise level at high frequencies without any loss of information. In order to experimentally confirm our result, we compared it to a very low noise scalar magnetometer: an optically pumped potassium magnetometer. In the frequency band from [0.03Hz to 0.5Hz] we found an rms-noise reduction from 80pT for the unprocessed fluxgate data to about 25pT for the processed data. We show how our method improves geomagnetic 1 sec observatory data for, e.g., the study of magnetospheric pulsations and EMIC waves.

[Development of methods for determining acrylamide in food products by gas-liquid chromatography].

PubMed

Bessonov, V V; Malinkin, A D; Perederiaev, O I; Bogachuk, M N; Volkovich, S V; Medvedev, Iu V

2011-01-01

The method of determination of acrylamide in various food (milk powder, potato chips, instant coffee) by gas-liquid chromatography after pre-bromination was developed. Studies have shown the possibility of using bromination of acrylamide to give it the necessary properties for better extraction, purification and detection. Also revealed the possibility of qualitative and quantitative determine a acrylamide in food by gas-liquid chromatography with detection by electron capture detector.