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  1. Indole-3-Acetic Acid is produced by Emiliania Huxleyi Coccolith-bearing cells and triggers a physiological response in bald cells [Download]

    Title: Indole-3-Acetic Acid is produced by Emiliania Huxleyi Coccolith-bearing cells and triggers a physiological response in bald cells
    Creator: Labeeuw, Leen
    Description: Indole-3-acetic acid (IAA) is an auxin produced by terrestrial plants which influences development through a variety of cellular mechanisms, such as altering cell orientation, organ development, fertility, and cell elongation. IAA is also produced by bacterial pathogens and symbionts of plants and algae, allowing them to manipulate growth and development of their host. They do so by either producing excess exogenous IAA or hijacking the IAA biosynthesis pathway of their host. The endogenous production of IAA by algae remains contentious. Using Emiliania huxleyi, a globally abundant marine haptophyte, we investigated the presence and potential role of IAA in algae. Homologs of genes involved in several tryptophan-dependent IAA biosynthesis pathways were identified in E. huxleyi. This suggests that this haptophyte can synthesize IAA using various precursors derived from tryptophan. Addition of L-tryptophan to E. huxleyi stimulated IAA production, which could be detected using Salkowski’s reagent and GC×GC-TOFMS in the C cell type (coccolith bearing), but not in the N cell type (bald). Various concentrations of IAA were exogenously added to these two cell types to identify a physiological response in E. huxleyi. The N cell type, which did not produce IAA, was more sensitive to it, showing an increased variation in cell size, membrane permeability, and a corresponding increase in the photosynthetic potential quantum yield of Photosystem II (PSII). A roseobacter (bacteria commonly associated with E. huxleyi) Ruegeria sp. R11, previously shown to produce IAA, was co-cultured with E. huxleyi C and N cells. IAA could not be detected from these co-cultures, and even when stimulated by addition of L-tryptophan, they produced less IAA than axenic C type culture similarly induced. This suggests that IAA plays a novel role signaling between different E. huxleyi cell types, rather than between a bacteria and its algal host.
    Subjects: Algae, Auxin, Cell Type, Coccolith, Emiliania Huxleyi, Haptophyte, Indole-3-Acetic Acid (Iaa), Phytohormone
    Date Created: 2016
  2. Classifying Crystal Structures of Binary Compounds AB through Cluster Resolution Feature Selection and Support Vector Machine Analysis [Download]

    Title: Classifying Crystal Structures of Binary Compounds AB through Cluster Resolution Feature Selection and Support Vector Machine Analysis
    Creator: Oliynyk, Anton O.
    Description: Partial least-squares discriminant analysis (PLS-DA) and support vector machine (SVM) techniques were applied to develop a crystal structure predictor for binary AB compounds. Models were trained and validated on the basis of the classification of 706 AB compounds adopting the seven most common structure types (CsCl, NaCl, ZnS, CuAu, TlI, β-FeB, and NiAs), through data extracted from Pearson’s Crystal Data and ASM Alloy Phase Diagram Database. Out of 56 initial variables (descriptors based on elemental properties only), 31 were selected in as unbiased manner as possible through a procedure of forward selection and backward elimination, with the quality of the model evaluated by measuring the cluster resolution at each step. PLS-DA gave sensitivity of 96.5%, specificity of 66.0%, and accuracy of 77.1% for the validation set data, whereas SVM gave sensitivity of 94.2%, specificity of 92.7%, and accuracy of 93.2%, a significant improvement. Radii, electronegativity, and valence electrons, previously chosen intuitively in structure maps, were confirmed as important variables. PLS-DA and SVM could also make quantitative predictions of hypothetical compounds, unlike semiclassical approaches. The new compound RhCd was predicted to have the CsCl-type structure by PLS-DA (0.669 probability) and, at an even stronger confidence level, by SVM (0.918 probability). RhCd was synthesized by reaction of the elements at 800 °C and confirmed by X-ray diffraction to adopt the CsCl-type structure. SVM is thus a superior classification method in crystallography that is fast and makes correct, quantitative predictions; it may be more broadly applicable to help identify the structure of unknown compounds with any arbitrary composition.
    Subjects: Automotive Gasoline Samples, Principal Component Analysis, Ionization-Potentials, Neural-Networks, Prediction, Scale, Electronegativity, Classification, Optimization, Informatics
    Date Created: 2016
  3. Unique Ion Filter: A data reduction tool for GC/MS data preprocessing prior to chemometric analysis [Download]

    Title: Unique Ion Filter: A data reduction tool for GC/MS data preprocessing prior to chemometric analysis
    Creator: Adutwum, Lawrence A.
    Description: Using raw GC/MS data as the X-block for chemometric modeling has the potential to provide better classification models for complex samples when compared to using the total ion current (TIC), extracted ion chromatograms/profiles (EIC/EIP), or integrated peak tables. However, the abundance of raw GC/MS data necessitates some form of data reduction/feature selection to remove the variables containing primarily noise from the data set. Several algorithms for feature selection exist; however, due to the extreme number of variables (106–108 variables per chromatogram), the feature selection time can be prolonged and computationally expensive. Herein, we present a new prefilter for automated data reduction of GC/MS data prior to feature selection. This tool, termed unique ion filter (UIF), is a module that can be added after chromatographic alignment and prior to any subsequent feature selection algorithm. The UIF objectively reduces the number of irrelevant or redundant variables in raw GC/MS data, while preserving potentially relevant analytical information. In the m/z dimension, data are reduced from a full spectrum to a handful of unique ions for each chromatographic peak. In the time dimension, data are reduced to only a handful of scans around each peak apex. UIF was applied to a data set of GC/MS data for a variety of gasoline samples to be classified using partial least-squares discriminant analysis (PLS-DA) according to octane rating. It was also applied to a series of chromatograms from casework fire debris analysis to be classified on the basis of whether or not signatures of gasoline were detected. By reducing the overall population of candidate variables subjected to subsequent variable selection, the UIF reduced the total feature selection time for which a perfect classification of all validation data was achieved from 373 to 9 min (98% reduction in computing time). Additionally, the significant reduction in included variables resulted in a concomitant reduction in noise, improving overall model quality. A minimum of two um/z and scan window of three about the peak apex could provide enough information about each peak for the successful PLS-DA modeling of the data as 100% model prediction accuracy was achieved. It is also shown that the application of UIF does not alter the underlying chemical information in the data.
    Subjects: Principle Component Analysis, Automotive Gasoline Samples, Gas Chromatography/Mass Spectrometry, Mass Spectrometry, Feature-Selection, Pattern-Recognition, Oil-Spill, Classification, Gc, Ms
    Date Created: 2014
  4. Determination of Hydrocarbon Group-Type of Diesel Fuels by Gas Chromatography with Vacuum Ultraviolet Detection [Download]

    Title: Determination of Hydrocarbon Group-Type of Diesel Fuels by Gas Chromatography with Vacuum Ultraviolet Detection
    Creator: Weber, Brandon M.
    Description: A GC-vacuum ultraviolet (UV) method to perform group-type separations of diesel range fuels was developed. The method relies on an ionic liquid column to separate diesel samples into saturates, mono-, di-, and polyaromatics by gas chromatography, with selective detection via vacuum UV absorption spectroscopy. Vacuum UV detection was necessary to solve a coelution between saturates and monoaromatics. The method was used to measure group-type composition of 10 oilsands-derived Synfuel light diesel samples, 3 Syncrude light gas oils, and 1 quality control sample. The gas chromatography (GC)-vacuum UV results for the Synfuel samples were similar (absolute % error of 0.8) to historical results from the supercritical fluid chromatography (SFC) analysis. For the light gas oils, discrepancies were noted between SFC results and GC-vacuum UV results; however, these samples are known to be challenging to quantify by SFC-flame ionization detector (FID) due to incomplete resolution between the saturate/monoaromatic and/or monoaromatic/diaromatic group types when applied to samples heavier than diesel (i.e., having a larger fraction of higher molecular weight species). The quality control sample also performed well when comparing both methods (absolute % error of 0.2) and the results agreed within error for saturates, mono- and polyaromatics.
    Subjects: Supercritical-Fluid Chromatography, Mass-Spectrometry, Middle Distillates, Petroleum, Separations, Aromatics, Hplc, Gc, Biodiesel, Products
    Date Created: 2016
  5. Energetics of intermolecular hydrogen bonds in a hydrophobic protein cavity [Download]

    Title: Energetics of intermolecular hydrogen bonds in a hydrophobic protein cavity
    Creator: Liu, Lan
    Description: This work explores the energetics of intermolecular H-bonds inside a hydrophobic protein cavity. Kinetic measurements were performed on the gaseous deprotonated ions (at the −7 charge state) of complexes of bovine β-lactoglobulin (Lg) and three monohydroxylated analogs of palmitic acid (PA): 3-hydroxypalmitic acid (3-OHPA), 7-hydroxypalmitic acid (7-OHPA), and 16-hydroxypalmitic acid (16-OHPA). From the increase in the activation energy for the dissociation of the (Lg + X-OHPA)7– ions, compared with that of the (Lg + PA)7– ion, it is concluded that the –OH groups of the X-OHPA ligands participate in strong (5 – 11 kcal mol–1) intermolecular H-bonds in the hydrophobic cavity of Lg. The results of molecular dynamics (MD) simulations suggest that the –OH groups of 3-OHPA and 16-OHPA act as H-bond donors and interact with backbone carbonyl oxygens, whereas the –OH group of 7-OHPA acts as both H-bond donor and acceptor with nearby side chains. The capacity for intermolecular H-bonds within the Lg cavity, as suggested by the gas-phase measurements, does not necessarily lead to enhanced binding in aqueous solution. The association constant (Ka) measured for 7-OHPA [(2.3 ± 0.2) × 105 M–1] is similar to the value for the PA [(3.8 ± 0.1) × 105 M–1]; Ka for 3-OHPA [(1.1 ± 0.3) × 106 M–1] is approximately three-times larger, whereas Ka for 16-OHPA [(2.3 ± 0.2) × 104 M–1] is an order of magnitude smaller. Taken together, the results of this study suggest that the energetic penalty to desolvating the ligand –OH groups, which is necessary for complex formation, is similar in magnitude to the energetic contribution of the intermolecular H-bonds.
    Subjects: Hydrogen bonds, Kinetics energetics, Hydrophobic, Protein–ligand complexes
    Date Created: 2014
  6. Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile [Download]

    Title: Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile
    Creator: Murase, Tomohiko
    Description: Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.
    Subjects: Mass Spectrometry (MS), Protein-Protein Interactions, X-ray Crystallography, Antibodies, Bacterial Toxins
    Date Created: 2014
  7. Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein [Download]

    Title: Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein
    Creator: Hug, Isabelle
    Description: Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications.
    Subjects: Bacterial Glycoproteins, Glycoengineering, Glycoprotein, Glycoconjugate, Fucosyltransferase, Helicobacter pylori, Glycosyltransferases, Glycosylation, Oligosaccharyltansferase
    Date Created: 2011
  8. Thermal dissociation of streptavidin homotetramer in the gas phase: Subunit loss versus backbone fragmentation [Download]

    Title: Thermal dissociation of streptavidin homotetramer in the gas phase: Subunit loss versus backbone fragmentation
    Creator: Kitova, Elena N.
    Description: The results of time-resolved blackbody infrared radiative dissociation experiments performed on gaseous protonated ions, at charge states +15, +16 and +17, of the homotetramer streptavidin (S4) are reported. Evidence is found for three dissociation pathways involving (i) the loss of a single subunit, (ii) covalent cleavage of the backbone of one of the subunits and ejection of the resultant b212+ ion (followed by loss of complementary y106 ions), and (iii) the direct loss of one or more water molecules. The contribution of the different dissociation channels was found to be dependent on temperature, with the loss of subunit dominating at higher reaction temperatures and backbone fragmentation dominating at lower temperatures, and reaction times, with longer times favoring covalent fragmentation. Analysis of the dissociation kinetics and the influence of reaction time on the relative abundance of product ions indicate that backbone fragmentation and subunit loss are not produced via parallel pathways from a single reactant. Instead, the results suggest the presence of multiple, non-interconverting structures, which contribute differentially to the backbone fragmentation and subunit loss pathways. The results of molecular dynamics simulations performed on S416+ ions with different charge configurations suggest that unfolding of the N-terminus of the subunit may be associated with the backbone fragmentation pathway.
    Subjects: Blackbody infrared radiative dissociation, Dissociation kinetics, Protein complexes, Dissociation pathways
    Date Created: 2013
  9. Dissociation of multisubunit protein-ligand complexes in the gas phase: Evidence for ligand migration [Download]

    Title: Dissociation of multisubunit protein-ligand complexes in the gas phase: Evidence for ligand migration
    Creator: Zhang, Yixuan
    Description: The results of collision-induced dissociation (CID) experiments performed on gaseous protonated and deprotonated ions of complexes of cholera toxin B subunit homopentamer (CTB5) with the pentasaccharide (β-D-Galp-(1→3)-β-D-GalpNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Galp-(1→4)-β-D-Glcp (GM1)) and corresponding glycosphingolipid (β-D-Galp-(1→3)-β-D-GalpNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Galp-(1→4)-β-D-Glcp-Cer (GM1-Cer)) ligands, and the homotetramer streptavidin (S4) with biotin (B) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (Btl), are reported. The protonated (CTB5 + 5GM1)n+ ions dissociated predominantly by the loss of a single subunit, with the concomitant migration of ligand to another subunit. The simultaneous loss of ligand and subunit was observed as a minor pathway. In contrast, the deprotonated (CTB5 + 5GM1)n- ions dissociated preferentially by the loss of deprotonated ligand; the loss of ligand-bound and ligand-free subunit were minor pathways. The presence of ceramide (Cer) promoted ligand migration and the loss of subunit. The main dissociation pathway for the protonated and deprotonated (S4 + 4B)n+/– ions, as well as for deprotonated (S4 + 4Btl)n– ions, was loss of the ligand. However, subunit loss from the (S4 + 4B)n+ ions was observed as a minor pathway. The (S4 + 4Btl)n+ ions dissociated predominantly by the loss of free and ligand-bound subunit. The charge state of the complex and the collision energy were found to have little effect on the relative contribution of the different dissociation channels. Thermally-driven ligand migration between subunits was captured in the results of molecular dynamics simulations performed on protonated (CTB5 + 5GM1)15+ ions (with a range of charge configurations) at 800 K. Notably, the migration pathway was found to be highly dependent on the charge configuration of the ion. The main conclusion of this study is that the dissociation pathways of multisubunit protein–ligand complexes in the gas phase depend, not only on the native topology of the complex, but also on structural changes that occur upon collisional activation.
    Subjects: Collision-induced dissociation, Dissociation mechanism, Multisubunit protein complex, Ligand migration
    Date Created: 2013
  10. Nonspecific interactions between proteins and charged biomolecules in electrospray ionization mass spectrometry [Download]

    Title: Nonspecific interactions between proteins and charged biomolecules in electrospray ionization mass spectrometry
    Creator: Sun, Nian
    Description: An investigation of the nonspecific association of small charged biomolecules and proteins in electrospray ionization mass spectrometry (ES-MS) is described. Aqueous solutions containing pairs of proteins and a small acidic or basic biomolecule that does not interact specifically with either of the proteins were analyzed by ES-MS and the distributions of the biomolecules bound nonspecifically to each pair of proteins compared. For the basic amino acid arginine and the peptide RGVFRR, nonequivalent distributions were measured in positive ion mode, but equivalent distributions were measured in negative ion mode. In the case of uridine 5′-diphosphate, nonequivalent distributions were measured in negative ion mode, but equivalent distributions observed in positive ion mode. The results of dissociation experiments performed on the gaseous ions of the nonspecific complexes suggest that the nonequivalent distributions result from differences in the extent to which the nonspecific complexes undergo in-source dissociation. To test this hypothesis, the distributions of nonspecifically bound basic molecules measured in the presence of imidazole, which protects complexes from in-source dissociation, were compared. In all cases, equivalent distributions were obtained. The results indicate that nonspecific binding of charged molecules to proteins during ES is a statistical process, independent of protein structure and size. However, the kinetic stabilities of the nonspecific interactions are sensitive to the nature of the protein ions. It is concluded that the reference protein method for correcting ES mass spectra for nonspecific ligand-protein binding can be applied to the analysis of ionic ligands, provided that in-source dissociation of the nonspecific interactions is minimized.
    Subjects: Affinities, Binding, Noncovalent complexes, Ligand complexes, Constants, Stability, Analogs, Nanoelectrospray ionization, Infrared radiative dissociation
    Date Created: 2010