Intervening with Urinary Tract Infections Using Anti-Adhesives Based on the Crystal Structure of the FimH–Oligomannose-3 Complex

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and alpha3beta1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.We demonstrate that alpha-D-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl alpha-D-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl alpha-D-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl alpha-D-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Man alpha1,3Man beta1,4GlcNAc beta1,4GlcNAc in an extended binding site. The interactions along the alpha1,3 glycosidic bond and the first beta1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl alpha-D-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for '' new '' microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-13), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l(-1) of FOS from a 500 g l(-1) sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates. (c) 2007 Elsevier B.V. All rights reserved.4941730434

Purification and characterisation of a fructosyltransferase from Rhodotorula sp.

The present work was devoted to investigations concerning the purification and characterisation of the fructooligosaccharide (FOS)-producing extracellular enzyme of Rhodotorula sp. LEB-V10. FOS are functional food ingredients showing prebiotic properties, meaning that it could stimulate selectively the growth and/or activity of probiotic bacteria in the gut. The purification of the enzyme was carried out according to the following sequential procedure: cell separation by centrifugation, recovering by ethanol precipitation and purification by anion exchange chromatography. The molecular weight was estimated to be 170 kDa by preparative gel filtration and 77 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, signifying that the native enzyme exists as a dimer. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, rather showing a sigmoid shape similar to that of the allosteric enzymes (cooperative behaviour), requiring high sucrose concentrations to obtain high reaction rates. The enzyme showed both fructofuranosidase (FA) and fructosyl-transferase (FTA) activities. The optimum pH and temperature for FA activity were found to be around 4.0 and 72-75 degrees C, respectively, while FTA showed optimum activity at pH 4.5 and 65-70 degrees C. Both activities were very stable at temperatures below 66 degrees C, while for FA, the enzyme was more stable at pH 4.0 and for FTA at pH 5.0.79458959

Properties of thermostable extracellular FOS-producing fructofuranosidase from Cryptococcus sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis-Menten behaviour, showing a substrate inhibitory model. The K (m), K (i) and v (max) values were shown to be 64 mM, 3 M and 159.6 mu mol mL(-1) min(-1) for FA and 131 mM, 1.6 M and 377.8 mu mol mL(-1) min(-1) for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 degrees C, while the best stability was achieved at pH 4.5 and temperatures below 60 degrees C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).228221322

Numerical Simulations to Evaluate the Performance of CERN PS Dummy Septum to Reduce Irradiation for the Multi-Turn Extraction

The losses created by the proposed Multi-Turn Extraction (MTE) at the CERN PS induce high activation of the magnetic extraction septum due to the de-bunched longitudinal beam structure requested to transfer the beam to the SPS. A mitigation measure is under study aiming at localizing the losses in a well-shielded area by shadowing the magnetic extraction septum thanks to a septum-like passive device. Such a solution is based on a so-called dummy septum, a blade which absorbs particles during the rise time of the extraction kickers for MTE beams. The efficiency of the scheme is presented in this paper. The quantitative estimate is based on detailed simulations that analyze the beam-matter interaction and provide a determination of the shadowing effect of the dummy septum

Effect Of Feeding Strategies On Lipid Production By Lipomyces Starkeyi

The aim of this study was to produce microbial oil from Lipomyces starkeyi DSM 70296 grown in hemicellulose hydrolysate (H-H). Glucose and xylose were used for batch, fed-batch, repeated fed-batch, and continuous cultures, and H-H was tested at continuous culture. The highest cell and lipid concentrations of 85.4 and 41.8g/L, respectively, were obtained using repeated fed-batch strategy. Continuous culture with dilution rate of 0.03h-1 presented the highest overall cell (0.443g/g) and lipid yields (0.236g/g). At 0.06h-1 were obtained the highest cell and lipid productivities. Continuous cultivation using H-H at 0.03h-1 resulted in higher cell productivity than that obtained using glucose:xylose. Gas chromatography analysis of the esterified lipids indicated that the major constituents of this complex are palmitic acid, stearic acid, oleic acid, and linoleic acid with an estimated cetane number (approximately 61) similar to that of palm biodiesel, which is important for biofuel production. © 2014.157214222Aggelis, G., Komaitis, M., Enhancement of single cell oil production by Yarrowia lipolytica growing in the presence of Teucrium polium L. aqueous extract (1999) Biotechnol. Lett., 21, pp. 747-749Evans, C.T., Ratledge, C., A comparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture (1983) Lipids, 18, pp. 623-629Galafassi, S., Cucchetti, D., Pizza, F., Franzosi, G., Bianchi, D., Compagno, C., Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis (2012) Bioresour. Technol., 111, pp. 398-403Gill, C.O., Hall, M.J., Ratledge, C., Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single stage continuous culture (1977) Appl. Eviron. Microb., 33, pp. 231-239Gray, J.V., Petsko, G.A., Johnston, G.C., Ringe, D., Singer, R.A., Werner-Washburne, M., "Sleeping beauty": quiescence in Saccharomyces cerevisiae (2004) Microbiol. Mol. Biol. Rev., 68, pp. 187-206He, M., Hu, Q., Gou, X., Liu, X., Li, Q., Pan, K., Screening of oleaginous yeast with xylose assimilating capacity for lipid and bio-ethanol production (2010) Afr. J. Biotechnol., 9, pp. 8392-8397Hu, C., Zhao, X., Zhao, J., Wu, S., Zhao, Z.K., Effects of biomass hydrolysis by-products on oleaginous yeast Rhodosporidium toruloides (2009) Bioresour. Technol., 100, pp. 4843-4847Hu, C., Wu, S., Wang, Q., Jin, G., Shen, H., Zhao, Z.K., Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum (2011) Biotechnol. Biofuels, 4, pp. 25-32Huang, C., Zong, M.H., Wu, H., Liu, Q.P., Microbial oil production from rice straw hydrolysate by Trichosporon fermentans (2009) Bioresour. Technol., 100, pp. 4535-4538Huang, C., Wu, H., Li, R.-F., Zong, M.-H., Improving lipid production from bagasse hydrolysate with Trichosporon fermentans by response surface methodology (2012) New Biotechnol., 29, pp. 372-378Kawaguchi, H., Vertes, A.A., Okino, S., Inui, M., Yukawa, H., Engineering of a xylose metabolic pathway in Corynebacterium glutamicum (2006) Appl. Environ. Microbiol., 72, pp. 3418-3428Klopfenstein, W.E., Effect of molecular weights of fatty acid esters on cetane numbers as diesel fuels (1985) J. Am. Oil Chem. Soc., 62, pp. 1029-1031Knothe, G., "Designer" biodiesel: optimizing fatty ester composition to improve fuel properties (2008) Energ. Fuel, 22, pp. 1358-1364Krisnangkura, K., A simple method for estimation of cetane index of vegetable oil methyl esters (1986) J. Am. Oil Chem. Soc., 63, pp. 552-553Kun, L.Y., Bioprocess technology (2006) Microbial Biotechnology Principles and Aplications, , World Scientific Publishing Co. Pte. Ltd., Singapore, (Chapter 2), L.Y. Kun (Ed.)Lewis, T., Nichols, P.D., McMeekin, T.A., Evaluation of extraction methods for recovery of fatty acids from lipid producing microheterotrophs (2000) J. Microbiol. Methods, 43, pp. 107-116Li, Y.H., Zhao, Z.B., Bai, F.W., High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture (2007) Enzyme Microb. Technol., 41, pp. 312-317Lin, J., Shen, H., Tan, H., Zhao, X., Wu, S., Hu, C., Zhao, Z.K., Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients (2011) J. Biotechnol., 152, pp. 184-188Liu, B., Zhao, Z., Biodiesel production by direct methanolysis of oleaginous microbial biomass (2007) J. Chem. Technol. Biotechnol., 82, pp. 775-780Liu, Y.F., Meng, Z., Zhang, F.Q., Shan, L., Wang, X.G., Influence of lipid composition, crystallization behavior and microstructure on hardness of palm oil-based margarines (2010) Eur. Food Res. Technol., 230, pp. 759-767Liu, J.X., Yue, Q.Y., Gao, B.Y., Ma, Z.H., Zhang, P.D., Microbial treatment of the monosodium glutamate wastewater by Lipomyces starkeyi to produce microbial lipid (2012) Bioresour. Technol., 106, pp. 69-73Lobo, I.P., Ferreira, S.L.C., Cruz, R., Biodiesel: quality parameters and analytical methods (2009) Quim. Nova, 32, pp. 1596-1608Maleki, E., Aroua, M.K., Sulaiman, N.M.N., Improved yield of solvent free enzymatic methanolysis of palm and jatropha oils blended with castor oil (2013) Appl. Energy, 104, pp. 905-909Norhaizan, M.E., Hosseini, S., Gangadaran, S., Lee, S.T., Kapourchali, F.R., Moghadasian, M.H., Palm oil: features and applications (2013) Lipid Technol., 25, pp. 39-42Palmqvist, E., Hahn-Hägerdal, B., Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition (2000) Bioresour. Technol., 74, pp. 25-33Papanikolaou, S., Aggelis, G., Lipid production by Yarrowia lipolytica growing on industrial glycerol in a single stage continuous culture (2002) Bioresour. Technol., 82, pp. 43-49Ratledge, C., Cohen, Z., Microbial and algal oils: do they have a future for biodiesel or as commodity oils? (2008) Lipid Technol., 20, pp. 155-160Sattur, A.P., Karanth, N.G., Production of microbial lipids 1. Development of a mathematical-model (1989) Biotechnol. Bioeng., 34, pp. 863-867Sharma, Y.C., Singh, B., Upadhyay, S.N., Advancements in development and characterization of biodiesel: a review (2008) Fuel, 87, pp. 2355-2373Shen, H., Gong, Z., Yang, X., Jin, G., Bai, F., Zhao, Z.K., Kinetics of continuous cultivation of the oleaginous yeast Rhodosporidium toruloides (2013) J. Biotechnol., 168, pp. 85-89Tapia, E.V., Anschau, A., Coradini, A.L., Franco, T.T., Deckmann, A.C., Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening (2012) AMB Expr., 2, pp. 64-71Wild, R., Patil, S., Popovic, M., Zappi, M., Dufreche, S., Bajpai, R., Lipids from Lipomyces starkeyi (2010) Food Technol. Biotechnol., 48, pp. 329-335Zhang, J., Fang, X.L., Zhu, X., Li, Y., Xu, H., Zhao, B., Chen, L., Zhang, X., Microbial lipid production by the oleaginous yeast Cryptococcus curvatus O3 grown in fed-batch culture (2011) Biomass Bioenergy, 35, pp. 1906-1911Zhao, X., Kong, X.L., Hua, Y.Y., Feng, B., Zhao, Z.B., Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi (2008) Eur. J. Lipid Sci. Technol., 110, pp. 405-412Zhao, X., Hu, C., Wu, S., Shen, H., Zhao, Z.K., Lipid production by Rhodosporidium toruloides Y4 using different substrate feeding strategies (2011) J. Ind. Microbiol. Biotechnol., 38, pp. 627-63

Optimization of Trehalose Production by Rhodotorula dairenensis Following a Sequential Strategy of Experimental Design

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Initially, a technical screening was applied to select yeasts with a potential for trehalose production using thermal stress for the induction of synthesis. The effects of the concentrations of sugar cane molasses, corn steep liquor and a commercial yeast extract Prodex Lac SD (R), and of pH and temperature on the biomass were studied using a fractional design followed by a central composite rotatable design (CCRD). The optimum values were 50 g/l for the molasses and corn steep liquor concentrations, initial pH of 5.5, and temperature of 30 degrees C, with no yeast extract. Under these conditions, the production of trehalose was studied using a CCRD to optimize the temperature (33 to 47 degrees C) and exposition time (60 to 120 min) of the cultures to the thermal stress. The maximum intracellular trehalose content reached was 20.5% (g trehalose/100 g dry cell) at temperatures of 35-40 degrees C and with 100-120 min of exposition time to the thermal stress.32265275Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CENAConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Modified Extraction Scheme for the CERN PS Multi-Turn Extraction

High-activation of the extraction magnetic septum of the CERN PS machine was observed due to the losses of the continuous beam extracted via the Multi-Turn Extraction (MTE) method. A possible mitigation measure consists of using an existing electrostatic septum, located upstream of the extraction magnetic septum, to deflect the beam. This would highly decrease the beam losses, and hence the induced activation, during the rise time of the MTE kickers due to the reduced thickness of the electrostatic septum with respect to the magnetic one. The layout of this new extraction will be described in detail and the results of beam measurements presented

Parametrizations of coupled betatron motion for strongly coupled lattices

The coupling of transverse motion is a natural occurrence inparticle accelerators, either in the form of a residual couplingarising from imperfections or originating by design from strongsystematic coupling fields. While the first can be treatedperturbatively, the latter requires a robust approach adapted tostrongly coupled optics, and a parametrization of the linear opticsmust be performed to explore beam dynamics in such peculiarlattices. This work highlights the key physical interpretations ofthe main parametrization formalisms to describe linear coupledoptics, along with explicit links and comparisons of theseparametrizations. Concepts rarely illustrated in other works, suchas forced mode flips and local coupling, are explored in detail,clarifying some anomalies that can arise in lattice functions. Theanalytical methods have been implemented in a reference Pythonpackage and connected with ray-tracing and integration codes toexplore examples of strongly coupled lattices, which are discussedin detail to highlight the key physical interpretations of theparametrizations and characteristics of the lattices.The coupling of transverse motion is a natural occurrence in particle accelerators, either in the form of a residual coupling arising from imperfections or originating by design from strong systematic coupling fields. While the first can be treated perturbatively, the latter requires a robust approach adapted to strongly coupled optics and a parametrization of the linear optics must be performed to explore beam dynamics in such peculiar lattices. This paper reviews the main concepts commonly put forth to describe coupled optics and clarifies the proposed parametrization formalisms. The links between the generalized Twiss parameters used by the different approaches are formally proven, and their physical interpretations are highlighted. The analytical methods have been implemented in a reference Python package and connected with a ray-tracing code to explore strongly coupled lattices featuring complex 3D fields. Multiple examples are discussed in detail to highlight the key physical interpretations of the parametrizations and characteristics of the lattices