21 research outputs found

    Potential for improvement of population diet through reformulation of commonly eaten foods

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    Food reformulation: Reformulation of foods is considered one of the key options to achieve population nutrient goals. The compositions of many foods are modified to assist the consumer bring his or her daily diet more in line with dietary recommendations. Initiatives on food reformulation: Over the past few years the number of reformulated foods introduced on the European market has increased enormously and it is expected that this trend will continue for the coming years. Limits to food reformulation: Limitations to food reformulation in terms of choice of foods appropriate for reformulation and level of feasible reformulation relate mainly to consumer acceptance, safety aspects, technological challenges and food legislation. Impact on key nutrient intake and health: The potential impact of reformulated foods on key nutrient intake and health is obvious. Evaluation of the actual impact requires not only regular food consumption surveys, but also regular updates of the food composition table including the compositions of newly launched reformulated foods

    小学校教科書語彙の研究

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    <div><p>Abstract</p><p><i>Leishmania</i> parasites replicate within the phagolysosome compartment of mammalian macrophages. Although <i>Leishmania</i> depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient <i>Leishmania major</i> mutant (<i>∆gnat</i>) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in <i>ex vivo</i> infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the <i>L</i>. <i>major ∆gnat</i> mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in <i>ex vivo</i> infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in <i>Leishmania</i>-induced skin lesions and is efficiently internalized into <i>Leishmania</i> containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides <i>Leishmania</i> with essential carbon sources, creating a uniquely favorable niche for these parasites.</p></div

    Hyaluronan localization to the <i>Leishmania</i> containing phagolysosome.

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    <p>RAW 264.7 macrophages were infected with <i>L</i>. <i>mexicana</i> promastigotes and then labelled with FITC conjugated hyaluronan (HA::FITC, 10μg/ml) for 24 hours. Macrophage lysosomes were stained with LysoTracker. Live-macrophages were analysed by fluorescence microscopy. Arrow indicates two <i>L</i>. <i>mexicana</i> amastigotes. Scale bar = 10μm.</p

    High molecular weight hyaluronan rescue intracellular ∆<i>gnat</i> parasites.

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    <p>RAW 264.7 macrophages were infected with (A) wild type (WT), (B) <i>∆gnat</i> and (C) <i>∆gnd</i> promastigotes in the absence of presence of different molecular weight HA. Intracellular parasites numbers at day 1 and 4 post-infection were determined by fluorescence microscopy after staining with Hoechst. Data represents mean and SD from three biological repeats and p-values were determined by the Student’s t-test.</p

    The <i>L</i>. <i>major ∆gnat</i> mutant is a GlcNAc auxotroph.

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    <p>(A) Wild type (WT) and ∆<i>gnat</i> promastigotes were cultivated in M199 medium supplemented with 50 μg/ml GlcN or GlcNAc and parasite growth monitored by measuring optical density at 600nm. (B) ∆<i>gnat</i> growth in the absence of GlcNAc was rescued by ectopic expression of GNAT from pXG-PURO plasmid, ∆<i>gnat</i> [<i>pX-PURO-GNAT</i>], in M199. ∆<i>gnat</i> mutant was incubated in (C) limiting or (D) increasing concentrations of GlcNAc and growth monitored over time. Data represents mean from three biological repeats.</p

    Schematic diagram of hexosamines biosynthesis and catabolism in <i>Leishmania</i>.

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    <p>Exogenous sugars, such as glucose (Glc), glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are taken up via the hexose transporters and phosphorylated within glycosomes by hexokinase. Fructose 6-phosphate (Fru-6P) can be used for <i>de novo</i> hexosamine biosynthesis via cytosolic glutamine:fructose-6-phosphate amidotransferase (GFAT) and N-acetylglucosamine acetyltransferase (GNAT), which are essential for the synthesis of glycoconjugates. In contrast, catabolism of GlcNAc-6P depends on the glycosomal glucosamine deaminase (GND) and GlcNAc deacetylase (GNAD), allowing the utilization of GlcNAc as major carbon source.</p

    Intracellular ∆<i>gnat</i> parasites are rescued by exogenous GlcNAc sources.

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    <p>(A) RAW 264.7 macrophages were infected with wild type (WT) and <i>∆gnat</i> stationary promastigotes in the presence of either GlcNAc, hyaluronic acid (HA) or chitin. Intracellular growth was determined after fixation and staining with the DNA dye, Hoechst, and is expressed relative to parasite numbers at day four of untreated cells (whereby WT contained 67 +/- 8 parasites and <i>∆gnat</i> 26 +/- 4 parasites). Error bars are the SD from three biological repeat experiments and p-values were calculated by the Student’s t-test. (B) WT and (C) <i>∆gnat</i> promastigotes were cultured in the presence of GlcNAc, HA or chitin as the sole hexose source and growth was determined by OD<sub>600</sub>.</p

    ∆<i>gnat</i> parasites lack GNAT activity and are defective in glycoconjugate biosynthesis.

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    <p>(A) Cytosolic extracts of wild type (WT), ∆<i>gnat</i> and ∆<i>gnat</i> [<i>pX-PURO-GNAT</i>] cell lines were incubated with 1 mM GlcN6P for indicated times at 27°C. The relative abundances of GlcN6P and GlcNAc6P were determined by direct infusion-mass spectrometry in negative ion mode. (B) WT and Δ<i>gnat</i> promastigotes were cultivated in hexosamine-free M199 medium for 24 hours and then pulse labelled with <sup>3</sup>H-Glc for 30 min. Parasite extracts containing total cellular lipids were analysed by HPTLC and the major phospholipids phosphatidylethanolamine (PE), phosphatidylinositol (PI), inositolphosphatidylcholine (IPC), phosphatidylcholine (PC), phosphatidylinositolphosphate (PIP) and glycolipids (GIPL1, 2 and 3). (C) LPG and gp63 were detected by Western blotting using anti-phosphoglycan repeat antibody, LT15 (top panel) or anti-gp63 (middle panel) antibodies. The flagellar protein, SMP1, was used as a loading control (bottom panel). (D) The neutral glycan mannan contains increasing levels of mannose (M) and was resolved by HPTLC. (E) WT or ∆<i>gnat</i> parasites cell lysates were labeled with GDP-[<sup>3</sup>H]Man and dolichol-P-mannose (Dol-P-Man), the GIPL precursor, M1, and a lipid-linked oligosaccharide (LLO) precursors detected by HPTLC.</p

    Targeted silencing of <i>vps26</i> in Δ<i>ku80-</i>DHFR <i>T</i>. <i>gondii</i> parasites.

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    <p>(A) Schematics of the genomic locus of <i>vps26</i> after single homologous integration of the XbaI linearised endogenous <i>ha</i> tagging and U1 gene silencing construct in absence and presence of rapamycin. A codon optimised DiCre cassette is cloned into the SpeI restriction site between the <i>hxgprt</i> selection cassette and the second loxP site. (B) and (C) Analytical PCR on genomic DNA extracted from clonal VPS26-HA-U1 parasites and the parental line Δ<i>ku80-</i>DHFR grown for 24 h in absence and presence of 50 nM rapamycin. (B) Construct integration was confirmed by using oligos indicated as orange arrows in (A). Theoretical fragment sizes of 2200 bp and 2500 bp for 5’ and 3’ integration were amplified respectively in VPS26-HA-U1 parasites independent of rapamycin. (C) Confirmation of Cre/loxP site specific recombination. Binding sites of primers used for analysis are indicated with purple arrows in (A) with the theoretical fragment sizes. Even though it was impossible to amplify the very large spacer (5900 bp) in absence of rapamycin the PCR product of 600 bp in presence of rapamycin confirms Cre/loxP site specific recombination. L, ladder. (D) Immunoblot analysis of clonal VPS26-HA-U1 parasites cultured for 48 h with or without 50 nM rapamycin. Membrane probed with anti-HA and anti-IMC1 as loading control. In presence of rapamycin no downregulation of VPS26-HA expression was observed. (E) Giemsa stain. Growth analysis over a 7 days period in absence and presence of 50 nM rapamycin shows no difference in plaque formation. Scale bar represents 500 μm. (F) Immunofluorescence analysis of clonal VPS26-HA-U1 parasites grown for 48 h in presence of 50 nM rapamycin. Green, HA; red, IMC1; blue, DAPI; scale bar represents 10 μM. Endogenous HA-tagged VPS26 localises apical to the nucleus in Golgi region. Parasites with silenced <i>vps26</i> are encircled in yellow. (G) Quantification of <i>vps26</i> silencing efficiency. The graph shows the percentage of HA positive and negative vacuoles determined by examination of 100 vacuoles per condition based on immunofluorescence analyses of clonal VPS26-HA-U1 parasites grown for 48 h in absence or presence of 50 nM rapamycin. Values are means ±SD (n = 3). Under rapamycin conditions a <i>vps26</i> downregulation of 12.6 ± 5.32 was obtained (***, p<0.001, Mann-Whitney test).</p

    U1 gene silencing strategy in <i>T</i>. <i>gondii</i>.

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    <p>(A) Alignment of the first 50 nucleotides of the U1 snRNAs of the indicated organisms. Shaded in green are nucleotides 2–11 complementary to the artificial U1 snRNA recognition sequence in (B). Nucleotides 3–11 are identical across the aligned species. <i>H</i>. <i>sapiens</i>, <i>Homo sapiens</i>; <i>A</i>. <i>thaliana</i>, <i>Arabidopsis thaliana</i>; <i>T</i>. <i>gondii</i>, <i>Toxoplasma gondii</i>; <i>P</i>. <i>falciparum</i>, <i>Plasmodium falciparum</i>. (B) Schematic of strategy. Modification of the target gene’s 3’ terminal exon by insertion of a U1 snRNA recognition sequence leads to recruitment of the U1 snRNP to the pre-mRNA of the target gene. Base pairing between these 10 complementary nucleotides of the U1 site and the U1 snRNA of the U1 snRNP occurs and the resulting U1 snRNP pre-mRNA complex inhibits polyadenylation and therefore pre-mRNA maturation. For simplification nucleotides are illustrated as black bars and the target gene is intron-less. UTR, untranslated region.</p
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