54 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

    A model for TgSORTLR functions in protein sorting and the biogenesis of apical secretory organelles.

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    <p>We propose that TgSORTLR has a distinct role as a type I transmembrane cargo-protein receptor for ROPs and MICs of apicomplexan parasites. We observed TgSORTLR-positive structures that could be transport vesicles destined for the endolysosomal system or they might be integral to the endolysosomal system, i.e., early (EE) and late (LE) endosomes. The model further proposes that the cytoplasmic tail of TgSORTLR binds to AP-1, Sec23/24, clathrin, clathrin-associated adaptor protein, and VPS9, and this defines it as a key receptor involved in the anterograde transport of cargo ROP and MIC proteins. The binding of TgSORTLR to the retromer VPS26/VPS35 also indicates that this receptor is also involved in the retrograde transport of components. <i>T. gondii</i> lysosome-like, acidic vacuolar compartment (VAC), also termed the Plant-Like Vacuole (PLV), contains cathepsin proteases implicated in the proteolytic maturation of proproteins targeted to MICs. Proteolytic maturation likely occurs in the LE where conditions are thought to be more conducive for limited proteolysis.</p

    Comparative bioinformatics analysis of genes coding components of vesicle-mediated trafficking and endosomal sorting in apicomplexan parasites, <i>Saccharomyces cerevisiae</i>, and <i>Homo sapiens</i>.

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    <p>Most of these genes and their corresponding accession numbers were collected from Eupathdb.org (for apicomplexan parasites) and uniprot.org (yeast and human cells). The data from apicomplexan parasites <i>Toxoplasma gondii</i> (<i>T. gondii</i>), <i>Plasmodium falciparum</i> (<i>P. falciparum</i>), <i>Theileria parva</i> (<i>T. parva</i>), and <i>Cryptosporidium parvum</i> (<i>C. parvum</i>) were compared with human (<i>H. sapiens</i>) and the yeast <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>). AP, adaptor protein; GGAs, Golgi-localized, γ-ear–containing, ADP-ribosylation factor binding protein; COPI, Coatomer complex I (retrograde transport from trans-Golgi apparatus to cis-Golgi and endoplasmic reticulum); COPII, Coatomer complex II (anterograde transport from ER to the cis-Golgi); ESCRT, Endosomal Sorting Complex Required for Transport.</p

    TgSORTLR co-localizes with TgVsp26 and Tgμ1-adpatin.

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    <p>(<b>A</b>) Confocal images of tachyzoites expressing endogenously tagged TgVps26-HA (green) that co-localizes with TgSORTLR (red). (<b>B</b>) Confocal images of tachyzoites expressing endogenously tagged Tgμ1adaptin-HA (red) and TgSORTLR (green). White circles indicate the zoomed areas showing co-distribution between TgSORTLR and Tgμ1adaptin-HA or TgVPS26-HA in the Golgi and post-Golgi. Scale bars, 5 µm.</p

    Ultrastructure of <i>Toxoplasma gondii</i>.

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    <p>(<b>A</b>) Intracellular <i>T. gondii</i> tachyzoite showing the MICs (Mn), ROPs (Rh), micropore (Mp), Golgi (G), nucleus (N), endoplasmic reticulum (ER), and dense granules (DG). (<b>B</b>) A schematic picture of <i>T. gondii</i> entering into a nucleated mammalian host cell. The apical exocytosis of MICs deploys onto the parasite surface MIC proteins required for parasite motility and the formation of moving junction. ROP secretion provides the ROP proteins that are involved in host cell invasion and modulation of immune responses. The constitutive secretion of dense granules (DG) is involved in the modification of the parasitophorous vacuole (PV). (<b>C</b>) Higher magnification of the single Golgi apparatus of <i>T. gondii</i>. (<b>D</b>) Higher magnification of the <i>T. gondii</i> ER. Scale bars, 1 µm.</p

    Ultrastructural analysis of CHC-HA expressing <i>T. gondii</i> parasites.

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    <p>Routine (<b>A</b>) and immuno (<b>B</b> and <b>C</b>) electron micrographs. (<b>A</b>) Section through the anterior of a tachyzoite showing nucleus (N), apicoplast (A), Golgi (G), rhoptries (R), micronemes (M), and mitochondrion (Mi). Scale bar is 100 nm. Insert: Enlargement of the enclosed area showing a coated vesicle. Bar is 50 nm. (<b>B</b>) Similar area to that in (<b>A</b>) of a parasite processed for immuno electron microscopy showing gold particles located predominately at the periphery of the Golgi (G). N, nucleus; A, apicoplast; DG, dense granules. Bar is 100 nm. Insert: Detail of the enclosed area showing a number of gold particles around a possible vesicle. Bar is 50 nm. (<b>C</b>) Section through a parasite undergoing endodyogeny showing the two partially formed daughters (D1, D2) with gold particles (arrowheads) associated with the periphery of the daughter Golgi bodies (G). N, nucleus. Bar is 500 nm. Insert: Enlargement of the enclosed area showing gold particles at the periphery of the Golgi body. Bar is 100 nm.</p

    CHC1 is essential for Golgi function and segregation.

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    <p>(<b>A</b>) Immunofluorescence analysis of DD-Hub expressing parasites stable transfected with the cis-medial Golgi marker GRASP-RFP cultured for 24 hr in absence or presence of 0.1 and 1 μM Shield-1. Only under high Shield-1 concentrations daughter parasites show abnormal Golgi morphology (white arrow heads) or no Golgi at all (yellow arrow heads, N highlighted in yellow). N, nucleus. (<b>B</b>-<b>D</b>) Immunofluorescence analysis of DD-Hub expressing parasites stable transfected with SAG1∆GPI-dsRed. After treatment for 24 hr with 0.1 and 1 μM Shield-1 DD-Hub expression causes a block in constitutive secretion of SAG1∆GPI-dsRed. (<b>C</b> and <b>D</b>) Overnight treatment with 5 μg/ml Brefeldin A (BFA) or 24 hr treatment with 1 μM Shield-1 respectively show an identical block in transport for SAG1∆GPI-dsRed and the micronemal protein MIC3 (arrow heads). Immunoflourescence images are representative of at least three independent experiments and depicted abnormalities have been observed in 100% of 200 random examined vacuoles compaired to controls.</p

    Clathrin interactome and localisation in <i>T. gondii</i>.

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    <p>(<b>A</b>) Putative clathrin interactome with green symbols conserved in <i>T. gondii</i>, orange symbols conserved in most eukaryotes and red symbols indicating fungi specific interactors. For details see also Table S1. (<b>B</b>) Schematics of the chc1 endogenous ha tagging construct and its integration into the genomic chc1 locus. UTR, untranslated region. (<b>C</b>) Analytical PCR on genomic DNA using oligos indicated as orange arrows in (<b>B</b>). The obtained 1484 bp confirms endogenous tagging of <i>chc1</i>. (<b>D</b>) Immunoblot of indicated parasite lines probed with anti-HA, anti-Catalase, and anti-Aldolase antibodies. Catalase and aldolase were used as loading controls. (<b>E</b> and <b>F</b>) Immunofluorescence analysis of endogenously <i>chc1</i> tagged parasites with indicated antibodies. CHC1-HA shows no colocalisation with IMC1 (<b>E</b>) and predominantly accumulates apical to the nucleus (<b>E</b> and <b>F</b>). It colocalises with DrpB and resides like DrpB adjacent to the trans-Golgi weakly overlapping with GRASP-RFP. (<b>F</b>). R, Pearson correlation coefficient ± SD averaged for the indicated number (n) of parasites. Scale bars represent 1 μm and 10 μm in (<b>E</b>) and 10 μm in (<b>F</b>). Inlets show twofold enlargements of the indicated area.</p

    Impact of DD-Hub expression on mitochondrial and apicoplast segregation.

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    <p>Immunofluorescence analysis of DD-Hub expressing parasites stable transfected with either HSP60-RFP (<b>A</b>) or FNR-RFP (<b>B</b>). Parasites were cultured for 24 hr in absence or presence of 0.1 and 1 μM Shield-1. In presence of high Shield-1 concentrations mutants show mitochondrial and apicoplast segregation defects. Scale bars represent 10 μm. Immunoflourescence images are representative of at least three independent experiments and depicted abnormalities have been observed in 100% of 200 random examined vacuoles compaired to controls.</p

    Rapid ablation of CHC1 function in <i>T. gondii</i>

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    <p>(<b>A</b>) Schematic of the dominant negative DD-Hub construct and of the chlathrin triskelion structure. The Hub fragment is highlighted in pink. <i>dd</i>, destabilization domain; <i>myc</i>, myc epitope tag; p, promoter; UTR, untranslated region. (<b>B</b>) Schematic (left) and immunofluorescence analysis (right) of Shield-1 dependent regulation of DD-Hub. Immunofluoresence has been performed with indicated antibodies 24 hours after incubation of parasites in presence or absence of 1 μM Shield-1. Hub fragments localise to the cytoplasm and their expression lead to deformed IMCs (<b>C</b>) Immunoblot of parasite lysates probed with indicated antibodies. Catalase and aldolase were used as loading controls. Intra- or extracellular parasites were treated with 0 μM, 0.1 μM and 1 μM Shield-1 for the indicated time. (<b>D</b>) In contrast to the controls DD-Hub expressing parasites show no plaque formation after 7 days incubation in presence of 1 μM Shield-1. Scale bar represents 500 μm. (<b>E</b>) For analysis of the invasion rate intra- or extracellular parasites were treated for 6 hr with or without 1 μM Shield-1 prior to the experiment. Data represent mean values of three independent experiments ±SD. (<b>F</b>) For analysis of the replication rate indicated parasite lines were cultured in absence or presence of 1 μM Shield-1 and fixed 24 hr post invasion. Data represent mean values of three independent experiments ±SD. (<b>G</b>) Immunofluorescence analysis with indicated antibodies of DD-Hub parasites treated with indicated Shield-1 concentrations for 24 hours. Scale bar represents 10 μm. Immunoflourescence images are representative of at least three independent experiments and depicted abnormalities have been observed in 100% of 200 random examined vacuoles compaired to controls.</p
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