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

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

Overexpression of ddFKBPmyc-Rab5A and ddFKBPmyc-Rab5C causes mislocalisation of only a subset of microneme proteins.

<p>(A) Growth analysis of parasites expressing indicated ddFKBPmyc-Rab constructs inoculated on HFF cells and cultured for 5 days +/− 1 µM Shld-1. Single plaques are indicated by black edging. The scale bar represents 1 mm. The depicted quantification of the plaque sizes is a representative of 3 independent experiments. In each case, the mean area and standard deviation of 10 plaques was determined. The data was normalised relative to the plaque size of the respective uninduced parasite strain. (B) Immunofluorescence analysis of intracellular parasites expressing indicated ddFKBPmyc-Rab constructs and wild type parasites RH^hxgprt−treated for 24 hrs with 1 µM Shld-1 and probed with α-MIC3, α-MIC2 or α-ROP2-4 antibody (red) and Dapi (blue). Only parasites overexpressing Rab5A and 5C show a mislocalisation effect on the secretory organelles (micronemes, rhoptries). Scale bars represent 5 µm.</p

Only a basic set of Rab-GTPases is present in apicomplexan parasites.

<p>(A) Unrooted neighbour joining tree of the GTPase domains identified from each conserved Rab sequence of the eight canonical species and <i>T.gondii</i>. Clades containing sequences derived from <i>T. gondii</i> are indicated by shaded wedges. Three major clades containing <i>T. gondii</i> sequences are evident containing sequences of a) Rab1 and18 b) Rab5,6,7 and 23 c) Rab2,4 and 11. The species origin of the nodes is shown by a colour code as indicated. For detailed subtrees of major clades and support values see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003213#ppat.1003213.s002" target="_blank">Figure S2</a>). (B) Immunofluorescence analysis of intracellular parasites expressing ddFKBPmyc-Rab1A,B,2,4,5A,5C,7 and 18 or Rab5B-ddFKBPHA-construct treated for 18 hrs with 1 µM Shld-1 prior to fixation. The left panel shows Rabs costained with ER/Golgi marker (TgERD2 and TgGalNAC in green), whereas the right panel shows co-staining with markers for ELCs (TgVP1 and proM2AP in red), the nucleus is shown in blue (Dapi). The scale bars represent 5 µm. 1 =  Nucleus, 2 =  Apicoplast, 3 =  Mitochondrium, 4 =  Dense Granule, 5 =  Endoplasmatic Reticulum (ER), 6 =  Golgi, 7 =  late endosome-like compartment (TgVP1 compartment), 8 =  early endosome like compartment (proM2AP), 9 =  Micronemes, 10 =  Rhoptries. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003213#ppat.1003213.s004" target="_blank">Figure S4</a>&S5 for more details.</p

Time course analysis of parasites expressing ddFKBPmyc-Rab5A(N158I).

<p>(A) Immunofluorescence analysis of intracellular parasites stably expressing ddFKBPmyc-Rab5A(N158I) treated for 0, 12, 18 and 24 hrs with 1 µM Shld-1 and co-stained with the indicated microneme antibodies (green/red) and Dapi (blue). (B) Quantification of localisations of Rop2-4 and indicated microneme proteins. 300–400 PVs of three independent experiments were analysed and normalised with RH ^hxgprt−parasites. Mean values and the respective standard deviation are presented. Note that MIC2 and M2AP is mainly mislocalised inside the parasite (arrowhead in A).</p

Microneme processing and organisation of endosomal-like compartments (ELCs) is unaffected in ddFKBPmyc-Rab5A(N158I) expressing parasites.

<p>(A) Parasites expressing ddFKBPmyc-Rab5A(N158I) have been grown for 24 hrs +/− 1 µM Shld-1 and analysed with the indicated antibodies. MIC3 and MIC11 are not transported to the micronemes, whereas the micronemal proteins PLP1 and M2AP as well as the marker for endosomal-like compartments (CPL and TgVP1) show normal localisation. The scale bars represent 5 µm. (B,C) Pulse-chase experiments of MIC3 processing in parasites expressing ddFKBPmyc-Rab5A(N158I). (B) Immunofluorescence analysis of ddFKBPmyc-Rab5A(N158I) parasites grown for 18 hrs +/− 1 µM Shld-1 and probed with proMIC3 (red) and CPL (green) antibodies. In presence of the inducer the pro-peptide of MIC3 is secreted into the PV (proMIC3), whereas no effect on endosomal-like compartments (CPL) is obvious. Dapi is shown in blue. The scale bar represents 5 µm. (C) Quantification and the respective western blots of MIC3 maturation in RH ^hxgprt− and ddFKBPmyc-Rab5A(N158I) parasites are shown. Mean values and the respective standard deviation of three independent experiments are presented.</p

Ultrastructural and STED analysis of parasites expressing ddFKBPmyc-Rab5A(N158I).

<p>(A) Electron microscopy analysis of the apical region of a tachyzoite expressing ddFKBPmyc-Rab5A(N158I) in the absence of 1 µM Shld-1 showing the natural morphological organisation of the apical organelles with various number of micronemes (arrowhead) associated with the limiting membrane complex. (B) Similar parasite stage from a culture incubated for 24 hrs with 1 µM Shld-1 showing a reduced number of micronemes (arrowheads). Scale bars represent 100 nm. (C,D) STED analysis of the same parasite strain grown in absence (C) or presence (D) of 1 µM Shld-1 for 18 hrs. Two-colour STED measurements were performed on MIC3 and M2AP, showing the absence of MIC3 within the parasites expressing ddFKBPmyc-Rab5A(N158I). (E) Electron microscopy quantification of micronemes present in longitudinal sections passing through the conoid and nucleus. 20 parasites per situation were quantified. Less micronemes are detected when ddFKBPmyc-Rab5A(N158I) is expressed. (F) Model of the vesicular traffic of secretory proteins to the rhoptries and micronemes. After modification at the Golgi secretory proteins are transported to the ELC's, which is regulated by VPS1 and VPS10. From the ELC's some of the microneme proteins (i.e. MIC3, MIC8, MIC11) and rhoptry proteins are transported to their target organelles in a Rab5A/C dependent manner. The regulation of the transport of other microneme proteins (AMA1, MIC2, M2AP, PLP1) remains unknown (?). VPS10 =  Sortilin (TgSORTLR), VPS1 =  dynamin related protein B (DrpB), Ap =  Apicoplast, C =  Conoid, ELC =  endosome like compartments, ER =  Endoplasmatic Reticulum, G =  Golgi, Mi =  Mitochondrium, M =  Micronemes, R =  Rhoptries.</p

Analysis of parasites expressing ddFKBPmyc-Rab5A(N158I) and ddFKBPmyc-Rab5C(N153I).

<p>(A) Immunofluorescence analysis of indicated parasites treated for 24 hrs with 1 µM Shld-1 and probed with indicated antibodies (green) and Dapi (blue). (B) Replication assay of indicated parasites grown for 24 hrs in presence, or absence of 1 µM Shld-1 prior to fixation. Average number of parasites per PV was determined. No significant differences in replication were detected. (C) Egress assay of indicated parasites grown for 36 hrs +/− 1 µM Shld-1 before egress was triggered with A23187. Host cell lysis was determined 8 min after induction of egress and normalised with RH ^hxgprt−parasites. For both mutants the egress is significantly decreased. (D) Invasion assay of indicated parasites treated for 24 h +/− 1 µM Shld-1, scratched and inoculated on fresh HFF cells. Subsequently invasion was determined and normalised with RH^hxgprt− parasites. For both mutants the invasion is significantly blocked. (B–D) Mean values and the respective standard deviation of three independent experiments are presented. (***indicates p-value of P≤0.01 and **indicates P≤0.02 in a two tailed Student's test).</p