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

Loss of AUK2 results in nuclear DNA damage.

<p><b>A.</b> Western blot analysis of γH2A in two <i>auk2</i> -/- mutants clones (CL1 and CL2) relative to an <i>AUK</i>+/- heterozygous mutant and wild type cells (WT427). Whole cell lysates were probed with anti-γH2A (below) and anti-EF1α (above; loading control) antisera. The graph shows levels of γH2A after normalisation by EF1α: γH2A levels in WT cells were set at 1 and fold change in the mutants relative to WT is shown. Data points represent means and SEM (n = 3). <b>B.</b> Immunofluorescence (IF) of RAD51 foci formation. Cells were harvested, fixed and RAD51 localised with anti-RAD51 antiserum. Representative IF images of <i>auk2</i>-/- mutants are shown in which DAPI stained DNA is in blue and RAD51 in red (cell morphology is shown by differential contrast imaging); the scale bar = 10 μm. The graph shows the percentage of WT cells with detectable RAD51 foci compared with <i>AUK2</i>+/- mutants and two <i>auk2</i>-/- clones. Cells with RAD51 foci are represented as a percentage of the total population of cells counted (n >200). Data points represent the mean from three independent experiments; errors bars show SEM. * denotes a significant difference from WT (P<0.05, Mann Whitney U test).</p

AUK2 displays dynamic nuclear localisation.

<p><b>A.</b> Western blot of whole cell extracts from wild type (WT) <i>T</i>. <i>brucei</i> and from two clones in which the <i>AUK2</i> ORF has been C-terminally fused to a tag encoding 12 myc epitopes (<i>AUK2</i>+/-::12myc). The blot was probed with anti-myc and anti-EF1α antiserum (as a loading control); a size marker is shown. <b>B.</b> Representative images of <i>AUK2</i>+/-::12myc cells from each cell cycle stage (denoted by N-K ratio). Anti-myc antiserum was used to visualise myc tagged AUK2 (green) and nDNA and kDNA were stained with DAPI (magenta); DC imaging shows cell shape; scale bars = 5 μm. <b>C.</b> Super resolution images of AUK2-12myc localisation. Only in the merged images are DAPI (blue) and anti-myc signals (green) shown in colour. Graphs show fluorescence intensity (arbitrary units; AU) over distance plotted for both the DAPI (blue) and anti-myc (green) signals. The white box represents the area from which the fluorescence intensity was measured; scale bar = 5 μm. <b>D.</b> 3D reconstruction of AUK2-12myc localisation in a 1N1K or 1N2K cell.</p

Schematic outline of the whole genome <i>T</i>. <i>brucei</i> MMS RIT-seq screen.

<p>A whole genome tetracycline (Tet) inducible RNAi library was established in BSF <i>T</i>. <i>brucei</i> cells as a pool, within which random RNAi fragments target potentially all genes and provide unique identifiers. Cells were induced by Tet addition (+) for a total of 5 days, during which cells targeting RNAi against important genes (red, green, blue) are lost from or reduced in the population. In parallel, Tet+ cells were grown in the presence of methyl methanesulphonate (MMS, 0.0003%), which was added 1 day after RNAi induction. Cells carrying an RNAi target for a gene necessary for repair of MMS damage (purple) are specifically lost or depleted in the Tet+, MMS+ population relative to the Tet+, MMS- population. PCR was used to amplify all RNAi target fragments after five days of RNAi with or without exposure to MMS; the amplicons were sequenced and mapped to the genome. Read depth mapping is shown schematically for a gene whose RNAi causes loss of fitness without MMS (red), and for a gene whose RNAi causes loss of fitness only after MMS exposure (purple).</p

<i>In vitro</i> growth of putative MMS damage response protein kinases identified by genome-wide MMS RIT-seq.

<p>Individual tetracycline (Tet) inducible RNAi cell lines were generated for five PK genes (identified by gene ID and name, if known) and their growth assessed by counting parasite density every 24 hrs for 96 hrs. Growth was assessed in the absence (-) and presence (+) of MMS (0.0003% v/v) and with (+) or without (-) Tet RNAi induction. The same analysis is shown for parental 2T1 cells, which do not induce gene-specific RNAi. Each data point displays the mean cell density from three independent biological replicates error bars represent SEM. Significant differences between the means of the Tet-, MMS+ sample relative to the Tet+, MMS+ were calculated using a Mann Whitney U test; (*) = p<0.05, which was considered significant. Within each graph, protein loss for 12myc-tagged PKs was tested by western blot analysis on whole cell extracts using anti-myc antiserum. Cells were harvested after 24 and 48 hrs growth with (+) or without RNAi induction by addition of Tet. Anti-EF1-α was used as a loading control. Beside each graph γH2A expression levels after RNAi against the PKs, with (MMS+) or without exposure to MMS (MMS-), are shown in western blots. Cells were induced (with 1 μg.ml^-1 tetracycline; Tet+) or left uninduced (Tet-) for 24 hrs. At 24 hrs, MMS (to a concentration of 0.0003% [v/v]) was added to an induced and an uninduced culture. Whole cell lysates were collected, separated on gels, blotted and γH2A was detected using anti-γH2A antiserum (anti-EF1α was used as a loading control). Gene IDs and names are provided for the PKs genes analysed; 2T1 is the parental cells, where Tet does not induce dsRNA.</p

MMS RIT-seq prediction of gene categories providing damage response functions.

<p>Scatter plots are shown of MMS+/MMS- read depth ratios for all <i>T</i>. <i>brucei</i> genes (grey dots), as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.g002" target="_blank">Fig 2</a>, highlighting individual genes within four functional categories (further details provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s002" target="_blank">S2 Table</a>). <b>A.</b> All predicted <i>T</i>. <i>brucei</i> helicase genes are separated into putative RNA (blue) and DNA helicases (red), or those whose substrate is unclear (dark grey). <b>B.</b> All genes encoding predicted DNA polymerase (Pol) activities are shown in red, with specific factors arrowed; putative DNA Pol kappa genes are highlighted blue and two Poly (ADP-ribose) Pol genes (PARP) in green. <b>C.</b> All genes with predicted involvement in DNA replication are in red; blue highlights Minichromosome Maintenance Complex (MCM)-related factors, and green denotes Origin Recognition Complex (ORC) factors. <b>D.</b> Genes with chromosome structure-associated functions are in red, with telomere factors in green and cohesin/condensin (SMC) factors in blue.</p

Analysis of the MMS RIT-seq screen.

<p><b>A, B.</b> Scatter plots showing the ratio of mapped RNAi target-specific reads for every gene (grey dots) in the RNAi-induced, MMS-treated population relative to the RNAi-induced, untreated population (MMS+/MMS-); gene location within the 11 megabase chromosomes is shown and dotted lines indicate 2-fold increase and decrease in MMS+/MMS- ratio. Genes are highlighted with roles in (A) homologous recombination (HR, red), mismatch repair (MMR, blue) and nucleotide excision repair (NER, green), or in (B) intraflagellar transport (IFT, red), mitochondrial replication (Mito rep, blue) and encoding histones (green). <b>C.</b> A pie chart of the distribution of all genes displaying an MMS+/MMS- ratio of less than 0.5, excluding 44 genes predicted to be VSGs. Hypothetical and hypothetical unlikely denotes genes for which there are currently no homology-predicted functions. Unknown denotes genes with homology-predicted functions that cannot be readily associated with the response to MMS damage. Finally, genes in seven classes of predicted functions with putative roles in responding to MMS are detailed. <b>D.</b> GO terms, within two headings, which show significantly increased frequency in the MMS+/MMS- <0.5 gene set relative to the whole GO gene set (IDs and further analysis are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s003" target="_blank">S3 Table</a>).</p

Damage response protein kinases predicted by whole-genome MMS RIT-seq.

<p><b>A.</b> Scatter plot of MMS+/MMS- read depth ratios for all <i>T</i>. <i>brucei</i> genes (grey dots). Protein kinase (PK) genes are highlighted in red and individual genes are further identified (arrows) by gene IDs and names, if known (further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s002" target="_blank">S2 Table</a>). <b>B.</b> Nine PK genes, including PK family and name, present in the MMS+/MMS- <0.5 gene set are listed. <b>(C</b> displays read mapping profiles for selected PKs (red) after RNAi and growth with (MMS+) or without (MMS-) 0.0003% MMS (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.g005" target="_blank">Fig 5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s011" target="_blank">S5</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.s012" target="_blank">S6</a> Figs for more detail).</p

Loss of AUK2 sensitises <i>T</i>. <i>brucei</i> to DNA damaging agents and results in altered cell cycle progression.

<p><b>A-D</b> Growth curves of one <i>auk2</i> -/- null mutant clone (CL1) compared with wildtype (WT427) cells; cell density was monitored every 24 hrs for 72 hrs in the presence (+) and absence of MMS (0.0003%), phleomycin (PHL; 0.1 μg ml^-1), hydroxyurea (HU; 0.6 mM; C) or after exposure to UV (1500 J/m²). All graphs show mean density from three experiments; error bars denote SEM. Significant differences are shown by * (P<0.05; Mann Whitney U test). <b>E.</b> Cell cycle analysis of <i>auk2</i> -/- mutants compared with WT cells. Cells were harvested, fixed and stained with DAPI for visualisation of the kinetoplast (k) and the nucleus (n). >200 cells were counted from three independent replicates of each cell type, and the n-k configuration of individual cells expressed as a percentage of the total population. Cells that did not show any of the expected N-K configurations (1N1K, 1N2K or 2N2K) were categorised as ‘other’. Error bars represent SEM. * P<0.05 (Mann Whitney U test; comparison between WT other cells and AUK2 -/- CL1 other cells).</p

<i>In vitro</i> growth of putative MMS damage response protein kinases identified by kinome-focused MMS RIT-seq.

<p>Individual RNAi cell lines were generated for four PK genes (identified by gene ID and name, if known) and their growth assessed by counting parasite density every 24 hrs for 96 hrs, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.g005" target="_blank">Fig 5</a>. Protein loss was tested by western blot analysis on whole cell extracts, as was γH2A expression level after RNAi (Tet+) against the PK, with (MMS+) or without exposure to MMS (MMS-); experimental details are as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006477#ppat.1006477.g005" target="_blank">Fig 5</a>.</p