Fungal strains and genotypes.

<p>Markers not separated by semi-colons are located on the same linkage group.</p><p>The ∼ symbol indicates the presence of the allele in the genome at an unknown location and/or copy number.</p

Northern analyses of gene expression.

<p><b>A</b> Total RNA was isolated from shake flask biomass grown in the presence of the following carbon sources (initial concentration): ethanol (0.5% v/v) a poor, derepressing source; glucose (0.5% w/v) a good, repressing source; and glycerol (0.5% w/v) a good, derepressing source. All sources were exhausted by 20 h. Biomass was harvested at the times indicated after inoculation of conidia. <b>B </b><i>mstC</i> mRNA accumulation during conidial germination in media containing glucose or lactose (each present at an initial concentration of 0.5% w/v). Total RNA was isolated from biomass harvested from shake-flask cultures at the times indicated after inoculation of conidia. <b>C </b><i>mstC</i> mRNA accumulation in two CCR mutants (<i>creA^d</i>) compared to wild type (wt). The two CCR mutants and the wild type strain were grown in the presence of glucose (glc) or galactose (gal), each initially present at 0.5% (w/v). Total RNA was isolated from biomass harvested from shake-flask cultures at the times indicated after inoculation of conidia. In all cases cultures were grown from conidia for the times indicated, and rRNA was visualised by methylene blue staining <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094662#pone.0094662-Herrin1" target="_blank">[39]</a>.</p

Structural organisation of the <i>mstA</i> and <i>mstC</i> genes and their translation products.

<p>The upper bars are schematic representations of gene structure with introns (A to E) in white and exons in black. cDNAs generated by RT/PCR from transcripts (dark grey bars) of each gene were sequenced and compared with the genomic sequences, thus confirming the intron/exon structures deduced for each gene. Proteins are shown as white bars within which the numbered grey-shaded boxes correspond to the TM domains predicted by TMHMM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094662#pone.0094662-Krogh1" target="_blank">[31]</a>. The locations of the mutations and the corresponding changes in the 1° structure of MstC found in the <i>sorA2</i> (#) and <i>sorA3</i> (*) mutants are also shown (see text for details). The <i>sorA2</i> mutation causes a change in reading frame resulting in a shorter and novel COOH-terminal sequence (shown in bold) within which resides a putative TM domain (marked with a circle). The annotation of the <i>A. nidulans</i> genome assigned the locus identities AN8737 and AN6669 to <i>mstA</i> and <i>mstC</i>, respectively.</p

Oligonucleotides used in this work.

<p>Oligonucleotides used in this work.</p

Energetics of glucose uptake.

<p>Relative glucose uptake rates for glycerol-germinated conidia expressing MstC (dark grey - V004) and MstA (light grey - V152) transporters assayed in the absence (−) and presence (+) of CCCP.</p

Characterisation of MstC.

<p><b>A</b> Marker rescue of a <i>sorA</i> mutant by <i>mstC</i>. Top panel: <i>sorA</i> mutant strains (V045 and G186) are able to grow in the presence of 50 µg/ml 2-DOG and 1% EtOH compared to wild type. Middle panel: A typical minimal medium plate lacking riboflavin but supplemented with 2-DOG used for identifying 2-DOG sensitive transformants (those that do not grow); V082 fails to grow as it is auxotrophic for riboflavin. Lower panel: typical PCR products specific to the wild type <i>mstC</i> allele amplified off gDNA templates. R = resistant to 2-DOG, S = sensitive to 2-DOG. T = transformant. T22 and T29 are asterisked to help their identification on the plate in the middle panel. T33 is a control transformed with pPL5 alone. <b>B</b> Typical Michaelis-Menten plots of glucose uptake rate <i>versus</i> glucose concentration for conidia of the two <i>ΔmstC</i> strains V109 (□) and V110 (▴), and the <i>mstC⁺</i> strain V111 (•) germinating for 4 h in appropriately supplemented minimal medium containing 1% glycerol as carbon source; non-linear regressions are shown as dashed, dotted and solid lines, respectively. Insert: Eadie-Hofstee plots. The plots for V109 and V110 are monophasic. Their displacement towards the y axis relative to the plot of V111 is indicative of the loss of high-affinity uptake. <b>C</b> Typical Michaelis-Menten plots of glucose uptake rate <i>versus</i> glucose concentration for conidia of strains V140 (<i>sorA2</i>) (□) and V045 (<i>sorA3</i>); non-linear regressions are shown as solid and dashed lines, respectively. (•) germinating for 4 h in appropriately supplemented minimal medium containing 1% glycerol as carbon source. The insert shows Eadie-Hofstee plots of the uptake data for both strains. <b>D</b> Relative ¹⁴C-glucose uptake rates in the presence of a 200-fold molar excess of competing compounds are expressed as a percentage of the non-competed (control) rate.</p

Unrooted phylogenetic tree of primary structures of Eurotiomycete proteins related to MstA (AN8737) and MstC (AN6669).

<p>The evolutionary history was inferred using the Maximum Likelihood method, and the percentages of replicate trees in which the associated sequences clustered together in the bootstrap test (50 replicates) are shown next to the branches (values below 80% are not included). Branch lengths correspond to the mean number of substitutions per site. Where known, genome locus identities are given; ‘Corr’ indicates that the gene model was corrected; unannotated sequences are given as ‘mstA-like’. The homologues in <i>A. flavus</i> and <i>A. oryzae</i> were found to be encoded by identical genomic DNA sequences (only <i>A. flavus</i> is shown), as expected for organisms that are believed to be variants of the same species <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094662#pone.0094662-Payne1" target="_blank">[33]</a>. <i>A. nidulans</i> is the only organism represented that possesses two very closely related proteins. MstA (AN8737) and MstC (AN6669) are shown in bold.</p

A Cocoa Peptide Protects <i>Caenorhabditis elegans</i> from Oxidative Stress and β-Amyloid Peptide Toxicity

<div><p>Background</p><p>Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases.</p><p>Methodology/Principal Findings</p><p>A bioactive peptide, 13L (DNYDNSAGKWWVT), was obtained from a hydrolyzed cocoa by-product by chromatography. The <i>in vitro</i> inhibition of prolyl endopeptidase (PEP) was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic <i>Caenorhabditis elegans</i> strain CL4176 expressing the human Aβ_1–42 peptide as a pre-clinical <i>in vivo</i> model for Alzheimer's disease. Among the peptides isolated, peptide 13L (1 µg/mL) showed the highest antioxidant activity (<i>P</i>≤0.001) in the wild-type strain (N2). Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24–47 h period after Aβ_1–42 peptide induction (<i>P</i>≤0.0001). This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide.</p><p>Conclusions/Significance</p><p>These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a <i>C. elegans</i> model of Alzheimer's disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.</p></div

<i>Lactobacillus rhamnosus</i> CNCM I-3690 has an anti-inflammatory effect <i>in vitro</i>.

<p>A. Relative IL-8, production and I-κB-luciferase detection from the HT-29-bacteria interaction assays. Each bar represent the mean value of three replicate samples and and error bars depict corresponding standard deviation. Black bars: I-kB-luciferase, grey bars: IL-8. **p-value ≤0.05. B. Phenotypical analysis of monocyte-derived DCs co-cultured with HT-29-NF-κB-luciferase cells. Cells were incubated with LPS or LPS+bacteria. CNCM I-3690 down-regulated the expression of HLA-DR and CD86 surface markers. Results were expressed according to the following equation [(bacteria+LPS)-LPS]/LPS-basal*100. Experiments were performed with two different donors. C. Cytokine ratios (IL-8/IL-10, IL-6/IL-10, IL-12/IL-10 and TNF-α/IL-10) of DCs from donor 2 in co-culture with HT-29-NF-κB-luciferase cells and bacteria w or w/o LPS. Experiments were performed with two different donors (data shown for donor 2). *p-value≤0.05.</p

Transcriptomic analysis of <i>C. elegans</i> N2 fed with CNCM I-3690 vs CNCM I-4317.

<p>Transcriptomic analysis of <i>C. elegans</i> N2 fed with CNCM I-3690 vs CNCM I-4317.</p