Odor exploration behavior in mice.

<p>Percentage of entries (A) and exploration time spent (B) by mice in zones A and B of the open field with food containers filled with freshly cut (0 h) WT or -PPO potato samples. Percentage of entries (C) and exploration time spent (D) by mice in zones A and B of the open field with food containers filled with oxidized (24 h) WT or -PPO potato samples. Stars represent statistically significant differences (<i>P≤0.03</i>) according to the one-sample <i>t</i>-test for difference from 50%. Error bars represent the ±95% confidence interval of eight independent experiments. (E) Hole-board experiment. Mean investigation times (s) ± SEM of six independent measurements are shown for each type of sample. The star represents statistically significant differences (<i>P≤0.05</i>) in investigation time according to the ANOVA test followed by the Newman-Keuls multiple comparison post-hoc test.</p

Mouse open field experimental design.

<p>(A) Schematic upper view of the open field activity boxes with zones A and B comprising food containers. (B) Diagram showing the open field experimental procedure. From day 1 to day 3 mice were habituated to the activity boxes with empty food containers. On day 4, mice exploration behavior was monitored with empty food containers and the data obtained was considered as a negative experimental control (−). On day 5, freshly cut potato samples (0 h) were randomly placed in containers A or B. On day 6, oxidized potato samples (24 h) were placed in the opposite positions with respect to day 5. On day 7, freshly cut WT potato samples were placed in one container while the other container remained empty and the data obtained was considered as a positive experimental control (+).</p

Humans sensory analyses.

<p>(A–B) Smell discrimination by humans. (A) Smell discrimination with all transgenic lines. A filled box indicates that the odor of the sample was described as more intense by the subject. (B) Triangle test with fresh and oxidized samples. The number of correct answers to be significant is 11 (<i>P≤0.03</i>) correct responses for the group tested with fresh samples (n = 19) and 25 (<i>P≤0.001</i>) correct responses for the group tested with oxidized samples (n = 42). The number of correct responses (+) was determined by counting the number of participants that chose the unique sample of the three. The number of incorrect responses (−) equals the number of participants not choosing the distinct sample of the three. The percentage of correct and incorrect responses is depicted and the number of the corresponding responses is shown inside each bar. Two stars represents statistically significant differences at <i>P≤0.03</i> and three stars represents statistically significant differences at <i>P≤0.001</i>. (C–E) Organoleptic evaluations. (C) Hedonic rating. Being 1: “not at all pleasant” and being 9: “very pleasant”. No statistical differences were found among samples according to the ANOVA test. (D) Comments describing that the odor of the sample was more intense. The percentage of comments is depicted and the number of the corresponding comments is shown inside or above each bar. (E) Comments describing that the odor of the sample evoked the sense-impression of a familiar vegetable. The percentage of comments is depicted and the number of the corresponding comments is shown inside or above each bar.</p

Mouse open field test experimental validation.

<p>Percentage of entries (A) and exploration time spent (B) by mice in zones A and B of the open field with both food containers empty. (C) Representative 5 min mouse trajectory of a negative experimental control where both food containers were empty. Percentage of entries (D) and exploration time spent (E) by mice in zones A and B of the open field with one container filled with freshly cut WT potato and the other one left empty. (F) Representative 5 min mouse trajectory of a positive experimental control where food container A was empty and food container B was filled with freshly cut WT potato samples. Central darker squares in Figures C and F represent zones A (on the left) and B (on the right). Stars represent statistically significant differences (<i>P≤0.01</i>) according to the one-sample <i>t</i>-test for difference from 50%. Error bars represent the ±95% confidence interval of eight independent experiments.</p

Amino acid sequence alignment of the PARG signature from different organisms.

<p>The multiple alignment of the PARG signature amino acid sequences corresponding to <i>T. cruzi</i> PARG (accession number ABG73229); <i>T. brucei</i> PARG (GeneDB Systematic Name: Tb09.211.3760); <i>C. elegans</i>_1 PARG (accession number NP_501496) and <i>C. elegans</i>_2 PARG (accession number NP_501508); <i>T</i><i>. thermophila</i> (accession number EAR94344); <i>A. thaliana</i>_1 PARG (accession number NP_973578); <i>A. thaliana</i>_2 PARG (accession number AAK72256); <i>D. discoideum</i> PARG (accession number XP_642024); <i>D. melanogaster</i> PARG (accession number NP_477321); <i>C</i><i>. quinquefasciatus</i> PARG (accession number XP_001853435); <i>A. aegypti</i> PARG (accession number XP_001659301); <i>D. rerio</i> PARG (accession number XP_001338257); <i>X. laevis</i> PARG (accession number NP_001089602); <i>G. gallus</i> PARG (accession number XP_421502); <i>B. taurus</i> PARG (accession number NP_776563); <i>R. norvegicus</i> PARG (accession number NP_112629); <i>M. musculus</i> PARG (accession number NP_036090); <i>H. sapiens</i> PARG (accession number NP_003622); <i>P. abel</i>ii PARG (accession number NP_001125086); <i>P</i><i>. troglodytes</i> PARG (accession number XP_001139727) was generated with the ClustalW2 program and edited with the BOXSHADE (3.21) software. Colors used for amino acids background are as follow: white for different residues, black for identical residues, gray for similar and conserved residues. Asterisk: essential acidic residues D-E-E, underlined: key residues, G and two consecutive E, and black diamond: important Y residue.</p

Role of TcPARG in <i>Trypanosoma cruzi</i> epimastigotes proliferation and cell cycle progression.

<p>A) Effect of the PARG inhibitors ADP-HPD and DEA on <i>T. cruzi</i> growth and survival was determined by incubating epimastigotes at an initial density of 10⁷ parasites/ml in the continuous presence of inhibitors at 1 µM. The number of epimastigotes was determined daily by counting formaldehyde-fixed parasites in a Neubauer chamber. All data points were determined in triplicates and shown as means with standard deviation. The significance of the results versus the control at day 4 was analyzed with t test and indicated in the figure (* p0.05). B) Effect of ADP-HPD at 1 µM concentration on cell cycle progression of epimastigotes was determined by adding the inhibitor at the indicated concentration to the culture media of hydroxyurea synchronized parasites after digitonin permeabilization. Samples were drawn every 2 hours for 14 hours and DNA content was determined by propidium iodide staining followed by flow cytometry analysis. The percentage of epimastigotes in each cell cycle phase was determined by setting gates according to the DNA content in the 0 hs of the control sample and maintained for all other samples. The data were analyzed using the Cyflogic software.</p

Expression of TcPARG throughout the <i>Trypanosoma cruzi</i> life-cycle.

<p>(A) Microarray expression data for TcPARG over the course of <i>T. cruzi</i> life-cycle. TcPARG mRNA relative abundance was evaluated by using the transcriptome analysis of different <i>T. cruzi</i> stages available at Gene Expression Omnibus database (<a href="http://www.ncbi.nlm.nih.gov/geo" target="_blank">www.ncbi.nlm.nih.gov/geo</a>, DataSets: GSE14641). Shown are mean microarray log₂ ratios (stage/reference) for TS significantly regulated in <i>Trypanosoma cruzi</i> amastigotes (AMA), trypomastigotes (TRYP), epimastigotes (EPI), and metacyclic trypomastigotes (META). (B) Western blot analysis of the three life-cycle stages of <i>T. cruzi</i>. Protein extracts (35 µg) of amastigote, epimastigote or trypomastigote stages of <i>T. cruzi</i> were solved in a 10% polyacrylamide gel, transfer to a nitrocellulose membrane and revealed with an anti-TcPARG (1:10000) specific antiserum. β-tubulin was used as loading control.</p

Effect of PARG inhibitors on <i>T. cruzi</i> infection on Vero or A549 host cells.

<p><i>T. cruzi</i> trypomastigotes were purified from the supernatant of previously infected cells and preincubated for 30 min in the respective culture medium in the absence (Control) or presence of 1 µM PARG inhibitor (DEA). Twenty-four hours Vero, A549 wild type or shPARG (hPARG silenced) cell monolayers were infected with 50 trypomastigotes/cell. The infection process was followed by microscopic direct visualization. At the indicated days (A and C) or at day 6 post-infection (B and D), percentage of infected cells and number of amastigotes intracellular were determined on May-Grünwald Giemsa stained samples. Amastigotes and cells were counted using the ImageJ software in at least 7 fields. The number of trypomastigotes/ml in the supernatant of infected cell cultures was determined by counting unfixed trypomastigotes in a Neubauer chamber at the indicated days (E) or at day 9 post-infection (F). All points were determined in triplicates and shown as means with standard deviation. Significance of the result versus the Control (***p0.001; two way ANOVA) or Wild Type Control (***p0.001; **, p0.01; two way ANOVA) is indicated.</p

Immunolocalization of PARG on <i>Trypanosoma cruzi</i>, CL Brener epimastigotes.

<p>The parasites were fixed for 25 min with 3.8% (W/V) formaldehyde in PBS at 4°C, permeabilized with fresh PBS - 0,1% Triton X-100 and blocked for 1 h at room temperature with 5% (W/V) BSA in PBS. (A) Differential interference contrast (DIC). (B) Cells were counterstained with DAPI to identify nuclear DNA and kinetoplastid DNA. (C) PARG was detected with 1:500 mouse polyclonal TcPARG antibody followed by 1:600 Alexa Fluor 488 goat anti-mouse IgG antibody. (D) Merge of PARG and DNA signals show the nuclear localization of this enzyme. Bar: 10 µm. (E–F) For electron microscopy, epimastigotes were fixed in PBS 2.5% glutaraldehyde, 4% formaldehyde, embedded in epoxy resin and PARG detected with 1:50 mouse polyclonal TcPARG antibody followed by 1:100 anti-mouse antibody conjugated with 10-nm gold particle. N: nucleus; K: kinetoplast. Bar: 0.2 µm.</p

Screening of TcPARP inhibitors.

<p>A) A panel of 32 compounds was evaluated for the ability to inhibit TcPARP activity <i>in vitro</i>. Compounds were tested at 10 µM and 1 µM concentrations, while protein concentration was 10 nM. The values obtained were converted to % of inhibition in relation to the control performed in the same plate. All data points were determined in duplicates and shown as mean values ±SD. B) Inhibition of TcPARP was confirmed by Western blot using a mixture of biotinylated NAD⁺ (1 µM) and regular NAD⁺ (4 µM) as a substrate. Lysozyme was used as a loading control. TcPARP (150 nM) was incubated in the assay buffer with a set of inhibitors (1 µM). Biotinylated protein was detected by using streptavidin-HRP.</p