Challenge of ookinete cultures with increasing concentrations of the ROS producing agent paraquat (PQ).

<p>RT-qPCR data showing dose-response modulation of target gene expression in ookinete cultures in the presence of increasing concentrations of the superoxide-producing compound Paraquat (PQ, Viologen, Aldrich). Increasing concentrations of PQ were added to 12-hours <i>P. berghei</i> ookinete cultures as indicated. Shown are the ratios of normalized relative transcript quantities (RQ) of treated vs. non-treated samples (non-treated RQ = 1, represented by the dashed line). All data were normalized against the expression of 18 s rRNA A-type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Yano1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Thompson1" target="_blank">[34]</a>. (*) indicates statistical significance (p<0.1). Shown are mean values of 3 independent experiments. Error bars indicate STDEV.</p

Candidate genes of the cytosolic thioredoxin system of <i>P. berghei</i>.

<p>Candidate genes of the cytosolic thioredoxin system of <i>P. berghei</i>.</p

Time dependent transcription profiles of the cytosolic thioredoxin system in culture-derived and mosquito-derived parasites.

<p>RT-qPCR data showing relative target gene expression as fold increase over time. The 3-hour time point was used as a reference. All data was normalized against the expression of 18 s rRNA A-type <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Yano1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003136#ppat.1003136-Thompson1" target="_blank">[34]</a>. The Mann-Whitney U test was conducted on each candidate gene from both mosquito-derived and from culture-derived parasites. Significance was assessed at p<0.1(*) due to the small sample sizes. Shown are mean values of 4 independent blood feeding experiments (n = 55 mosquitoes/time point/experiment) and 3 independent ookinete culture setups, respectively. Error bars show STDEV.</p

Expression of the Trx-1, TPx-1 and 1-Cys Prx in developing ookinetes from culture or from mosquito bloodmeal.

<p>Samples were isolated from ookinete cultures and blood fed mosquitoes at indicated time points and prepared as described in material and methods. Fixed samples were probed with <b>A</b>) anti-PbTrx-1, <b>B</b>) anti-Pf TPx-1 or <b>C</b>) anti-PbTPx-1. Primary antibodies were probed with secondary antibody coupled to AF 488 (Molecular Probes). Samples were counterstained with the nuclear dye TO-PRO-3 (Life technologies). The source of the ookinetes from either ookinete cultures or bloodfed mosquito midguts is indicated. Images are merged and overlaid onto the respective DIC image. White arrows indicate accumulation of Trx-1, TPx-1 or 1-Cys Prx at the apical ends of developing ookinetes. Scale bar = 5 µM. Quantitative <i>Relative Fluorescence Analysis</i>: Shown is the median boxed by the first and third quartiles with minimum and maximum values displayed as whiskers. Mann-Whitney U-Test was performed with samples of culture-derived ookinetes compared to mosquito-derived ookinetes (p = 0.4551 for Trx-1; p = 0.1061 for TPx-1; p = 0.0413 for 1-Cys Prx). Below each graph are representative images from IFA-epifluorescence experiments.</p

Comparison of <i>Haemophilus</i> across all subject groups.

<p>The relative abundance of <i>Haemophilus</i> for each group is plotted. Box represents median and 25^th and 75^th percentile (interquartile range, IQR) and whiskers represent 1.5x IQR. Individual samples outside the 1.5x IQR are marked as open circles.</p

Subject Characterization Summary.

<p>Subject Characterization Summary.</p

Principal Components Analysis (PCA) of Normal and EoE samples.

<p>PCA using normal esophagus (n = 25) and EoE samples (n = 11 untreated and n = 26 treated). The biplot displays the first two principal components which explains 29% of the variability across genera, as well as vectors corresponding to the weight and direction of the loadings for the highest weighted genera. Vectors pointing in the same direction are genera that are positively correlated; those going in opposite directions are negatively correlated.</p

Manhattan plot of the two part statistical analysis of Aurora, CO (n = 17) versus Chicago, IL (n = 20) microbiome samples.

<p>The plot shows the negative log₁₀ p-value for each taxon identified. There are six significant taxa identified. The peaks correspond to 1. <i>Actinomyces</i> (p = 0.048), 2. <i>Prevotella</i> (p = 0.016), 3. <i>Streptococcus</i> (p = 0.016), 4. <i>Parvimonas</i> (p = 0.022), 5. <i>Fusobacterium</i> (p = 0.009) and 6. <i>Aggregatibacter</i> (p = 0.027). All significantly different taxa were elevated in the Aurora relative to Chicago group except for <i>Streptococcus</i>.</p

Esophageal phyla are similar in EoE compared to normal esophagus, and treatment affects the genus abundance in EoE and GERD.

<p>Abundance of esophageal phyla and genera as captured using the EST. A. Bar graphs present the aggregate of the relative phylum abundance on the ESTs of Normal subjects (n = 25), EoE subjects untreated (n = 11), treated (n = 26), and GERD subjects untreated (n = 4), PPI treated (n = 4). Each phylum is indicated in a different color. The width of the bar corresponds to the relative phylum abundance. B. Bar graphs present the aggregate of the relative genus abundance on the ESTs of Normal subjects (n = 25), EoE subjects untreated (n = 11), treated (n = 26), and GERD subjects untreated (n = 4), treated (n = 4). Each genus is indicated in a different color. The width of the bar corresponds to the relative genus abundance. The Other category includes taxa with <1% relative abundance in all samples.</p

The esophageal bacterial load is increased in subjects with EoE and GERD: Analysis by 16S Q-PCR.

<p>The esophageal bacterial load was captured using the EST as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128346#pone.0128346.ref005" target="_blank">5</a>]. A. The copy number of bacteria per ng of DNA is significantly increased in subjects with EoE, and GERD as compared to Normal. B. Treatments (Trt) do not significantly change the load of bacteria in each disease. C. The disease activity or response to treatment does not significantly change the load of bacteria in EoE. D. The effective number of taxa is similar in Normal, EoE and GERD. The mean ± SEM are shown. Normal n = 25, EoE n = 37, GERD n = 8. Mean and SEM, two-sample t-test * P<0.<i>05</i>, ** P<0.<i>01</i>, *** P<0.<i>001</i>, **** P<0.<i>0001</i>.</p