Enrichment of reciprocal <i>C. elegans BLAST hits</i> among tissue-overexpressed genes.

*<p>34.9% of all genes expressed in this study had reciprocal <i>C. elegans</i> BLAST hits.</p

Summary of predicted <i>Ascaris suum</i> peptidases identified in Concanavalin A (ConA) binding and intestinal perfusate fractions.

1<p>Proteins listed as ConA and detected in Perfusates are listed in the ConA column. ²Proteins unique to Perfusates are listed in the Perfusates column. ³A. suum proteins predicted as intestinal peptidases in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#pntd-0003375-t001" target="_blank">Table 1</a> are highlighted in bold-italics</p><p>Summary of predicted <i>Ascaris suum</i> peptidases identified in Concanavalin A (ConA) binding and intestinal perfusate fractions.</p

Gene Ontology (GO) terms significantly (p≤0.01, FDR corrected) enriched among genes overexpressed in non-reproductive tissues according to FUNC.

<p>GO root terms were abbreviated (MF = Molecular Function, BP = Biological Process, CC = Cellular component) and terms are sorted according to descending enrichment significance.</p

Gene Ontology (GO) terms significantly (p≤0.01, FDR corrected) enriched among genes overexpressed in reproductive tissues according to FUNC.

<p>GO root terms were abbreviated (MF = Molecular Function, BP = Biological Process, CC = Cellular component), and terms are sorted according to descending enrichment significance.</p

Peptidase intestinal gene expression and presence among <i>A. suum</i> proteomic datasets.

1<p>Protease classes identified using MEROPS web server <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#pntd.0003375-Rawlings1" target="_blank">[27]</a>; ²Intestinal Expression and overexpression based on previous study <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#pntd.0003375-Rosa1" target="_blank">[30]</a>; ³Data collected from this study.</p><p>Peptidase intestinal gene expression and presence among <i>A. suum</i> proteomic datasets.</p

(A) Simplified diagram of the tissues selected for deep RNA sequencing in male and female adult <i>A. suum</i> worms.

<p>(B) Hierarchical clustering of <i>A. suum</i> RNA-seq samples based on gene expression data across all expressed genes. Numbers above lines represent the number of genes overexpressed in each branch of the clustering and numbers below lines in parentheses represent the number of genes overexpressed in both of its child branches.</p

Concanavalin A (ConA) binding proteins from Adult <i>A. suum</i> intestine.

<p><b>A, B</b>. Histological sections were treated without (A) or with sodium periodate (B), as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#s2" target="_blank">Methods</a>, and then incubated with the ConA-horse radish peroxidase conjugate (ConA-HRPO). Binding was localized by histochemical detection. Black arrows point to intestinal microvilli, the white arrow points to material extending from microvilli into the lumen. <b>C</b>. Intestinal proteins separated by non-reducing SDS-PAGE were transferred to nitrocellulose filters, and then untreated (NP) or treated (P) with sodium periodate. Filters were incubated with Con A-HRPO and ConA binding bands localized by chemiluminescence. Molecular weight markers are indicated on the left of the panel. <b>D</b>. Peptidase activity was evaluated for intestinal proteins bound to ConA-agarose beads, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#s2" target="_blank">Methods</a>, and conducted at the pH conditions indicated on the x axis. Relative fluorescence units (RFU) µg-protein⁻¹ from hydrolysis of Bodipy casein is indicated on the y axis. A 95% confidence interval was constructed for group means at each pH tested, which in each case was greater than zero. <b>E</b>. Inhibition of peptidase activity isolated on ConA agarose beads. Assays were conducted at pH 5.0. Percentage inhibition indicated on the y axis was determined with inhibitors, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#pntd-0003375-g001" target="_blank">Figure 1</a>, and means for each inhibitor treatment tested were significantly lower (p<0.05) than the uninhibited control group. All peptidase assays were conducted with three replicates. <b>F</b>. Coomassie blue stained SDS PAGE gel, as in panel C, of intestinal proteins isolated on ConA agarose beads. ConA, indicates heavy ConA proteins released from the beads. No bands were excised for mass spectrometry at or below this position in the gel.</p

Transcription factor binding motif enrichment in the 5′ untranslated regions (UTRs) of genes overexpressed in specific tissues.

<p>Transcription factor binding motif enrichment in the 5′ untranslated regions (UTRs) of genes overexpressed in specific tissues.</p

RNA-seq statistics for tissues-specific samples.

<p>RNA-seq statistics for tissues-specific samples.</p

Intestinal peptidase activity.

<p><b>A</b>. Whole intestinal cell fractionation of peptidase activity. Peptidase activity was determined using a Bodipy casein substrate with 4 µg of sample, as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003375#s2" target="_blank">methods</a>, for whole intestinal homogenates (WL), 5,000×<i>g</i> pellet (P1) and supernatant (S1), and a further 50,000×<i>g</i> pellet (P2) and supernatant (S2) derived from S1. All samples were solubilized in 1% TX-100 prior to running assays. pH at which reactions were run is indicated on the x axis. Peptidase activity (y axis) is reported as Relative Fluorescent Units (RFU) µg-protein⁻¹. Means that differ from one another (p<0.05) are indicated by different letter designations (A, B, C, D) below the x axis, and were analyzed according to pH conditions. <b>B, C</b>. Inhibition of peptidase activity in the whole lysate (B) and P2 (C) fractions by pepstatin (aspartic proteases, Pep), phenylmethylsulfonyl chloride (serine peptidases, PMSF), 1,10 phenanthroline (metallopeptidases, 1, 10 Phen) and <i>trans</i>-epoxysuccinyl-L-leucylamido(4-guanidino)butane, L-<i>trans</i>-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (C1 cysteine peptidases (including cathepsin B) cysteine peptidases, E-64). Percentage inhibition (y axis) of the mean activity uninhibited compared to mean activity of the inhibited is shown for each pH (x axis) tested. An asterisk denotes treatments that were significantly different (p<0.05) from the untreated control group for each pH indicated. All assays were conducted with three replicates.</p