Scatter plots of signature proteins for specific worm tissues or processes to illustrate the extent of variation between heads and tails.

<p>The format of the plot is the same as for <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004272#pntd.0004272.g001" target="_blank">Fig 1</a> Those genes with a > fourfold difference in the heads are demarcated by an ellipse. A, nervous system; B, tegument; C, Gut secreted proteins and saposins; D, Muscle and cytoskeleton; E, Parenchyma (glycogen metabolism) and cytosol; F, Glycosyl transferases. Key for B, tegument: black circle, enzymes and transporters; green circle, ATPases; red circle, annexins and tetraspanins.</p

Expression of selected genes in the esophageal region of male and female worms detected by WISH.

<p>VAL-7⁺ is a positive control andVAL-7^- sense strand is a negative control for the technique. All the selected genes that were abundant and differentially expressed in the heads are localised specifically to either the anterior or posterior cell masses of the glands, as revealed by the bluish-purple stain. The intense brown-black colour is hemozoin pigment in the worm gut. Key: Asp Prot, Aspartyl protease; β13glyc, Beta 1,3-galactosyltransferase; PIA2, Phospholipase A2. Palthio, Palmitoyl thioesterase. Scale bar = 24um.</p

Bioinformatic analysis of MEG properties.

<p><b>A)</b> Schematic representation of protein products from MEG-8 family members. Bar represent the full coding sequence of each protein with colours representing different properties of each region: green, intrinsically unstructured hydrophilic region; red, signal peptide; yellow, hydrophobic region. Stars ind icate points of predicted O-glycosylation (black) or N-glycosylation (red). Hatched bar below each protein indicates the C-terminal conserved region. <b>B)</b> Multiple alignment of the conserved C-terminus of MEG-8 family proteins. <b>C)</b> Schematic representation of protein products from MEG-12 gene: pale blue, regions with predicted alpha-helical structure. <b>D)</b> Diagram of the central helix region of MEG-12 showing its amphipathic character. Colors indicate amino acids with: yellow, hydrophobic, purple, polar, blue, positively charged and small side chains. The hydrophobic face of the helix comprises the amino acids LFFIFF.</p

Summary of signature protein expression.

<p>Data from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004272#pntd.0004272.s007" target="_blank">S4 Table</a> plotted to illustrate mean values for signature categories. A,Transcript abundance in the heads expressed relative to that in the nervous system as unity. B, Bias in signature gene expression plotted as the mean RPKM heads/mean RPKM tails. Apart from the nervous system and glycosyl transferases, the bias is distinctly towards the tails, but nowhere large.</p

Scatter plot of all MEGs identified in the study.

<p>The format of the plot is the same as for Figs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004272#pntd.0004272.g001" target="_blank">1</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004272#pntd.0004272.g002" target="_blank">2</a>. The MEGs found by Cufflinks mapping (black circle) are distinguished from those located only by Trinity de novo assembly (red circle). The MEGs fall into three distinct populations: (i) solid line, fourfold in the heads > tails; (ii) dashed line, heads = tails; (iii) dotted line, tails > heads.</p

Exon structure of newly designated MEG genes.

<p>Exons are colour coded as follows: red, symmetrical micro-exons; grey, coding region of the flanking exons; black, non-coding regions; yellow, non-symmetrical micro exons; purple, regular sized symmetrical exons. Other features: black circle, exons that code for transmembrane anchors; black triangle, exons that code for signal peptides. In the case of MEGs 26 and 30 part of the signal peptide is coded by the first micro-exon in addition to the terminal exon.</p

Scatter plot of all genes expressed in heads and tails mapped by Tophat/Cufflinks to the genome.

<p>A RPKM cutoff of 16 (log2 = 4) was first applied to all data (mean of two replicates) which were then sorted by RPKM score into two populations, Heads>Tails and Tails>Heads. Abundance on the y axis is the log2 RPKM score of each head-enriched gene (right) or tail-enriched gene (left). Difference on the x axis = the log 2 ratio of mean RPKM scores. The demarcated areas enclose genes that are expressed at fourfold higher level in either the heads or the tails.</p

ClustalX multiple sequence alignment of the mature protein sequence (excluding the signal peptide) of TFPDs from platyhelminthes, CD59 and Ly6.

<p>The regions with high identity and similarity between sequences are shown as black and gray columns, according to the ClustalX algorithm. Arrows indicate highly conserved Cysteines and Asparagines with a C and an N, respectively. Dashed lines represent pairs of cysteine residues forming disulfide bonds determined from Hs-CD59 (red) and predicted for SmCD59.2 (black). Only for SmCD59 sequences, potential sites for N-glycosylation are shown in blue with asparagine (N) in red, and potential sites for GPI anchor are shown in yellow. Human CD59 active sites are shaded in red. The sequences abbreviation are: <i>Schistosoma mansoni</i> (SmCD59.1-7), <i>Schistosoma japonicum</i> (Sj1, Sj2.3, Sj3, Sj4.1, Sj6), <i>Schistosoma hematobium</i> (Sh1-3 and Sh5-7), <i>Clonorchis sinensis</i> (Cs-757, Cs-8328, Cs-8627), <i>Opisthorchis viverrini</i> (Ov-8524, Ov-3995 and Ov-6738), <i>Fasciola hepatica</i> (Fh-6273), <i>Fasciola gigantica</i> (Fg-25430 and Fg-15245), <i>Schmidtea mediterranea</i> (Smed), <i>Equus caballus</i> (Ec-Ly6), <i>Pongo abelii</i> (Pa-Ly6 and Pa-CD59), <i>Macaca mulatta</i> (Mam-Ly6), <i>Mus musculus</i> (Mm-Ly6 and Mm-CD59), <i>Monodelphis domestica</i> (Md-Ly6), <i>Ornithorhynchus anatinus</i> (Oa-Ly6), <i>Homo sapiens</i> (Hs-Ly6 and Hs-CD59), <i>Saimiriine herpesvirus</i> (Sah-CD59), <i>Rattus norvegicus</i> (Rn-CD59) (the accession numbers are listed in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002482#pntd.0002482.s006" target="_blank">Table S2</a>).</p

Evaluation of the ability of SmCD59.1 and SmCD59.2 to modulate complement activity.

<p>Hemolytic assays were performed after incubating normal human serum (NHS) with different amounts of SmCD59.1 (produced in <i>P. pastoris</i>), SmCD59.2 (produced in <i>P. pastoris</i> or <i>E. coli</i>), or BSA (negative control). (A) Treated NHS was then incubated with rabbit erythrocytes (Alternative Pathway) or (B) antibody-sensitized sheep erythrocytes (Classical Pathway). The percentage of hemolysis was calculated in comparison with erythrocytes suspensions completely lysed with water (100% lysis). The volume of NHS used in these assays corresponds to the amount that promotes 50% lysis of erythrocytes. Each column represents the mean of three independent experiments ± SD.</p

Immunoblotting of protein extracts from <i>S. mansoni</i> stages using anti-rSmCD59.1 or anti-rSmCD59.2 polyclonal antibodies.

<p>Protein extracts (20 µg) from different <i>S. mansoni</i> stages: EGG (eggs), MIR (miracidia), CER (cercariae), SCH (<i>in vitro</i> 7-day-old schistosomula), ♂ (male adult worm), ♀ (female adult worm) and P (positive control, 100 ng of rSmCD59.1) were analyzed using (A) anti-rSmCD59.1 antiserum; or (B) anti-rSmCD59.2 antiserum, P (positive control, 100 ng of rSmCD59.2 expressed in <i>E. coli</i>). Extracts of stripped worms (Strip) and Tegument (Teg) of adult worms were probed with (C) anti-rSmCD59.1 or (D) anti-rSmCD59.2 antisera. Insoluble (Ins) and soluble (Sol) protein extracts of stripped worms; (Tsm) enriched tegument surface membranes fraction; (Twm) tegument extract without surface membranes. Positions of molecular mass standard are indicated.</p