Variation in the relative numbers of satellite DNA copies per genome.

<p>Results are expressed as arbitrary units, obtained calculating the ratio of copy numbers of Cl Brener satellite/ ribosomal-P2α gene DNA targets.</p

Follow-up of Chronic Chagas heart disease patients after heart transplantation.

<p>Parasitic loads in peripheral blood samples of Chronic Chagas heart disease patients with clinical reactivation due to immunosupression after heart transplantation. * Time of diagnosis of clinical reactivation and etiological treatment.</p

Reproducibility of the Q-PCR and IS-PCR assays in clinical samples of Chagas disease patients.

<p>The <i>T.cruzi</i> lineage group was assessed by Lg-PCR as <i>T.cruzi</i> II in all tested patients. Parasitic Loads were calculated as shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000419#pntd-0000419-t003" target="_blank">Table 3</a>.</p

Translation inhibition.

<p><b>A.</b> Crude extracts from <i>T. cruzi</i> (lane 1), <i>T. brucei</i> (lane 2), <i>C. fasciculata</i> (lane 3) and <i>R. norvegicus</i> (lane 4) were analyzed by SDS-PAGE and Western blot with scFv C5. The position of previously characterized P proteins from <i>T. cruzi</i> (Tc) is shown on the left. The image is representative of three independent assays. <b>B.</b> Dose-response effect of scFv C5 on protein synthesis by ribosome extracts from <i>R. norvegicus</i> and <i>T. cruzi</i>. <b>C.</b> Effect of scFv C5 160 nM on <i>in vitro</i> protein synthesis in ribosome extracts from <i>R. norvegicus</i>, <i>C. fasciculata</i> and <i>T. cruzi</i> compared with the translation inhibitor emetine at 0.1 mg/mg. Average values for control assays were 80,000 cpm, 48,000 cpm and 43,000 cpm for <i>R. norvegicus</i>, <i>C. fasciculata</i> and <i>T. cruzi</i>, respectively. The scFv C5 significantly inhibited protein synthesis by <i>C. fasciculata</i> and <i>T. cruzi</i> ribosomes (*, p<0.05; ***, p<0.001). <b>D.</b> Effect of the preincubation with R13 or H13 peptide on the translation inhibition by scFv C5 (**; p<0.01).</p

Melting curve analysis of satellite amplicons from reference stocks.

<p>Group I satellite amplicons show melting temperatures above 85°C, whereas Group II render amplification products with melting temperatures below 85°C.</p

scFv C5 Epitope specificity.

<p><b>A.</b> Sequence aligment of <i>T. cruzi</i> R13 and P015 peptides with the mammalian counterpart H13 peptide. White letters correspond to residues necessary for antibody recognition, as identified by alanine scanning. Grey background corresponds to those residues conserved in the other two peptides. <b>B.</b> Inhibition of the interaction between scFv C5 and TcP2β protein by R13, P015 and H13 peptides, using surface plasmon analysis. The figure corresponds to one representative result out of 3 independent assays. <b>C.</b> Crystal structure of the complex mAb 17.2-R13 (PDB 3SGE). <b>D.</b> Model of the interaction of mAb 17.2 with the H13 peptide. The interaction energy is indicated on each case. <b>E.</b> NMR solution structure of H13 peptide (PDB 1S4J) in comparison with the antibody-bound peptide structure modeled.</p

P proteins phylogeny and epitope analysis.

<p><b>A.</b> Phylogeny inference of ribosomal P proteins. Red, branch of trypanosomatid P1α proteins. Blue, branch of trypanosomatid P1β proteins. Magenta, branch of trypanosomatid P2α proteins. Light blue, branch of trypanosomatid P2β proteins. Brown, branch of non-trypanosomatid P2 proteins. Violet, branch of non-trypanosomatid P1 proteins. Yellow, <i>Arabidopsis thaliana</i> P3. Green, branch of P0 where has been grouped separated the trypanosomatids P0 (light green). Bootstrap values above 70 are shown. <b>B.</b> The sequences of the C-terminal residues of mammalian P-proteins and the C-terminal region of the small protozoan P-proteins were aligned using WebLogo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036233#pone.0036233-Crooks1" target="_blank">[31]</a>. Arrows indicate critical residues for scFv C5 recognition. The region boxed corresponds to the key difference between mammal and protozoan epitopes.</p

Parasitic loads in peripheral blood samples from pediatric patients.

<p>(A) Association between basal parasitic loads and patients' ages in 43 pediatric cases. Coefficient of correlation: −0.5832; P<0.05. (B) Monitoring of parasitological response to benznidazole therapy in 38 pediatric patients. The evolution of the parasitic loads for patients with more then one positive sample are depicted. Samples were withdrawn at time of diagnosis (t1), after 7 (t2) and 30 (t3) days of treatment, as well as at the end of treatment (t4, 60 days). Only the PCR positive samples are shown. The horizontal line represents the lower limit of the dynamic range of Q-PCR.</p

Examples of the calculation of parasitic loads in pediatric and transplanted patients.

<p>In the samples from transplanted patients (Tx) whose infecting lineages could not be determined, they were presumed from the data obtained from other samples of the same patient, assuming that the lineage is persistent during reactivation <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000419#pntd.0000419-Altcheh1" target="_blank">[13]</a>. ND: Not determined.</p>#<p>In case Pd6 no presumption could be made about the parasite lineage.</p>‡<p>Samples diluted before Lg-PCR.</p

scFv C5 intrabody expression in T. <i>brucei</i>.

<p><b>A.</b> Western blot detection of C5 intrabody expression at 0, 24 and 72 hours in six different clones. C5 was detected by an anti-Myc antibody. The loading control ALD was detected by an anti-aldolase antibody. <b>B.</b> Averaged densitometry analysis of intrabody expression (***; p<0.001). <b>C.</b> Averaged growth rate curve of the six different clones induced and un-induced C5 intrabody parasites (***; p<0.001).</p