Z-Stack images (interval = 0.2 µm) of an erythrocyte infected with two parasites (<i>P. falciparum</i>).

<p>The first image is an overlay of the DIC image, the fluorescence signal from the <b>BtC</b> probe (pseudo-colored green) and the Hoechst dye (pseudo-colored blue). [<b>BtC</b>] = 35 µmol L^–1; bar = 2 µm.</p

Labeling of live <i>P. falciparum</i>-infected erythrocytes with BtC.

<p><b>BtP</b> was used as the betalain control. Panels show representative wide-field images of three independent experiments. The arrow indicates the non-fluorescent hemozoin crystal inside the DV of a mature trophozoite. [<b>BtC</b>] = 28 µmol L^–1; [<b>BtP</b>] = 170 µmol L^–1; incubation = 2 min at 25°C; bar = 10 µm.</p

Claudin-10 expression on B-1 lymphocytes mediates the alterations in B16F10 melanoma cell behavior.

<p><b>A)</b> Expression of claudin-10 on B-1 lymphocytes from peritoneal cell culture transfected for 24 h with stealth claudin-10 siRNA1 (B-1 cldn-10 siRNA1), siRNA2 (B-1 cldn-10 siRNA2), siRNA3 (B-1 cldn-10 siRNA3). <b>B)</b> The number of lung colonies in mice injected with B16F10 melanoma cells from single culture (B16F10) or after co-cultivation with B-1 lymphocytes (B16F10+B1), transfected or not with claudin-10 siRNA1 (B16F10 + B-1 cldn-10siRNA1), siRNA2 (B16F10 + B-1 cldn-10 siRNA2), siRNA3 (B16F10 + B-1 cldn-10 siRNA3). <b>C)</b> Inhibition of claudin-10 expression impairs the activation of ERK pathway and the claudin-10 expression in B16F10 cells after B-1 contact. GAPDH was used as internal control. Bars represent the mean number of lung colonies per experimental condition ±SD. *p < 0.05; **p < 0.01;***p < 0.001, using one-way ANOVA with Tukey’s post hoc test.</p

Endogenous IL-10 drives the influence of B-1 lymphocytes in B16F10 behavior.

<p><b>A)</b> The deficiency in IL-10 production did not affect the phenotype and the number of B-1 lymphocytes recovered from the culture of peritoneal cavity cells; <b>B)</b> but affected their ability to increase the metastatic potential of B16F10 melanoma cells. Bars represent the mean number of lung colonies per experimental condition ±SD. **p <0.001, using one-way ANOVA with Tukey’s post hoc test.</p

IL-10 deficiency is associated with impaired expression of claudin-10 in B-1 lymphocytes.

<p>Total RNA of B-1 lymphocytes enriched culture from wild-type (B-1WT) or IL-10^-/- knockout (B-1IL-10^-/-) mice was submitted to microarray analysis. <b>A)</b> Heatmap showing differential expression of eleven transcripts between B-1WT and B-1IL-10^-/- lymphocytes, and the down-regulation of claudin-10 (cldn-10) in B-1IL-10^-/- samples. <b>B)</b> Western blot analysis of cldn-10 protein in whole-cell extracts from B-1WT and B-1IL-10^-/-. β-actin protein was used as internal control.</p

B-1 lymphocytes, but not other peritoneal cells, induce the pro-metastatic effect on B16F10 melanoma cells.

<p><b>A)</b> Adherent peritoneal cells (apc) from wild-type mice, but not from B cell-deficient mice (BKO), obtained from enriched peritoneal cell cultures increases B16F10 metastatic potential upon cell-to-cell contact. <b>B)</b> The same results were obtained with cell-sorting technique, confirming that B-1 lymphocytes are responsible for changes in the melanoma behavior. (Bars represent the mean number of lung tumor per experimental condition ±SD (<i>n</i> = 4); **p < 0.01; ***p < 0.001, using one-way ANOVA with Tukey’s post hoc test.</p

Inhibition of claudin-10 expression in B16F10 inhibits the influence of B-1 lymphocytes in melanoma aggressiveness.

<p>B16F10 cells were transfected for 24h with stealth claudin-10 siRNA1. The inhibition of claudin-10 expression in B16F10 blocks <b>A)</b> activation of ERK pathway and claudin-10 expression and, <b>B)</b> impairs the B16F10 aggressiveness induced by the contact with B-1 lymphocytes. (Bars represent the mean number of lung tumor per experimental condition ±SD (<i>n</i> = 3). **p < 0.01; ***p < 0.001, using one-way ANOVA with Tukey’s post hoc test.</p

Interclonal Variations in the Molecular Karyotype of <i>Trypanosoma cruzi</i>: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

<div><p><i>Trypanosoma cruzi</i> comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the <i>T. cruzi</i> G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for <i>Leishmania infantum,</i> have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the <i>T. cruzi</i> arsenal for responding to environmental pressure.</p></div

Identification of homologous chromosomal bands of similar molecular sizes in the G strain and clone D11.

<p>Hybridization profile of specific chromosomal markers hybridized to one or more bands of similar molecular size in both isolates after chromosome separation by PFGE and Southern-blot hybridization. The markers used are TEUF0099, rDNA18S, TEUF0242 and ADC. Gene identification and GenBank accession number of each marker are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063738#pone-0063738-t001" target="_blank">Table 1</a>.</p

Allele sizes (bp) for each microsatellite locus amplified for the G strain and clone D11.

a<p>Microsatellite loci with the same alleles in the G strain and D11 clone.</p>b<p>Microsatellite loci with a common allele in the G strain and D11 clone.</p>c<p>Microsatellite loci with different alleles in the G strain and D11 clone.</p