Aptamers ApPABP#3, ApPABP#7 and ApPABP#11 show a high affinity for LiPABP.

<p>(<b>A</b>) Analysis of the binding capability of the individual aptamers by ELONA. rLiPABP was plated at 1 μg/well (15 pmol/well) and incubated with the digoxigenin-labeled SELLiPABP aptamer population at 80 nM and, finally, incubated with anti-digoxigenin-POD antibodies and revealed with ABTS solution at 405 nm (a, p<0.001; c, p<0.05 versus RND40 value; ***, p<0.001 versus SELLiPABP value). (<b>B</b>) Binding affinity of ApPABP#3, ApPABP#7 and ApPABP11 to LiPABP by ELONA. Recombinant protein LiPABP was plated at 500 ng/well (7.5 pmol/well), incubated with the digoxigenin-labeled aptamers at the concentrations between 0 and 200 nM and revealed as above. All the experiments were made in triplicate and average of four different experiments is shown. (<b>C</b>) BSA and rLiPABP at the concentration indicated in the figure were fixed under vacuum to a nitrocellulose membrane. Membranes were blotted with digoxigenin-labeled ApPABP#3, ApPABP#7 and ApPABP#11 aptamers at 50 nM concentration. Afterwards, the membranes were probed with anti-digoxigenin-POD antibodies, developed with enhanced chemiluminescence’s kits and exposed to hyperfilm. The experiments shown are representative of at least three different experiments. (<b>D</b>) Sensitivity of the aptamers to recognize LiPABP from <i>L</i>. <i>infantum</i> promastigotes was analyzed by ELONA. Total lysates corresponding to increasing amounts of parasites (10²-5x10³) were plated and incubated in the presence of digoxigenin-labeled ApPABP#3, ApPABP#7 and ApPABP#11 at 10 nM and assays were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140048#pone.0140048.g001" target="_blank">Fig 1</a>. All the experiments were made in triplicate and average of 3–4 different experiments is shown. Statistical significance was calculated between each parasite number and the value obtained for 0 parasites (b, p<0.01; c, p<0.05).</p

Ultrastructural studies of antibiotics effects.

<p>A. CM17 untreated control; B. Silvio/X10 untreated control; C. CM17 PIM 0.4 µM for 4 hours; D and E. Silvio/X10 PIM 0.4 µM for 4 hours; F. CM17 PIM 4 µM for 4 hours; G. Silvio/X10 CE108B 4 µM for 4 hours; H. CM17 PIM 0.4 µM for 24 h; I. Silvio/X10 PIM 0.4 µM for 24 hours; Bars in white represent 10 µm in F and I, and 5 µm in all others figures.</p

Activity on metacyclic tripomastigotes.

<p>The table shows the effective concentration causing 50% growth inhibition at 72 hours compared to untreated control culture (IC₅₀) in micromolar.</p

Summary of antibiotic treatment effect on the mitochondrial membrane potential.

<p>IV: Index of variation as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040901#s2" target="_blank">Materials and Methods</a>.</p

Predicted structural folding of selected ssDNA aptamers for LiPABP.

<p>The DNA sequences of ApPABP#3, ApPABP#7 and ApPABP#11 aptamers were analyzed using the program Rapidshare. The resultant predicted secondary structures of the three selected aptamers are shown. The nucleotides labeled in red are those conserved in the sequence of the three aptamers.</p

Trypanocidal activity of antibiotics and Benznidazol (BZN) on epimastigote form.

<p>Trypanocidal activity of antibiotics and Benznidazol (BZN) on epimastigote form.</p

Representative dose-viability curves of PIM and CE108B on different DTUs after 72 hours incubation.

<p>Epimastigotes of indicated DTUs were incubated with the drug for 72 hours in the microtiter plate assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040901#s2" target="_blank">Materials and Methods</a>. It shows the growth inhibition compared to control cultures without any drug (shown media of three experiments in triplicate).</p

Cluster tree build from accumulated drug sensitivity as described in Materials and Methods.

<p>Cluster tree build from accumulated drug sensitivity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040901#s2" target="_blank">Materials and Methods</a>.</p

Binding capability and specificity of the SELLiPABP aptamer population for LiPABP.

<p>ELONA (<b>A, B</b> and <b>D</b>) and slot-blot (<b>C</b>) assays were performed as described in “Materials and methods” section. (<b>A</b>) Recombinant LiPABP was plated at 500 ng/well (7.5 pmol/well) and incubated with the digoxigenin-labeled SELLiPABP aptamer population at the concentrations indicated (0–80 nM). All the experiments were made in triplicate and average of four different experiments is shown. (<b>B</b>) Recombinant LiPABP was plated to several concentrations between 0–5 μg/well (0–75 pmol/well) and incubated with 80 nM of digoxigenin-labeled SELLiPABP aptamer population. All the experiments were made in triplicate and average of four different experiments is shown. Statistical significance between each concentration of LiPABP protein and the value obtained for the blank **, p<0.01. (<b>C</b>) BSA and rLIPABP at the concentration indicated were fixed under vacuum to a nitrocellulose membrane. Membranes were blotted with digoxigenin-labeled SELLiPABP aptamer population at several concentrations. Afterwards, the membrane was probed with anti-digoxigenin-POD antibodies, developed with enhanced chemiluminescence’s kits and exposed to hyperfilm. The experiment shown is representative of at least three different experiments. (<b>D</b>) Recombinant proteins LiPABP, LiIF2α, LiIF2β, LiIF2γ, LiH2A, LiP2a or eukaryotic eIF2 protein were plated at 2 μg/well and incubated with 40 nM of digoxigenin-labeled SELLiPABP aptamer population. Afterwards, anti-digoxigenin-POD antibody was added and the plates were developed using ABTS solution. All the experiments were made in triplicate and average of four different experiments is shown (a, p<0.001 versus LiPABP value; ***, p<0.001 versus value obtained for the blank).</p

Effect of antibiotics on <i>in vitro</i> infection and intracellular amastigotes.

<p>LLC-MK2 cells were infected with bloodstream trypomastigotes of the indicated DTUs as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040901#s2" target="_blank">Materials and Methods</a>. Two different protocols were used, PRE: the drugs were added during the four hours infection period and kept during the proliferation of amastigotes intracellularly. In the second protocol (POST), the drugs were added to the culture medium 24 h after the infection. After four days, the cultures were processed as described and not less than 300 cells were counted by two independent observers. A.- Shows the percentage of infected cells. B.- Shows the number of amastigotes per infected cell.</p