Additional file 1: Figure S1. of Differential cytokine profiling in Chagasic patients according to their arrhythmogenic-status

One phase association fit curves for pattern curve data. Axes represent association between mean fluorescence (MFI) values obtained for different know cytokine with results expressed in pg/ml (y axis). Y=Y0 + (Plateau-Y0)*(1-exp.(-K*x)) equation was used for calculating cytokine concentration for patient samples. Figure S2. High producer frequencies for Lown stratified Chagasic patients. Bar graph shows the percentage of high producers for the different cytokines studied for Lown clinical scoring. Control high producer’s percentages is showed as dotted line and Chagasic frequencies (high & low SD risk) as bars. Threshold to determining high producers was calculated on ROC curve values for patients included in this group. Figure S3. High producer frequencies for blood pressure stratified Chagasic patients. Bar graph shows the percentage of high producers for the different cytokines studied for blood pressure clinical scoring. Control high producer’s percentages is showed as dotted line and Chagasic frequencies (normotensive & hypertensive) as bars. Threshold to determining high producers was calculated on ROC curve values for patients included in this group. Figure S3. High producer frequencies for amiodarone treatment stratified Chagasic patients. Bar graph shows the percentage of high producers for the different cytokines studied for blood pressure clinical scoring. Control high producer’s percentages is showed as dotted line and Chagasic frequencies (untreated & treated) as bars. Threshold to determining high producers was calculated on ROC curve values for patients included in this group. (XLSX 980 kb

Evidence of Reversible Bradycardia and Arrhythmias Caused by Immunogenic Proteins Secreted by <i>T. cruzi</i> in Isolated Rat Hearts

<div><p>Rationale</p><p>Chagas cardiomyopathy, caused by the protozoan <i>Trypanosoma cruzi</i>, is characterized by alterations in intracellular ion, heart failure and arrhythmias. Arrhythmias have been related to sudden death, even in asymptomatic patients, and their molecular mechanisms have not been fully elucidated.</p><p>Objective</p><p>The aim of this study is to demonstrate the effect of proteins secreted by <i>T. cruzi</i> on healthy, isolated beating rat heart model under a non-damage-inducing protocol.</p><p>Methods and Results</p><p>We established a non-damage-inducing recirculation-reoxygenation model where ultrafiltrate fractions of conditioned medium control or conditioned infected medium were perfused at a standard flow rate and under partial oxygenation. Western blotting with chagasic patient serum was performed to determine the antigenicity of the conditioned infected medium fractions. We observed bradycardia, ventricular fibrillation and complete atrioventricular block in hearts during perfusion with >50 kDa conditioned infected culture medium. The preincubation of conditioned infected medium with chagasic serum abolished the bradycardia and arrhythmias. The proteins present in the conditioned infected culture medium of >50 kDa fractions were recognized by the chagasic patient sera associated with arrhythmias.</p><p>Conclusions</p><p>These results suggest that proteins secreted by <i>T. cruzi</i> are involved in Chagas disease arrhythmias and may be a potential biomarker in chagasic patients.</p></div

List of some proteins secreted by <i>T</i>. <i>cruzi</i>.

<p>List of some proteins secreted by <i>T</i>. <i>cruzi</i>.</p

(A) Perfusion circuit with recirculation/ reoxygenation.

<p>The black ovals represent the two peristaltic pumps used to perfuse solutions. The magenta oval represents the device on which the cannula is inserted to perfuse the heart and red oval represents the heart. The black lines show the connections that allow the entry of oxygenated solution (O₂T) in the heart. (B) Protocol of recirculation/reoxygenation. Rc: Recirculation and Ro: Reoxygenation. The red and diagonal black lines indicate the parallel perfused Conditioned medium or MEM.</p

Canonical Biplot of the measured in three perfusion conditions.

<p>Comparing QT interval, PR interval, and heart rate in time. The axis 1 explain 91.55% and the axis 2 explain 8.45% of the variance with a Global contrast based on Wilk’s Lambda of 132.0474 and a p-value of 4.3475e-31. With the three (A, B & C) amplified regions we showed the convex hulls of the three groups and the calculated centroid. Due to scale differences between variables, each one were standardize separately. Analysis carried out with MULTBIPLOT software.</p

Determination of Aspartate aminotransferase activity (AST) in coronary effluent during the recirculation-reoxygenation cycles.

<p>Rc: recirculation cycle and Ro: reoxigenation cycle. The arrows identify the times at which the samples were taken of the recirculation-reoxigenation stages and are identified as Rc1 (recirculation 1), Rc2 (recirculation 2), Rc3 (recirculation 3), Rc1 (reoxygenation 1), Rc2 (reoxygenation 2) and Rc3 (reoxygenation 3). The graph shows the enzyme activity averages of the perfusato samples from hearts treated with CM control (n = 4) and CM infected (n = 5).</p

Western blotting analysis of conditioned medium.

<p>Protein fraction > 50 Kda obtained from CMi and CMc were resolved by gel electrophoresis and probed with sera samples (dilution 1:100) from seropositive class II chagasic patients that exhibited moderate symptoms of clinical disease. As controls it was assayed a no cardiomyopathy Chagasic patient serum.</p

Additional file 4: of Can the intake of antiparasitic secondary metabolites explain the low prevalence of hemoparasites among wild Psittaciformes?

Scripts and combined dataset to analyse the presence of hemoparasites in Psittaciformes. Analyses and the combined dataset for the effects of diet, habitat, climate, screening method (as factors) and species (as a random variable) on the presence of parasites in the studied individuals using a binomial General Lineal Mixed-Effects Model and model averaging based on Akaike information criterion (AIC) with R. Scripts for the 10-fold cross validation and the calculations of parasite detection probability are also provided. (TXT 34 kb

Additional file 2: of Can the intake of antiparasitic secondary metabolites explain the low prevalence of hemoparasites among wild Psittaciformes?

Figure S1. Locations of the sampled population at Rasa I., Palawan, Philippines, in the Indo-Malayan zoogeographical region. Figure S2. Locations of the sampled populations in New Caledonia, Australasian zoogeographical region. Figure S3. Locations of the sampled population in the Chatham Is., Australasian zoogeographical region. Figure S4. Locations of the sampled populations in New Zealand, Australasian zoogeographical region. Figure S5. Locations of the sampled populations in the Neotropical zoogeographical region. (PDF 1271 kb

Additional file 1: of Can the intake of antiparasitic secondary metabolites explain the low prevalence of hemoparasites among wild Psittaciformes?

Table S1. Hemoparasites in wild Psittaciformes. Malaria parasites (Plasmodium), related intracellular haemosporidians (Haemoproteus and Leucocytozoon), the unicellular parasitic flagellate protozoans (Trypanosoma), and microfilaria reported in wild populations of Psittaciformes. The probability of detection for adults is based on a simulation (see Additional file 4) of the probability that the parasites will actually be detected given the sample size and an expected true prevalence based on the prevalences observed in wild Psittaciformes. The habitat and climate classification follow the references in Table 1. (XLSX 34 kb