Algunos aspectos no cartesianos del racionalismo de Spinoza y de Leibniz. Sobre la naturaleza de los cuerpos

Tras recordar en la primera parte algunos vestigios de la herencia neoplatónica y cabalística de la tradición animista en las obras de Spinoza y del joven Leibniz, analizo en la segunda parte aspectos del conatus, que son comunes a ambos filósofos: mens idea corporis y mens harmonia conatuum respectivamente. Por fin, en una tercera parte, describo la «transvaloración » que del conatus de Hobbes y de Spinoza, así como de las regulae motus de Huygens, trató de hacer Leibniz: por una parte, de la resistencia o inertia naturalis de los cuerpos a la vis insita rebus: i. e. potentia absoluta corporum; y por otra parte, de la relatividad del movimiento de los experimentos mecánicos a la universalidad del principio de la equipolencia de la causa plena y del efecto entero, cuyo último fundamento es el principio de individuación: la universalidad de todas las leyes mecánicas y la singularidad de cada suceso del universo.Després de recordar en la primera part alguns vestigis de l'herència neoplatònica i cabalística de la tradició animista en les obres de Spinoza i del jove Leibniz, analitzo en la segona part aspectes del conatus, que són comuns a ambdós filòsofs: mens idea corporis i mens harmonia conatuum respectivament. Finalment, en una tercera part, descric la «transvaloració» que del conatus de Hobbes i de Spinoza, així com de les regulae motus de Huygens, va tractar de fer Leibniz: d'una banda, de la resistència o inertia naturalis dels cossos a la vis insita rebus: i. e. potentia absoluta corporum; i de l'altra, de la relativitat del moviment dels experiments mecànics a la universalitat del principi de l'equipol·lència de la causa plena i de l'efecte enter, l'últim fonament del qual és el principi d'individuació: la universalitat de totes les lleis mecàniques i la singularitat de cada succés de l'univers.Remembering in the first part some vestiges of the neoplatonic and kabbalistic heritage of the animistic tradition in the work of Spinoza and the young Leibniz, I analyze in the second part the common aspects of the conatus in both philosophers: mens idea corporis and mens harmonia conatuum, respectively. Finally, in the third part, I describe the "transvaloration" of Hobbes's and Spinoza's conatus and Huygens's regulae motus Leibniz intended to add: from the "resistance or inertia naturalis" of bodies to the vis insita rebus (i.e., potentia absoluta corporum) and from the "relativity" of motion of mechanical experiments to the "universality" of the principle of equipolentia causae plenae et effectus integri grounded in the principle of individuation, that is, the universality of all mechanical rules and the singularity of every fact of the universe

Application of Computer-Aided Drug Repurposing in the Search of New Cruzipain Inhibitors: Discovery of Amiodarone and Bromocriptine Inhibitory Effects

Cruzipain (Cz) is the major cystein protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. From a 163 compound data set, a 2D-classifier capable of identifying Cz inhibitors was obtained and applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. Fifty-four approved drugs were selected as candidates, four of which were acquired and tested on Cz and <i>T. cruzi</i> epimastigotes. Among them, the antiparkinsonian and antidiabetic drug bromocriptine and the antiarrhythmic amiodarone showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite

Effect of polyamines on <i>T</i>. <i>cruzi</i> autophagy.

<p>Y strain <i>T</i>. <i>cruzi</i> epimastigotes were incubated under control conditions in the absence or the presence of 100 μM of spermidine (Spd) or 100 μM of spermine (Spm); or under starvation conditions in either the absence or the presence of 1 mM of DFMO for 2 h and then processed for autophagosome detection through <i>Tc</i>Atg8.1 by IIF. <b>A:</b> Confocal images depict autophagosomes labeled in red at the indicated conditions. Scale bar: 10 μm. <b>B:</b> Percentage of parasites with more than two Atg8.1 positive vesicles. Number of counted cells: 100. Data shown represent the mean +/- SE from 3 independent experiments. ***p < 0.001 (Tukey’s test)<b>. C:</b> Expression of <i>Tc</i>Atg8.1 by western blot. Protein extracts were obtained from parasites maintained under the indicated conditions and used to detect <i>Tc</i>Atg8.1 by western blot (see details in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006049#sec002" target="_blank">Methods</a>). The expression of <i>Tc</i>Atg8.1 was normalized to tubulin and expressed as AU. Data shown represent the mean +/- SE from 3 independent experiments ** p < 0.05 (Tukey’s test). <b>D:</b> Mutant Y-GFP-ODC epimastigotes were incubated in SDM or TAU media in either the absence or the presence of 1 mM of DFMO for 2 h and processed as above. Confocal images depicting the level of autophagosomes labeled with the <i>Tc</i>Atg8.1 protein (red) at the indicated conditions. <b>E:</b> Percentage of parasites with more than two Atg8.1 vesicles. Number of counted cells: 100. Data shown represent the mean +/- SE from 3 independent experiments. * p < 0.01, ***p < 0.001 (Tukey’s test).</p

Effect of autophagy modulators on <i>T</i>. <i>cruzi</i> autophagy.

<p>Y strain <i>T</i>. <i>cruzi</i> epimastigotes were incubated in control medium in the absence or the presence of 50 ng/μl rapamycin (Rap) at 28°C or in TAU medium in the absence or the presence of 100 nM wortmannin (Wort) at 37°C for 2 h. After these treatments, parasites were processed to detect autophagosomes through the presence of <i>Tc</i>Atg8.1 <b>(A)</b>, MDC staining showing the presence of acidic autophagosomes <b>(B),</b> DQ-BSA staining depicting degradative compartments by confocal microscopy <b>(C)</b> (see details in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006049#sec002" target="_blank">Methods</a>). The percentage of parasites labeled with these markers under each condition was quantified. Number of counted cells: 100. Data shown represent the mean +/- SE from 3 independent experiments. * p < 0.05, ** p < 0.01, ***p < 0.001 (Tukey’s test).</p

Effect of bafilomycin on parasites after the first period of metacyclogenesis.

<p>Y strain <i>T</i>. <i>cruzi</i> epimastigotes were incubated under either control (Control) or starvation (TAU) conditions in the absence or the presence of 100 nM of bafilomycin (Baf) for 2 h and then processed to observe autophagosomes for IIF using the <i>Tc</i>Atg8.1 antibody. <b>A:</b> Confocal images depict autophagosomes labeled in red. Scale bar: 10 μm. <b>B:</b> Percentage of parasites with more than two Atg8.1 positive vesicles under each condition. Number of counted cells: 100. Data shown represent the mean +/- SE from 3 independent experiments ** p < 0.05, ***p < 0.001 (Tukey’s test).</p

Autophagy induction promotes metacyclogenesis in <i>T</i>. <i>cruzi</i>.

<p>Y-GFP strain <i>T</i>. <i>cruzi</i> epimastigotes were subjected to the first (F) or the total (T) period of metacyclogenesis in control medium in either the absence or the presence of 50 ng/μl of rapamycin (Rap) or in TAU medium in either the absence or the presence of 100 nM of wortmannin at 37°C for 2 h. <b>A:</b> Trypomastigotes generated under each condition. Number of counted cells: 15 x 10⁶ cells. Data shown represent the mean +/- SE from 3 independent experiments. ** p < 0.01, ***p < 0.001 (Tukey’s test). <b>B:</b> Equivalent samples were placed over Vero cell monolayers for 24 h to allow infection and then fixed and processed as described in Methods. Confocal images show cells <b>i</b>nfected with MT generated under different conditions (actin was visualized in red by phalloidin-rhodamine staining, whereas <i>T</i>. <i>cruzi</i> amastigotes were observed in green). Scale bar: 10 μm <b>C:</b> Percentage of infected cells at the indicated conditions. Number of counted cells: 100. Data shown represent the mean +/- SE from 3 independent experiments. * p < 0.05, ** p < 0.01, ***p < 0.001 (Tukey’s test).</p

Flavins stimulate while analogs retard progression through <i>T</i>. <i>cruzi</i> life cycle.

<p>(A) <i>In vitro</i> metacyclogenesis assay scheme (for more details, see ‘<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005513#sec005" target="_blank">Material and methods</a>‘ section). Percentage of MT obtained in differentiation media (TAU3AAG) supplemented with (B) flavins (riboflavin: RF, FMN or FAD), or (C) chemical analogs (roseoflavin: RoF, lumiflavin: LF, or lumichrome: LC), at the indicated concentrations at 48 h. (D) <i>In vitro</i> infection assay scheme (for more details, see ‘<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005513#sec005" target="_blank">Material and methods</a>‘ section). Effect of 10 μM analogs on (E) cellular invasion at 48 h, expressed as percentage of H9C2 host cells infected with <i>T</i>. <i>cruzi</i> Y-GFP (∼400 cells counted) or (F) amastigote proliferation at 48 h, expressed as number of <i>T</i>. <i>cruzi</i> Y-GFP amastigotes per infected H9C2 host cell. Values are expressed as mean ± SD. Statistical analysis was performed by a Kruskal-Wallis non-parametric test followed by a post-hoc Dunn's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.005).</p

Characterization of acidic and degradative compartments during the first period of metacyclogenesis.

<p>Y strain <i>T</i>. <i>cruzi</i> epimastigotes were incubated under control (BHT medium at 28°C) or starvation (TAU medium at 37°C) conditions for 2 h and then processed to detect acidic or degradative compartments by microscopy. <b>A:</b> Parasites were stained with MDC and then analyzed by confocal microscopy <i>in vivo</i>. Acidic compartments were visualized in blue. Scale bar: 10 μm. <b>B:</b> Percentage of MDC fluorescent parasites incubated under each condition. Number of counted cells: 100. Data shown represent the mean +/- SE from 5 independent experiments. ***p < 0.001 (Tukey’s test). <b>C:</b> Parasites were stained with DQ-BSA and then analyzed by confocal microscopy <i>in vivo</i>. Degradative vesicles were visualized in red. Scale bar: 10 μm. <b>D:</b> Percentage of DQ-BSA positive parasites incubated under each condition. Number of counted cells: 100. Data shown represent the mean +/- SE from 3 independent experiments. *p < 0.05 (Tukey’s test). <b>E:</b> After the first period of metacyclogenesis, parasites were fixed and processed to detect Atg8.1 and DQ-BSA positive compartments as described in Methods. Confocal images depict the partial colocalization of autophagosomes (in green) and lysosomes (in red). Scale bar: 5 μm.</p

<i>Tc</i>RibJ and <i>Tb</i>RibJ display flavin transport activity in <i>E</i>. <i>coli</i>.

<p>The <i>E</i>. <i>coli</i> ∆<i>ribB</i> strain was transformed with expression plasmids coding for either RibM, <i>Tc</i>RibJ, <i>Tb</i>RibJ, <i>Lmi</i>RibJ or <i>Tc</i>PAT12, or with an empty vector. Strains were (A) plated on LB agar (left: strain plating scheme) with the addition of 0, 5 or 750 μM riboflavin (RF) or (B) cultured at 37°C for 24 h in liquid LB supplemented with 0.02–100 μM FMN or FAD, or 0.02–20 μM riboflavin. (C) [³H]-riboflavin uptake (2 μM) was measured from 0 to 180 min in bacteria expressing RibM, <i>Tc</i>RibJ or <i>Tb</i>RibJ or containing an empty vector. (D) Displacement assays performed with 0.3 μM [³H]-riboflavin in the absence of competitors (Ctrl: control) or in the presence of 3–30 μM of unlabelled riboflavin; aliquots were sampled at 0 and 120 min after the addition of the radioactive material. Values are expressed as mean ± SD. Statistical analysis was performed by one way ANOVA test followed by a post-hoc Tukey's multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.005).</p

<i>Tc</i>RibJ functions as flavin transport <i>in vivo</i> in <i>T</i>. <i>cruzi</i>.

<p>Epimastigotes transfected with pRIBOTEX-GFP (wt) or pRIBOTEX-<i>Tc</i>RibJ (<i>Tc</i>RibJ) were incubated in fresh SDM-20-10% FBS in the presence of (A) 20 nM riboflavin (RF), FMN or FAD, or (B) roseoflavin (RoF), lumiflavin (LF), or lumichrome (LC), at the indicated concentrations. Parasites were counted daily using a hemocytometer chamber. <i>T</i>. <i>cruzi</i> proliferation (%) was calculated at the third culture round, where the control condition (20 nM flavin) was referenced as 100%. (C) [³H]-riboflavin (100 nM) uptake measurements in control and over-expressing <i>Tc</i>RibJ epimastigotes. Values are expressed as mean ± SD. (A-B) Statistical analysis was performed by a two-tailed unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.005).</p