Novel Cell Wall Antifungals Reveal a Special Synergistic Activity in pbr1 Mutants Resistant to the Glucan Synthesis Antifungals Papulacandins and Echinocandins.

A series of 4-(arylmethylene)-3-isochromanones have been prepared with base-catalyzed Knoevenagel condensation starting from 3-isochromanone and aromatic aldehydes. The outcome of the reaction- the isomeric composition of the products depends on the aromatic aldehyde applied. These reactions afforded mostly the more stable E-diastereoisomer, but some condensations resulted in the Z-diastereoisomer or mixture of the stereoisomers (1-16). The products showed antifungal effect against some pathogenic fungi. We wanted to extend this study and to synthesize a new generation of 4-(arylmethylene)-3-isochromanones. These condensations led mostly to E-diastereoisomers (17-30). The structure verifications were performed by FT IR, 1H and13C NMR methods. Both the 1-16 and the novel 17-30 compounds have been screened against the three yeast models, fission yeast Schizosaccharomyces pombe (wild-type, and pbr1-6 and pbr1-8 mutants resistant to specific cell wall synthesis inhibitors), budding yeast Saccharomyces cerevisiae (wild-type and pbr1-1) and pathogenic yeast Candida albicans (wild-type, ATCC 26555, 90028 and SC5314). Osmotic protection with sorbitol attenuated the in vivo inhibition in living cells suggesting a cell wall-specific antifungal effect. Moreover, the S. pombe wild-type and mutant strains were tested for their resistant or sensitive in vitro ß(1,3)-glucan synthase (GS) activity. We found both in vivo in living cells and in vitro in the enzymatic GS assay a synergistic effect of higher sensitivity of the pbr1 mutants resistant to the specific GS inhibitors papulacandins and echinocandins. These results may provide new insights into new strategies of combined antifungal therapy of GS inhibitors directed against spontaneous mutants resistant to echinocandins.

First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease.

Real-Time PCR (qPCR) testing is recommended as both a diagnostic and outcome measurement of etiological treatment in clinical practice and clinical trials of Chagas disease (CD), but no external quality assurance (EQA) program provides performance assessment of the assays in use. We implemented an EQA system to evaluate the performance of molecular biology laboratories involved in qPCR based follow-up in clinical trials of CD. An EQA program was devised for three clinical trials of CD: the E1224 (NCT01489228), a pro-drug of ravuconazole; the Sampling Study (NCT01678599), that used benznidazole, both conducted in Bolivia; and the CHAGASAZOL (NCT01162967), that tested posaconazole, conducted in Spain. Four proficiency testing panels containing negative controls and seronegative blood samples spiked with 1, 10 and 100 parasite equivalents (par. eq.)/mL of four Trypanosoma cruzi stocks, were sent from the Core Lab in Argentina to the participating laboratories located in Bolivia and Spain. Panels were analyzed simultaneously, blinded to sample allocation, at 4-month intervals. In addition, 302 random blood samples from both trials carried out in Bolivia were sent to Core Lab for retesting analysis. The analysis of proficiency testing panels gave 100% of accordance (within laboratory agreement) and concordance (between laboratory agreement) for all T. cruzi stocks at 100 par. eq./mL; whereas their values ranged from 71 to 100% and from 62 to 100% at 1 and 10 par. eq./mL, respectively, depending on the T. cruzi stock. The results obtained after twelve months of preparation confirmed the stability of blood samples in guanidine-EDTA buffer. No significant differences were found between qPCR results from Bolivian laboratory and Core Lab for retested clinical samples. This EQA program for qPCR analysis of CD patient samples may significantly contribute to ensuring the quality of laboratory data generated in clinical trials and molecular diagnostics laboratories of CD.

Subtelomeric I-SceI-Mediated Double-Strand Breaks Are Repaired by Homologous Recombination in Trypanosoma cruzi.

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

Uroguanylin regulates net fluid secretion via the NHE2 isoform of the Na/H+ exchanger in an intestinal cellular model.

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na(+)/H(+) exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na(+)/H(+) exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (J(v)) was studied by optic techniques and intracellular pH (pH(i)) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na(+) absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pH(i). In the assayed conditions, cell exposure to apical UGN increases net secretory J(v), without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net J(v) was produced mainly by a reduction in normal Na(+) absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pH(i) is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pH(i) regulation.

Metabolic and functional consequences of cytosolic 5'-nucleotidase-IA overexpression in neonatal rat cardiomyocytes.

Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5'-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5'-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 +/-3 vs. 100 +/-4 beats/min) was inhibited (107 +/-3 vs. 94 +/-3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-(p-sulfophenyl)theophilline (120 +/- 3 vs. 90 +/- 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.