The dual pathway in action: decoupling parallel routes for CO2 production during the oscillatory electro-oxidation of methanol

As in the case of most small organic molecules, the electro-oxidation of methanol to CO2 is believed to proceed through a so-called dual pathway mechanism. The direct pathway proceeds via reactive intermediates such as formaldehyde or formic acid, whereas the indirect pathway occurs in parallel, and proceeds via the formation of adsorbed carbon monoxide (COad). Despite the extensive literature on the electro-oxidation of methanol, no study to date distinguished the production of CO2 from direct and indirect pathways. Working under, far-from-equilibrium, oscillatory conditions, we were able to decouple, for the first time, the direct and indirect pathways that lead to CO2 during the oscillatory electro-oxidation of methanol on platinum. The CO2 production was followed by differential electrochemical mass spectrometry and the individual contributions of parallel pathways were identified by a combination of experiments and numerical simulations. We believe that our report opens some perspectives, particularly as a methodology to be used to identify the role played by surface modifiers in the relative weight of both pathways-a key issue to the effective development of catalysts for low temperature fuel cells.

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract Introduction Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway. Methods During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay. Results Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α. Conclusion We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.