Examinando por Autor "Collao, B."
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Ítem Biological phosphorylated molecules participate in the biomimetic and biological synthesis of cadmium sulphide quantum dots by promoting H2S release from cellular thiols(Royal Society of Chemistry, 2017-08) Venegas, F.A.; Saona, L.A.; Monrás, J.P.; Órdenes-Aenishanslins, N.; Giordana, M.F.; Ulloa, G.; Collao, B.; Bravo, D.; Pérez-Donoso, J.M.Developing methods with a low environmental impact for nanoparticle synthesis remains one of the greatest challenges in nanotechnology. In this context, biomimetic and biological methods have emerged as green chemistry alternatives, and also contribute to our understanding of how nanomaterials interact with cellular molecules. Here, we report a phosphate-dependent biomimetic method to synthesize of cadmium sulphide (CdS) QDs at low temperatures, physiological pH and aerobic conditions, using CdCl2 and thiols (l-cysteine, glutathione or mercaptosuccinic acid). Inorganic phosphate (Pi) and cellular phosphorylated intermediates such as adenosine monophosphate, glucose-6-phosphate, glycerol-2-phosphate and fructose-1,6-biphosphate, can trigger CdS QDs synthesis. The produced QDs are cubic phase nanocrystals with a tunable fluorescence (450-700 nm), small size (4-12 nm), and are composed of thiols and Pi. In CdS synthesis, the importance of the phosphate is related to its capacity to release H2S from thiols, a phenomenon associated with its base-properties. Based on the biomimetic method, we developed a Pi-based procedure to synthesize CdS QDs in Escherichia coli. As in the biomimetic procedure, Pi favors QDs-biosynthesis not only by mediating biological generation of H2S, but also by improving Cd2+ cellular uptake. A role for phosphates in the cellular interaction and green synthesis of metal QDs is discussed.Ítem Hypochlorous acid and hydrogen peroxide-induced negative regulation of Salmonella enterica serovar Typhimurium ompW by the response regulator ArcA(2012) Morales, E.; Calderán, I.; Collao, B.; Gil, F.; Porwollik, S.; McClelland, M.; Saavedra, C.Hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) are reactive oxygen species that are part of the oxidative burst encountered by Salmonella enterica serovar Typhimurium (S. Typhimurium) upon internalization by phagocytic cells. In order to survive, bacteria must sense these signals and modulate gene expression. Growing evidence indicates that the ArcAB two component system plays a role in the resistance to reactive oxygen species. We investigated the influx of H2O2 and HOCl through OmpW and the role of ArcAB in modulating its expression after exposure to both toxic compounds in S. Typhimurium. Results: H2O2 and HOCl influx was determined both in vitro and in vivo. A S. Typhimurium ompW mutant strain (δompW) exposed to sub-lethal levels of H2O 2 and HOCl showed a decreased influx of both compounds as compared to a wild type strain. Further evidence of H2O2 and HOCl diffusion through OmpW was obtained by using reconstituted proteoliposomes. We hypothesized that ompW expression should be negatively regulated upon exposure to H2O2 and HOCl to better exclude these compounds from the cell. As expected, qRT-PCR showed a negative regulation in a wild type strain treated with sub-lethal concentrations of these compounds. A bioinformatic analysis in search for potential negative regulators predicted the presence of three ArcA binding sites at the ompW promoter region. By electrophoretic mobility shift assay (EMSA) and using transcriptional fusions we demonstrated an interaction between ArcA and one site at the ompW promoter region. Moreover, qRT-PCR showed that the negative regulation observed in the wild type strain was lost in an arcA and in arcB mutant strains. Conclusions: OmpW allows the influx of H2O2 and HOCl and is negatively regulated by ArcA by direct interaction with the ompW promoter region upon exposure to both toxic compounds.Ítem Probing the ArcA regulon under aerobic/ROS conditions in Salmonella enterica serovar Typhimurium(BMC, 2013-09) Morales, E.; Collao, B.; Desai, P.; Calderón, I.; Gil, F.; Luraschi, R.; Porwollik, S.; McClelland, M.; Saavedra, C.Background: Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS), which is part of the oxidative burst encountered upon internalization of Salmonella enterica serovar Typhimurium (S. Typhimurium) by phagocytic cells. It has previously been established that, the ArcAB two-component system plays a critical role in ROS resistance, but the genes regulated by the system remained undetermined to date. We therefore investigated the ArcA regulon in aerobically growing S. Typhimurium before and after exposure to H2O2 by querying gene expression and other physiological changes in wild type and ΔarcA strains.Results: In the ΔarcA strain, expression of 292 genes showed direct or indirect regulation by ArcA in response to H2O2, of which 141were also regulated in aerobiosis, but in the opposite direction. Gene set enrichment analysis (GSEA) of the expression data from WT and ΔarcA strains, revealed that, in response to H2O2 challenge in aerobically grown cells, ArcA down regulated multiple PEP-PTS and ABC transporters, while up regulating genes involved in glutathione and glycerolipid metabolism and nucleotide transport. Further biochemical analysis guided by GSEA results showed that deletion of arcA during aerobic growth lead to increased reactive oxygen species (ROS) production which was concomitant with an increased NADH/NAD+ ratio. In absence of ArcA under aerobic conditions, H2O2 exposure resulted in lower levels of glutathione reductase activity, leading to a decreased GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio.Conclusion: The ArcA regulon was defined in 2 conditions, aerobic growth and the combination of peroxide treatment and aerobic growth in S. Typhimurium. ArcA coordinates a response that involves multiple aspects of the carbon flux through central metabolism, which ultimately modulates the reducing potential of the cell.Ítem Synthesis of salt-stable fluorescent nanoparticles (quantum dots) by polyextremophile halophilic bacteria(Nature Publishing Group, 2019-12) Bruna, N.; Collao, B.; Tello, A.; Caravantes, P.; Díaz-Silva, N.; Monrás, J. P.; ÓrdenesAenishanslins, N.; Flores, M.; Espinoza-Gonzalez, R.; Bravo, D.; Pérez-Donoso, J. M.Here we report the biological synthesis of CdS fluorescent nanoparticles (Quantum Dots, QDs) by polyextremophile halophilic bacteria isolated from Atacama Salt Flat (Chile), Uyuni Salt Flat (Bolivia) and the Dead Sea (Israel). In particular, a Halobacillus sp. DS2, a strain presenting high resistance to NaCl (3–22%), acidic pH (1–4) and cadmium (CdCl 2 MIC: 1,375 mM) was used for QDs biosynthesis studies. Halobacillus sp. synthesize CdS QDs in presence of high NaCl concentrations in a process related with their capacity to generate S 2− in these conditions. Biosynthesized QDs were purified, characterized and their stability at different NaCl concentrations determined. Hexagonal nanoparticles with highly defined structures (hexagonal phase), monodisperse size distribution (2–5 nm) and composed by CdS, NaCl and cysteine were determined by TEM, EDX, HRXPS and FTIR. In addition, QDs biosynthesized by Halobacillus sp. DS2 displayed increased tolerance to NaCl when compared to QDs produced chemically or biosynthesized by non-halophilic bacteria. This is the first report of biological synthesis of salt-stable QDs and confirms the potential of using extremophile microorganisms to produce novel nanoparticles. Obtained results constitute a new alternative to improve QDs properties, and as consequence, to increase their industrial and biomedical applications. © 2019, The Author(s).