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Applied Molecular Biosciences Unit
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Dry powder formulations containing bioactive compounds from marine Actinobacteria
Publication . Almeida, Vanessa Clemente; Aguiar-Ricardo, Ana
Lower respiratory infections were the leading cause of sickness and mortality in 2013.1 The treatment of such infections relies on antibiotic therapy. However, antibiotic resistance to human pathogens and the prevalence of new cancer types continues to increase, so it is imperative not only to discover new lead-like drugs agents, but also develop new drug delivery systems for pulmonary diseases.2,3 In order to achieve such goal, it was isolated and elucidated antibacterial compounds from a marine-sediment-derived Actinobacteria, collected along Madeira archipelago. These bioactive compounds were obtained from Streptomyces aculeolatus, PTM-029, belonging to the MAR4 group. The structures of these compounds were elucidated by 1D and 2D NMR, HR-MS and other spectroscopic data. The antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium EF82 (VRE) were determined for the most promising compound isolated from PTM-029, with a minimum inhibitory concentration (MIC) of 1,95 μg/mL for both bacterial pathogens. Subsequently, chitosan and cholesterol-based dry powder formulations were manufactured, containing encapsulated POxylated polymer, efficiently synthesized using a supercritical-assisted polymerization in carbon dioxide (CO2), end-capped to a model drug ibuprofen (IBP) and a marine bioactive compound, PTM-029, F4, F39. The dry powder formulations (DPF) were then synthesized through the Supercritical Assisted Spray-Drying (SASD) technique. All the produced DPFs were characterized in detail in relation to their morphology, physical-chemical properties and aerodynamic performance. The resulting particles showed good aerodynamic diameters between the 1 and 7 μm, yields up to 45% and FPF percentages rounding the 71%, as well as the required morphology to make them suitable for pulmonary delivery.
Characterization of extracellular electron transfer components of Geobacter bacteria
Publication . Fernandes, Tomás Monteiro; Morgado, Leonor; Salgueiro, Carlos
Characterization of extracellular electron transfer networks in Geobacter sulfurreducens, a key bacterium for bioremediation and bioenergy applications
Publication . Dantas, Joana Margarida Franco; Salgueiro, Carlos
Geobacter bacteria have awakened significantly attention because of their impact on natural environments and biotechnological applications that include the bioremediation of organic and inorganic contaminants, bioenergy production and bioelectronics. In addition to electron transfer towards extracellular terminal acceptors, Geobacter cells can also accept electrons from electrodes, in currentconsuming biofilms, a process that is currently explored in microbial electrosynthesis. These practical applications rely on an efficient transfer of electrons between the cell and its exterior, a process
designated extracellular electron transfer (EET). However, the precise mechanisms underlying EET processes are still under debate. Genetic and proteomics studies have identified several c-type cytochromes as key components for EET in G. sulfurreducens. These proteins are located at the innermembrane (IM), periplasm and outer-membrane (OM). Examples of such cytochromes include the IMassociated cytochrome MacA, periplasmic cytochromes PpcA-E and PccH, as well as, the OM cytochrome OmcF, which were studied in this Thesis.
Molecular interactions between PpcA-E and their putative redox partners, including a humic substance analogue molecule, MacA or PccH, were probed by NMR spectroscopy, stopped-flow kinetics and molecular docking. For the interacting pairs, their binding affinity was also determined by NMR chemical shift perturbation experiments. The results obtained showed that the interacting molecules establish reversible low-binding affinity complexes in specific regions of the proteins to warrant a rapid and selective electron transfer, a typical feature observed for electron transfer reactions between redox partners.
In addition, NMR spectroscopy was also used to determine the solution structure of OmcF in the reduced state, its pH-dependent conformational changes and backbone dynamics.
A biochemical and structural characterization of the cytochrome PccH was also carried out using circular dichroism, UV-visible and NMR spectroscopic techniques. The structure of PccH determined by X-ray crystallography showed that it is unique among the monoheme c-type cytochromes. The reduction potentials determined for PccH at different pH values by visible redox titrations are unusually low compared to those reported for other monoheme c-type cytochromes. Considering the structural and
functional features of PccH it was proposed that this protein represents a first characterized example of a new subclass of monoheme c-type cytochromes.
Overall, the results obtained constitute an important contribute to the current understanding of the G. sulfurreducens extracellular electron transfer mechanisms.
Characterization and interaction studies of triheme cytochromes from Geobacter: a contribution to the elucidation of extracellular electron transfer pathways
Publication . Ferreira, Marisa Raquel Martins de Brito; Salgueiro, Carlos
Geobacter species are frequently the most abundant Fe(III)-reducing microorganism in soils and sediments. They can also reduce other metals in the same type of environments and, in addition, make electrical connections with electrodes to produce electricity from waste organic matter or to drive anaerobic process with electrical energy. Proteomic and genetic studies have identified several multiheme cytochromes as essential for Fe(III) reduction. From all the cytochromes that were shown to be involved in the reduction of Fe(III), the best characterized to date are five periplasmic triheme cytochromes from Geobacter sulfurreducens, which constitute the so-called PpcA-family. The members of this family are designated PpcA, PpcB, PpcC, PpcD, PpcE. A similar family was found in Geobacter metallireducens (PpcA, PpcB, PpcC, PpcE and PpcF) but none of these proteins was characterized to date. When compared to the other homologs found in G. sulfurreducens, PpcF differs the most and for this reason was targeted in the present work. To characterize this cytochrome, PpcF was firstly expressed and purified. The yield obtained was approximately 1 mg/L of cell culture. The molecular mass of the protein was confirmed by mass spectroscopy (9737.13 Da). The molar extinction coefficient was determined (87.4 mM-1cm-1). The UV-visible spectral characteristics of PpcF are consistent with low-spin heme groups with His-His axial coordination, a feature that was further confirmed by Nuclear Magnetic Resonance spectroscopy. The assignment of the heme substituent signals of PpcF in both reduced and oxidized states together with the analysis of their NOE connectivities showed that the heme core structure is similar to those of the PpcA family cytochromes in G. sulfurreducens. The reduction potentials of PpcF were determined at pH 7 and 8 (-56 mV and – 64 mV versus the standard hydrogen electrode, respectively). Lastly 2D-1H NMR exchange spectroscopy was used to determine the order of oxidation of the heme groups in PpcF: IV-I-III.
In the second part of this thesis it was analyzed the possible molecular interaction between cytochromes PpcA, PpcB and PpcE from G. sulfurreducens and Fe(III) citrate. This molecule can be utilized as terminal electron acceptor by this bacterium and PpcA, PpcB and PpcE were shown to be crucial in this electron transfer pathway. For these purpose isotopic 15N-labeled cytochromes were expressed and purified. NMR spectroscopy enabled us to assign the protein NH backbone and heme methyl proton signals, as well as to probe the interaction regions between each cytochrome and Fe(III) citrate. The chemical shift perturbation studies showed that in all cytochromes the interaction region is located in the vicinity of heme IV.
Tilapia fish microbial spoilage monitored by a single optical gas sensor
Publication . Semeano, Ana T. S.; Maffei, Daniele F.; Palma, Susana; Li, Rosamaria W. C.; Franco, Bernadette D. G. M.; Roque, Ana C. A.; Gruber, Jonas; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier Science B.V., Amsterdam.
As consumption of fish and fish-based foods increases, non-destructive monitoring of fish freshness also becomes more prominent. Fish products are very perishable and prone to microbiological growth, not always easily detected by organoleptic evaluation. The analysis of the headspace of fish specimens through gas sensing is an interesting approach to monitor fish freshness. Here we report a gas sensing method for monitoring Tilapia fish spoilage based on the application of a single gas sensitive gel material coupled to an optical electronic nose. The optical signals of the sensor and the extent of bacterial growth were followed over time, and results indicated good correlation between the two determinations, which suggests the potential application of this simple and low cost system for Tilapia fish freshness monitoring.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UID/Multi/04378/2013