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Projeto de investigação
2013 - Strategic Project
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Publicações
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.
Lanthanide-Based Organic Salts
Publication . Forte, Andreia; Gago, Sandra; Alves, Celso; Silva, Joana; Alves, Joana; Pedrosa, Rui; Laia, César A. T.; Marrucho, Isabel M.; Branco, Luís C.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); LAQV@REQUIMTE; MDPI - Multidisciplinary Digital Publishing Institute
The formulation of magnetic ionic liquids (MILs) or organic salts based on lanthanides as anions has been explored. In this work, a set of choline-family-based salts, and two other, different cation families, were combined with Gadolinium(III) and Terbium(III) anions. Synthetic methodologies were previously optimized, and all organic salts were obtained as solids with melting temperatures higher than 100 °C. The magnetic moments obtained for the Gd(III) salts were, as expected, smaller than those obtained for the Tb(III)-based compounds. The values for Gd(III) and Tb(III) magnetic salts are in the range of 6.55–7.30 MB and 8.22–9.34 MB, respectively. It is important to note a correlation between the magnetic moments obtained for lanthanides, and the structural features of the cation. The cytotoxicity of lanthanide-based salts was also evaluated using 3T3, 293T, Caco2, and HepG2 cells, and it was revealed that most of the prepared compounds are not toxic.
Estudo comparativo dos proteomas extra e intracelular das actinobactérias Streptomyces aculeolatus PTM-29 e PTM-129
Publication . Costa, Lígia Francisca Rodrigues; Almeida, Maria Gabriela; Gaudêncio, Susana
As actinobactérias marinhas, particularmente a linhagem MAR4, um novo grupo de actinobactérias relacionadas ao género Streptomyces, tem se revelado uma fonte rica de isoprenóides híbridos, uma vasta família de metabolitos com um largo espetro de bioatividades. A integração de tecnologias Omics, como genómica, proteómica, transcriptómica e metabolómica, têm desempenhado um papel fundamental no acesso ao potencial de produção destes microrganismos, permitindo o entendimento das vias moleculares envolvidas na biossíntese de metabolitos.
Neste trabalho adotou-se uma abordagem proteómica diferencial, baseada em 2DE, para o estudo comparativo dos proteomas de duas estirpes de actinobactérias marinhas da linhagem MAR4 pertencentes à espécie Streptomyces aculeolatus. Codificadas por PTM-29 e PTM-129 estas estirpes partilham 99% de identidade com base na sequência do gene 16S rRNA. Contudo, as estirpes exibem grandes diferenças ao nível do seu metaboloma e, consequentemente, ao nível do seu perfil de bioatividade, sendo que a PTM-29 está envolvida na síntese de napiradiomicinas e marinonas com atividades antimicrobianas contra MRSA COL (Staphylococcus aureus resistente à meticilina) e VRE EF82 (Enterococcus resistente a vancomicina) enquanto que a PTM-129 não exibiu, até à data, qualquer tipo de bioatividade. Assim, estabeleceu-se a estirpe PTM-129, como a estirpe de referência, com o objetivo de se identificarem biomarcadores proteicos da síntese de metabolitos excretados pela estirpe PTM-29. Adicionalmente, realizou-se um estudo aos exoproteomas das referidas estirpes, utilizando a mesma abordagem, com o objetivo de se identificarem novos metabolitos bioativos, nomeadamente péptidos não ribossomais, frequentemente sintetizados por estes microrganismos. Os proteomas foram analisados num intervalo de pH 4-7, e a recolha das células das referidas estirpes, para os estudos proteómicos, foi efetuada durante a fase estacionária do crescimento, uma vez que esta potencia o metabolismo secundário e, por conseguinte, a expressão das proteínas biossintéticas.
Os proteomas totais das estirpes PTM-29 e PTM-129, apresentaram padrões de expressão semelhantes, com 203 spots comuns. No entanto, por outro lado, foi detetado um grande número de spots exclusivos de cada uma das estirpes, que representam possíveis candidatos a biomarcadores da síntese dos metabolitos encontrados no metaboloma da estirpe PTM-29. No que diz respeito aos exoproteomas, as estirpes em estudo apresentaram perfis bastante distintos, com apenas 25 spots comuns e um grande número de spots exclusivos de cada estirpe. A grande diversidade de espécies proteicas observadas ao nível dos exoproteomas constitui uma boa oportunidade de investigação para o screening de novos metabolitos bioativos. Neste contexto, o potencial antimicrobiano destes extratos foi testado contra duas bactérias multirresistentes a MRSA COL e VRE EF82, e resultados preliminares revelaram bioatividade das estirpes PTM-29 e PTM-129 contra MRSA, com valores de MIC de 62,5 μɡ/mL e 125 μɡ/mL, respetivamente.
Sol-gel entrapped biosystems: enzyme(s) and whole cells
Publication . Vaz, Michael Martins; Barreiros, Susana; Sá-Nogueira, Isabel; Cabrita, Eurico
In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells.
CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor β-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC).
The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to “in-cell” Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6820 - DCRRNI ID
Número da atribuição
PEst-C/EQB/LA0006/2013