CIIMAR@E2S
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Os laboratórios CIIMAR@E2S resultam da integração do P.PORTO como membro associado do Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR). Esta integração tem vindo a ser progressivamente consolidada na E2S através do envolvimento de longa data de investigadores integrados no CIIMAR. A criação dos laboratórios CIIMAR@E2S reforçou a colaboração entre as instituições e potenciou o aumento da capacidade de investigação nas áreas da biotecnologia azul, saúde, ambiente e sustentabilidade. A investigação desenvolvida centra-se principalmente no desenvolvimento de soluções funcionais, inovadoras, seguras e sustentáveis, com aplicações nos domínios da saúde e do ambiente. Na área da biotecnologia aplicada à saúde, a investigação incide na descoberta de compostos naturais com potencial de intervenção em doenças neurodegenerativas e no cancro, bem como no desenvolvimento de alternativas naturais para dermocosméticos e nutracêuticos. No domínio da sustentabilidade ambiental, a investigação está centrada no desenvolvimento de pigmentos derivados de microrganismos para aplicações industriais e na valorização de resíduos têxteis e agroalimentares.
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- Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebratesPublication . Martins, Rosario; Fernandez, Nuria; Beiras, Ricardo; Vasconcelos, VítorAmong the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.
- Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcusPublication . Martins, Rosario; Ramos, Miguel; Herfindal, Lars; Sousa, José; Skærven, Kaja; Vasconcelos, VítorAqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.
- Are known cyanotoxins involved in the toxicity of picoplanktonic and filamentous north atlantic marine cyanobacteria?Publication . Frazão, Bárbara; Martins, Rosario; Vasconcelos, VítorEight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.
- Primary screening of the bioactivity of brackishwater cyanobacteria: toxicity of crude extracts to artemia salina larvae and paracentrotus lividus embryosPublication . Lopes, Viviana; Fernandez, Nuria; Martins, Rosario; Vasconcelos, VítorCyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, plants, microalgae, fungi, bacteria, viruses and cell lines. The aim of this study was to assess the toxic effects of aqueous, methanolic and hexane crude extracts of benthic and picoplanktonic cyanobacteria isolated from estuarine environments, towards the nauplii of the brine shrimp Artemia salina and embryos of the sea urchin Paracentrotus lividus. The A. salina lethality test was used as a frontline screen and then complemented by the more specific sea urchin embryo-larval assay. Eighteen cyanobacterial isolates, belonging to the genera Cyanobium, Leptolyngbya, Microcoleus, Phormidium, Nodularia, Nostoc and Synechocystis, were tested. Aqueous extracts of cyanobacteria strains showed potent toxicity against A. salina, whereas in P. lividus, methanolic and aqueous extracts showed embryo toxicity, with clear effects on development during early stages. The results suggest that the brackishwater cyanobacteria are producers of bioactive compounds with toxicological effects that may interfere with the dynamics of invertebrate populations.
- Morphological, toxicological and molecular characterization of a benthic Nodularia isolated from Atlantic estuarine environmentsPublication . Lopes, Viviana R.; Antunes, Agostinho; Welker, Martin; Martins, Rosário; Vasconcelos, Vítor M.A polyphasic study of a benthic Nodularia isolate (LEGE06071) from an Atlantic environment, specifically salt pans, was performed. LEGE06071 resembled both type strains of Nodularia sphaerocarpa and Nodularia harveyana, while ACOI00729 (purchased isolate) was identified as N. sphaerocarpa. The length and width of vegetative cells varied from 3.10 to 3.15 mu m and from 3.71 to 4.25 mu m, respectively, while heterocyts were 3.91-4.89 mu m long and 4.20-4.74 mu m, wide. None of the isolates had aerotopes. The sequencing of the 16S rRNA gene from the two Nodularia isolates indicated that they belonged neither to Nodularia spumigena nor N. harveyana. Nodularin and other cyanotoxin synthesis-associated genes could not be detected, nor could nodularin production be detected by ELISA. However, MALDI-TOF analysis of LEGE06071 revealed the presence of other compounds, namely, glycolipids. Hence, toxicological screenings against Artemia nauplii, Escherichia coli and Salmonella typhimurium were performed. Toxic effects could only be observed against Artemia, with 48 h-LC50 values for the aqueous and crude extract of methanol of 53.21 mg ml(-1) and 17.81 mg ml(-1), respectively. This study presents the first evidence of a non-nodularin-producing Nodularia isolate in Atlantic salt pan ecosystems and its potential ecotoxicity against Artemia sp. (C) 2009 Elsevier Masson SAS. All rights reserved.
- Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extractsPublication . Lopes, Viviana R.; Schmidtke, Michaela; Fernandes, Maria Helena; Martins, Rosario; Vasconcelos, VítorThe cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL−1) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48–72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.
- Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosisPublication . Costa, Margarida; Costa-Rodrigues, Joao; Fernandes, Maria Helena; Barros, Piedade; Vasconcelos, Vítor; Martins, RosarioMarine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed.
- Cytotoxicity of marine cyanobacteria extracts on osteosarcoma cellsPublication . Costa-Rodrigues, João; Costa, M. M.; Costa, S.; Garcia, M.; Fernandes, M. H.; Vasconcelos, V.; Barros, Piedade; Martins, RosárioMarine cyanobacteria have been identified as a rich source of secondary metabolites with potential pharmacological applications. Anti-inflammatory, antibacterial and anticancer activities are some examples of properties described for cyanobacteria compounds, being the cytoxicity against cancer cell lines one of the most documented. The Laboratory of Ecotoxicology Genomics and Evolution (LEGE) — CIIMAR, Porto, Portugal, possesses a large collection of cyanobacteria strains isolated from the Portuguese coast. In order to investigate the interest of genera such as Cyanobium, Synechocystis, Synechococcus, Leptolyngbya and Pseudoanabaena which are genera that have been largely overlooked in terms of bioactivity, we have been screening their ability to induce cytotoxicity on human cancer cell lines. Assays have been conducted with a crude extract obtained by a dichloromethane and methanol extraction of freeze dried biomass and three fractions obtained using Si column chromatography with a gradient from 100% hexane, to 100% ethyl acetate to 100% methanol. The cytotoxicity of cyanobacteria crude extract and fractions has been evaluated by the MTT assay at 24, 48 and 72 h. Here we present the results concerning the cytotoxicity of 24 cyanobacteria strains on the osteosarcoma cell line MG63. The results show both inhibitory and stimulatory effects on cell growth within the same cyanobacteria strain. However, five cyanobacteria strains were found to induce a decrease in cell viability that reached the 80% within the ethyl acetate fraction, which makes this fraction interesting for the isolation and characterization of new bioactive compounds.
- Citotoxicity of marine cyanobacteria from the portuguese coast agains breast cancer cellsPublication . Costa, Margarida; Leão, Pedro; Ribeiro, Maria João; Costa-Rodrigues, João; Garcia, Mónica; Barros, Piedade; Fernandes, Helena; Vasconcelos, Vítor; Martins, RosárioThe marine environment possesses an unmeasured chemical and biological diversity, being considered an extraordinary resource for the discovery of new therapeutic drugs. Between others, marine cyanobacteria have emerged as a prominent source of bioactive secondary metabolites with a promising anticancer potential, as the described and intensively studied dolastatins.
- Embryotoxicity assessment of a Microcystis extract with Daphnia in vitro egg testPublication . Barros, Piedade; Martins, RosárioMicrocystis aeruginosa is a colonial unicellular cyanobacteria, usually found in eutrophic and hipereutrophic waters. Microcystins are the main toxins produced. As chronic toxicity test for daphnid is time consuming, the developmental stages of Daphnia eggs in vitro has been used in several works as an alternative to the 21 days reproduction test.