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- Hierridin B Isolated from a Marine Cyanobacterium Alters VDAC1, Mitochondrial Activity, and Cell Cycle Genes on HT-29 Colon Adenocarcinoma CellsPublication . Freitas, Sara; Martins, Rosario; Costa, Margarida; Leão, Pedro; Vitorino, Rui; Vasconcelos, Vitor; Urbatzka, RalphHierridin B was isolated from a marine cyanobacterium Cyanobium sp. strain and induced cytotoxicity selectively in HT-29 adenocarcinoma cells. The underlying molecular mechanism was not yet elucidated.
- Structure of Hierridin C, Synthesis of Hierridins B and C, and Evidence for Prevalent Alkylresorcinol Biosynthesis in PicocyanobacteriaPublication . Costa, Margarida; Sampaio-Dias, Ivo E.; Castelo-Branco, Raquel; Scharfenstein, Hugo; Rezende de Castro, Roberta; Silva, Artur; Schneider, Maria Paula C.; Araújo, Maria João; Martins, Rosario; Domingues, Valentina F.; Nogueira, Fátima; Camões, Vera; Vasconcelos, Vitor M.; Leão, Pedro N.Small, single-celled planktonic cyanobacteria are ubiquitous in the world's oceans yet tend not to be perceived as secondary metabolite-rich organisms. Here we report the isolation and structure elucidation of hierridin C, a minor metabolite obtained from the cultured picocyanobacterium Cyanobium sp. LEGE 06113. We describe a simple, straightforward synthetic route to the scarcely produced hierridins that relies on a key regioselective halogenation step. In addition, we show that these compounds originate from a type III PKS pathway and that similar biosynthetic gene clusters are found in a variety of bacterial genomes, most notably those of the globally distributed picocyanobacteria genera Prochlorococcus, Cyanobium and Synechococcus.
- Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collectionPublication . Ramos, Vitor; Morais, João; Castelo-Branco, Raquel; Pinheiro, Ângela; Martins, Joana; Regueiras, Ana; Pereira, Ana L.; Lopes, Viviana R.; Frazão, Bárbara; Gomes, Dina; Moreira, Cristiana; Costa, Maria Sofia; Brûle, Sébastien; Faustino, Silvia; Martins, Rosario; Saker, Martin; Osswald, Joana; Leão, Pedro N.; Vasconcelos, Vitor M.Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.
- Wild mushrooms for Alzheimer’s diseasePublication . Silva, Margarida Lobo; Reis, Filipa S.; Grosso, Clara; Pérez, Diana Valverde; Barros, Lillian; Martins, RosárioAlzheimer’s disease (AD) is among the most incident, debilitating, and widespread forms of dementia. AD causes the decline of cognitive function, making overall day-to-day tasks difficult and even impossible. Despite extensive research, its origin and progression are still unclear, and no efficient treatment is available to prevent disease progression or mitigate symptoms. Due to the low efficiency in the available treatments, natural compounds have arisen as an alternative. These are gaining crescent relevance and have been explored, given their promising results as immunomodulatory, anti-inflammatory, or neuroprotective factors. Mountain natural resources, specifically mushrooms, are among the most studied sources of bioactive molecules. The Montesinho Natural Park, located in northeast Portugal, has incredible mushroom biodiversity, which has been exploited to obtain health-beneficial compounds, namely with anti-inflammatory potential. This work aimed to study different mushroom extracts and isolated compounds for the prevention and progression of AD. So far, our work aimed at bringing insight into the effect of extract administration in neuroblastoma and endothelial cell lines through the MTT viability assay, as well as studying their effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), enzymes known to be involved in AD pathogenesis through a modified Ellman’s method: dry extracts were resuspended in tris-HCl 50mM + 10% DMSO to concentrations between 2 and 0.03125 mg/mL for enzymatic inhibition assays. To be considered promising, extracts must not cause cellular death of over 15-20% and cause enzymatic inhibition. So far, our results using nine mushroom extracts show low-to-no cytotoxicity in concentrations between 100 and 6.25 µg/mL in endothelial and neuroblastoma cell lines. High concentrations of Lycoperdon umbrinum extract caused cytotoxicity levels of approximately 35% in endothelial cells after 24h treatment; different extracts caused cell proliferation in both cell lines, the most evident being 128% in the lowest concentrations of Russula delica and Boletus fragrans. The most promising results were obtained for Boletus aereus, which, while not cytotoxic, inhibited both enzymes by 60%. Around 30% AChE inhibition was achieved for the two highest concentrations of Agaricus silvicola and Boletus fragrans making both extracts good candidates for further studies.
- A natural oxadiazine isolated from cyanobacteria kills cancer cells in multicellular culture systems by impairing cellular respirationPublication . Sousa, Maria Lígia; Preto, Marco; Martins, Rosário; Vasconcelos, Vítor; Linder, Stig; Urbatzka, RalphCyanobacteria are versatile microorganisms that ubiquitously inhabit terrestrial and aquatic ecosystems. They adapt to external threats by mainly producing secondary metabolites. Therefore, cyanobacteria have been recognized as producer of natural products with potential biotechnological applications, such as bioplastics, antifouling, antibiotics, antiprotozoal or anticancer treatment.
- Using early life stages of marine animals to screen the toxicity of priority hazardous and noxious substancesPublication . Cunha, Isabel; Torres, Tiago; Oliveira, Helena; Martins, Rosário; McGowan, Thomas; Sheahan, David; Santos, Miguel MachadoThis study provides toxicity values for early life stages (ELS) of two phylogenetically distinct marine animal taxa, the sea urchin (Paracentrotus lividus), a deuterostome invertebrate, and the turbot (Scophthalmus maximus), a vertebrate (teleost), when challenged by six hazardous and noxious substances (HNS): aniline, butyl acrylate, m-cresol, cyclohexylbenzene, hexane and trichloroethylene. The aim of the study was to provide preliminary information on toxic effects of representative and relevant priority HNS to assess the risk posed by spills to marine habitats and therefore improve preparedness and the response at the operational level. Selection criteria to include each compound in the study were (1) inclusion in the HASREP (2005) list; (2) presence on the priority list established by Neuparth et al. (2011); (3) paucity of toxicological data (TOXnet and ECOTOX) for marine organisms; (4) behaviour in the water according to the categories defined by the European Behaviour classification system (GESAMP 2002), by selecting compounds with different behaviours in water; and (5) physicochemical and toxicological properties, where available, in order to anticipate the most toxic compounds. Aniline and m-cresol were the most toxic compounds with no observed apical effect concentration (NOAEC) values for sea urchin ranging between 0.01 and 0.1 mg/L, followed by butyl acrylate and cyclohexylbenzene with NOAECs ranging between 0.1 and 1.0 mg/L and trichloroethylene with NOAEC values that were in the range between 1 and 10 mg/L, reflecting their behaviour in water, mostly vapour pressure, but also solubility and log Kow. Hexane was toxic only for turbot embryos, due to its neurotoxic effects, and not for sea urchin larvae, at concentrations in the range between 1 and 10 mg/L. The concentrations tested were of the same order of magnitude for both species, and it was observed that sea urchin embryos (length of the longest arm) are more sensitive than turbot eggs larvae (hatching and cumulative mortality rates) to the HNS tested (except hexane). For this specific compound, concentrations up to 70 mg/L were tested in sea urchin larvae and no effects were observed on the length of the larvae. Both tests were found to be complementary depending on behaviour in water and toxicity target of the compounds analysed.
- Zebrafish and Nuclear Medicine methods and techniques: an each-day improving partnershipPublication . Lemos, Joana; Metello, Luís F.; Caires, Hugo; Costa, Pedro; Martins, Rosario; Bravo, Isabel; Carvalho, António Paulo; Vasconcelos, VítorAims: This work aims the introduction of zebrafish as a very promising and each day more interesting animal model in radiobiology. It will be briefly discussed the most relevant advantages and disadvantages related with the use of zebrafish, mentioning some of the previous studies using this model in Nuclear Medicine and concluding with its potential applications.
- Picocyanobacteria from a clade of marine cyanobium revealed bioactive potential against microalgae, bacteria, and marinePublication . Costa, Maria Sofia; Costa, Margarida; Ramos, Vitor; Leão, Pedro N.; Barreiro, Aldo; Vasconcelos, Vítor; Martins, RosarioThe production of bioactive compounds either toxic or with pharmacological applications by cyanobacteria is well established. However, picoplanktonic forms within this group of organisms have rarely been studied in this context. In this study, the toxicological potential of picocyanobacteria from a clade of marine Cyanobium strains isolated from the Portuguese coast was examined using different biological models. First, strains were identified by applying morphological and molecular approaches and cultured under lab conditions. A crude extract and three fractions reflecting a preliminary segregation of lipophilic metabolites were tested for toxicity with the marine microalga Nannochloropsis sp., the bacteria Pseudomonas sp., the brine shrimp Artemia salina, and fertilized eggs of the sea urchin Paracentrotus lividus. No significant apparent adverse effects were noted against Artemia salina. However, significant adverse effects were found in all other assays, with an inhibition of Nannochloropsis sp. and Pseudomonas sp. growth and marked reduction in Paracentrotus lividus larvae length. The results obtained indicated that Cyanobium genus may serve as a potential source of interesting bioactive compounds and emphasize the importance of also studying smaller picoplanktonic fractions of marine cyanobacteria.
- Neofiscalin A and fiscalin C are potential novel indole alkaloid alternatives for the treatment of multidrug-resistant Gram-positive bacterial infectionsPublication . Bessa, Lucinda J.; Buttachon, Suradet; Dethoup, Tida; Martins, Rosario; Vasconcelos, Vitor; Kijjoa, Anake; Martins da Costa, PauloTen indole alkaloids were obtained from the marine sponge-associated fungus Neosartorya siamensis KUFA 0017. We studied the antimicrobial properties of these and of three other compounds previously isolated from the soil fungus N. siamensis KUFC 6349. Only neofiscalin A showed antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE); with a minimum inhibitory concentration (MIC) of 8 μg mL(-1) against both strains. Another compound, fiscalin C, presented synergistic activity against MRSA when combined with oxacillin, although alone showed no antibacterial effect. Moreover, neofiscalin A, when present at sub-MICs, hampered the ability of both MRSA and VRE strains to form a biofilm. Additionally, the biofilm inhibitory concentration values of neofiscalin A against the MRSA and VRE isolates were 96 and 80 μg mL(-1), respectively. At a concentration of 200 μg mL(-1), neofiscalin A was able to reduce the metabolic activity of the biofilms by ∼50%. One important fact is that our results also showed that neofiscalin A had no cytotoxicity against a human brain capillary endothelial cell line.
- Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic studyPublication . Brito, Ângela; Ramos, Vitor; Seabra, Rui; Santos, Arlete; Santos, Catarina; Lopo, Miguel; Ferreira, Sérgio; Martins, António; Mota, Rita; Frazão, Bárbara; Martins, Rosario; Vasconcelos, Vítor; Tamagnini, PaulaCyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N2-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.