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- Analysis of the Use of Cylindrospermopsin and/or Microcystin-Contaminated Water in the Growth, Mineral Content, and Contamination of Spinacia oleracea and Lactuca sativaPublication . Llana-Ruiz-Cabello, Maria; Jos, Angeles; Cameán, Ana; Oliveira, Flavio; Barreiro, Aldo; Machado, Joana; Azevedo, Joana; Pinto, Edgar; Almeida, Agostinho; Campos, Alexandre; Vasconcelos, Vitor; Freitas, MarisaCyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxins.
- Analysis of the use of microcystin-contaminated water in the growth and nutritional quality of the root-vegetable, Daucus carotaPublication . Machado, J.; Azevedo, J.; Freitas, Marisa; Pinto, Edgar; Almeida, A.; Vasconcelos, V.; Campos, A.Toxic cyanobacterial blooms are often observed in freshwaters and may reflect the increased eutrophication of these environments and alterations in climate. Cyanotoxins, such as microcystins (MCs), are an effective threat to many life forms, ranging from plants to humans. Despite the research conducted to date on cyanotoxins, the risks associated to the use of contaminated water in agriculture require further elucidation. To tackle this aim, a research was conducted with the root-vegetable Daucus carota. The specific aims of this work were the following: (i) to evaluate the effects of MC-LR on the plant growth and photosynthesis; (ii) to evaluate the nutritional quality of carrot roots; and (iii) to measure bioaccumulation. To this purpose, young carrots were grown in soil during 1 month in natural conditions and exposed to Mycrocystis aeruginosa aqueous extracts containing environmentally realistic concentrations of MC-LR (10 and 50 MC-LR μg/L). The results showed that MC-LR may decrease root growth after 28 days of exposure to 50 μg/L and increase photosynthetic efficiency. We also observed changes in mineral and vitamin content in carrots as a result of the exposure to contaminated water. Moreover, MC-LR was detected in carrot roots by ELISA at very low concentration 5.23 ± 0.47 ng MC eq./g FW. The soil retained 52.7 % of the toxin potentially available for plants. This result could be attributed to MC-LR adsorption by soil particles or due to microbial degradation of the toxin. We conclude that the prolonged use of MC-LR-contaminated water may affect crop growth, alter the nutritional value of vegetable products, and potentiate contamination.
- 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.
- Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strainsPublication . Leão, Pedro N.; Costa, Margarida; Ramos, Vitor; Pereira, Alban R.; Fernandes, Virgínia C.; Domingues, Valentina F.; Gerwick, William H.; Vasconcelos, Vítor; Martins, RosárioCyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.
- Application of real-time PCR in the assessment of the toxic cyanobacterium cylindrospermopsis raciborskii abundance and toxicological potentialPublication . Moreira, Cristiana; Martins, António; Azevedo, Joana; Freitas, Marisa; Regueiras, Ana; Vale, Micaela; Antunes, Agostinho; Vasconcelos, VítorCyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.
- 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.
- Bibliometric analysis towards industrial-scale use of Marine Algae and Lichens as soil amendments and plant biofertilizers for sustainable agriculturePublication . Ouala, Oumaima; Essadki, Yasser; Oudra, Brahim; Khalloufi, Fatima El; Martins, Rosário; Martins, RosarioThe nutrient-rich composition of seaweeds and lichens makes them well-suited for agricultural applications. Their use as alternatives to synthetic fertilizers contributes to sustainable agricultural production, enabling farmers to adopt ecological practices while maintaining or increasing crop productivity. This review aims to highlight the status and trends of research, along with a literature analysis on the application of these biomasses in sustainable agriculture. A bibliometric analysis was performed based on two databases (Scopus and Web of Science) to overview the main research topics regarding the use of biomasses studied in agriculture, thus providing useful information for future research. The biochemical composition and agricultural applications of these biomasses have been highlighted. The analysis shows that these biomasses are rich of nutrient compounds, revealing their roles and mechanisms of action on the chemical, nutritional properties, and soil microbial activities and their effect on plant growth, using various extraction and application methods. It also highlighted the potential of seaweeds for protection against biotic and abiotic stresses. In light of all the data presented in this review, it is possible to stimulate farmers’ interest in using seaweeds and lichens as natural fertilizers, with a focus on sustainable and ecological agriculture mainly in developing countries.
- Bioaccessibility and changes on cylindrospermopsin concentration in edible mussels with storage and processing timePublication . Freitas, Marisa; Azevedo, Joana; Carvalho, António Paulo; Mendes, Vera M.; Manadas, Bruno; Campos, Alexandre; Vasconcelos, VitorThe alkaloid cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that bivalves can accumulate high levels of cylindrospermopsin. Based on the potential for human health risks, a provisional tolerable daily intake of 0.03 μg/kg-body weight has been recommended. However, the human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. Thus, this study aimed to assess the changes on cylindrospermopsin concentration in edible mussels with storage and processing time as well as cylindrospermopsin bioaccessibility. Mussels, (Mytilus galloprovincialis) fed cylindrospermopsin-producing C. raciborskii, were subjected to the treatments and then analyzed by LC-MS/MS. Mussels stored frozen allowed a significantly higher recovery of cylindrospermopsin (52.5% in 48 h and 57.7% in one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in the mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin. The in vitro digestion considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility of cylindrospermopsinin in the human health risk assessment.
- Bridging cyanobacteria to neurodegenerative diseases: a new potential source of bioactive compounds against Alzheimer’s diseasePublication . Castaneda, Andrea; Ferraz, Ricardo; Vieira, Mónica; Cardoso, Isabel; Vasconcelos, Vítor; Martins, RosárioNeurodegenerative diseases (NDs) represent a drawback in society given the ageing population. Dementias are the most prevalent NDs, with Alzheimer’s disease (AD) representing around 70% of all cases. The current pharmaceuticals for AD are symptomatic and with no effects on the progression of the disease. Thus, research on molecules with therapeutic relevance has become a major focus for the scientific community. Cyanobacteria are a group of photosynthetic prokaryotes rich in biomolecules with confirmed activity in pathologies such as cancer, and with feasible potential in NDs such as AD. In this review, we aimed to compile the research works focused in the anti-AD potential of cyanobacteria, namely regarding the inhibition of the enzyme β-secretase (BACE1) as a fundamental enzyme in the generation of β-amyloid (Aβ), the inhibition of the enzyme acetylcholinesterase (AChE) lead to an increase in the availability of the neurotransmitter acetylcholine in the synaptic cleft and the antioxidant and anti-inflammatory effects, as phenomena associated with neurodegeneration mechanisms.
- Carotenoids from cyanobacteria modulate iNOS and inhibit the production of inflammatory mediators: Promising agents for the treatment of inflammatory conditionsPublication . Morone, Janaína; Vasconcelos, Vitor; Hentschke, Guilherme; Rosário Martins, Maria; Pinto, Eugénia; Lopes, GracilianaCyanobacteria are green multiproduct refineries of increasing interest for different industrial prospects. In this work, eleven cyanobacteria strains isolated from the Cape Verde archipelago were explored for their biotechnological applications in the field of inflammation. A biorefinery approach was employed to produce carotenoidtargeted extracts, further profiled by HPLC-PDA and explored for their ability to i) scavenge important physiological free radicals of oxygen (superoxide anion radical, O 2 •) and nitrogen (nitric oxide, • NO) involved in the inflammatory process ii) slow-down post-inflammatory hyperpigmentation and iii) modulate the activity of inf lammatory cytokine-producing enzymes, in enzymatic and cell systems comprising RAW 264.7 cells. The studied strains turned out to be important carotenoid producers (70.47–186.71 μ g mg 1 dry extract), mainly represented by β-carotene and zeaxanthin. The targeted-extracts stood-out for their potential to slow-down the inflammatory process through a multitarget approach: scavenging • NOandO 2 •, reducing inflammatory cytokines production through lipoxygenase inhibition, and modulating the inducible nitric oxide synthase in LPSstimulated RAW 264.7 cells, with strains of the order Nodosilineales revealing to be worth of further biotechnological exploitation.