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- Anticancer potential of spores of the soil fungus Pisolithus tinctoriusPublication . Oliveira, Rui S.; Alves, Ricardo; Preto, Marco; Martins, RosárioCancer is one of the major causes of death worldwide being the search for new anticancer drugs essential for the treatment of this disease. The sporocarps of the soil fungus Pisolithus tinctorius contain pisosterol, a triterpene that has been shown to have antitumor activity against some cancer cell lines. Nevertheless, no studies have focused on the anticancer potential of other structures such as spores, and so the anticancer potential of P. tinctorius, remains largely unknown. The main objective of this study was to evaluate the potential of P. tinctorius spores as a source of anticancer compounds.
- Assessment of cyanobacterial biomass as sustainable agricultural fertilizer: soil experiment with plants in Pot †Publication . Massa, Anabella; Azevedo, Joana; Azevedo, Rui; Pinto, Edgar; Costa, Anabela; Vasconcelos, Vitor; Campos, Alexandre; Freitas, MarisaProviding food to the growing human population in a sustainable way is one of the greatest challenges of modern society. In this context, cyanobacterial biomass (CB) can function as a source of macronutrients to increase soil productivity. These organisms can be collected from the environment in considerable amounts, since they tend to grow in large blooms. However, some of these cyanobacterial strains produce toxins that need to be carefully monitored to avoid food accumulation. The objective of this work was to evaluate the possible use of toxic and non-toxic strains of CB as fertilizer supplement in the growth of economically relevant vegetables. One-month old Raphanus sativus (radish) and Spinacia oleracea (spinach) plants were grown in pots in indoor controlled conditions. Six experimental conditions were set: (1) a control with no nutrient addition, (2) a recommended dose of a NK commercial fertilizer (CF), 0.6g of lyophilized CB of (3) a non-toxic strain of Cylindrospermopsis raciborskii, (4) a toxin-producing strain of C. raciborskii, (5) Microcystis aeruginosa, and (6) Anabaena sp. Several variables were estimated: in CB, this included the NPK dose addition, and in plants, the height, dry weight (dw) of the shoot and root, and the mineral content of plant edible parts. The mineral content in CB was estimated and compared with the recommended dose of CF, according to the information given by the fabricant label. We found no significative differences in N composition; nevertheless, there was a significative higher content in P and significative lower content in K in the CB. In the plants, we found no significative statistical differences between the treatments for the dw of radish root and spinach height. In spinach, the dw of the shoot in the M. aeruginosa treatment was significantly lower than the control, CF, and both the toxic and non-toxic C. raciborskii biomass. Additionally, in radish, the plant height and dw of the shoot M. aeruginosa treatment were significantly lower than in the toxic strain of C. raciborskii treatment. When analyzing mineral content in edible parts, we found that spinach treated with control and CF showed a higher content of Ca, Mo, N, P, and K, while in radish, the same two treatments plus the C. raciborskii toxic had higher Co and Fe content. M. aeruginosa amendment seems to impair shoot growth in both plant species. On the contrary, the toxic C. raciborskii CB seems to have a beneficial effect on growth and in mineral uptake on radish plants.
- Bioaccessibility and changes on cylindrospermopsin concentration in edible mussels over storage and processing time.Publication . Freitas, Marisa; Azevedo, Joana; Carvalho, António Paulo; Mendes, Vera; Manadas, Bruno; Campos, Alexandre; Vasconcelos, VítorThe cyanotoxin cylindrospermopsin has been recognized of increased concern due to the global expansion of its main producer, Cylindrospermopsis raciborskii. Previous studies have shown that aquatic organisms, especially 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, human exposure assessment has been based on the cylindrospermopsin concentration in raw food items. This study aimed to assess the changes on cylindrospermopsin concentration in edible mussels over 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%/48 h and 57.7%/one week). The cooking treatments did not produce significant differences in cylindrospermopsin concentration in mussel matrices (flesh), however, cylindrospermopsin was found in the cooking water, suggesting that heat processing can be used to reduce the availability of cylindrospermopsin in this food item. The in vitro digestion with salivary and gastrointestinal juices considerably decreased the cylindrospermopsin availability in uncooked and steamed mussels, highlighting the importance in integrating the bioaccessibility in the human health risk assessment.
- Bioprospecting for cellulose-degrading microorganismsPublication . Almeida, Liliana; Alexandrino, Diogo; Oliveira, Rui S.; Carvalho, M. Fátima; Freitas, Marisa; Freitas, MarisaCellulose, a complex polysaccharide, is one of the main components of plant and algae biomass and therefore the most abundant polymer found in nature, serving as a significant carbon source in various ecosystems. Bioprospecting for cellulolytic microbes is a promising strategy for discovering potential biocatalysts for the hydrolysis of lignocellulosic materials, including residues. This work aims to assess the cellulolytic potential of microbial isolates (bacteria and fungi) sourced from diverse environments and to establish a synthetic microbial consortium capable to enhance the management of cellulosic waste. The experimental approach encompasses biodegradation assays coupled with optimized colorimetric methods, to estimate cellulose consumption (Congo Red assay) and the concomitant production of oligomeric/monomeric sugars (Dinitrosalicylic acid assay). For bacterial strains, these biodegradation assays involve batch incubations in minimal liquid medium supplied with cellulose as the sole carbon source, while solid medium is being used for fungi. Both assays are being run under mesophilic temperatures (28-30 ºC) and aerobic conditions. Upon achieving a final selection, strains exhibiting the best cellulose degradation performances will be assembled into a synthetic consortium that will be evaluated for their cellulose degradation capability. Future steps will also include phylogenetic identification of the cellulose degrading isolates through 16S and ITS rRNA gene sequence analysis. This study has the potential to identify novel cellulolytic microbes, highlighting the substantial role microorganisms play in ecosystem processes and their diverse applications in biotechnology.
- 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.
- Cyanobacteria for skin care and cosmeceutical formulationsPublication . Melo, Luísa; Morone, Janaína; Silva, Margarida; Ferraz, Ricardo; Martins, RosárioSkin acts as a barrier from external stimulus such as pollutants and ultraviolet radiation. Disturbs in the skin are involved in skin aging, which mostly results on a thinner epidermis, dryness, wrinkles, and loss of elasticity. Synthetic ingredients in cosmetics are known to be more toxic and to cause negative impact on the environment. In a society increasingly worried with human and environment health, natural skin care products gain special attention and boosted the search for natural sources. Due to the production of bioactive compounds, cyanobacteria emerged as an excellent source for cosmetic ingredients. Cyanobacteria strains of CIIMAR culture collection (LEGE-CC) have already proven to be interesting for cosmetic purposes [1,2]. In this project we aimed to evaluate the potential of four LEGE-CC cyanobacteria strains for skin care purposes. The cytotoxicity of acetonic and aqueous extracts was evaluated in keratinocytes (HaCAT), fibroblasts (3T3L1) and endothelial cells (hCMEC/D3) along with the total phenolic content and antioxidant properties. Cytotoxic assays revealed toxicity of acetonic extracts to keratinocytes in the higher concentrations tested. Aqueous extracts revealed low or non-cytotoxicity. Mainly in low concentrations of extracts, cellular proliferation was registered. Aqueous extracts from strain JM/RS021A and acetone extracts from strain JM/RS035B revealed higher values for total phenolic compounds. The acetone extracts of strain JM/RS021A showed the greatest scavenging activity values on the DPPH assay.
- Cyanobacterial biomass used as biofertilizer in lettuce plants: effects on growth and cyanotoxin accumulation †Publication . Santos, Érica; Massa, Anabella; Azevedo, Joana; Martins, Diogo; Reimão, Mariana; Vasconcelos, Vitor; Campos, Alexandre; Freitas, MarisaThe use of cyanobacterial biomass as a biofertilizer is promising in terms of sustainable agriculture. Nevertheless, cyanobacteria can be considered a threat to human and environmental health due to the potential presence of cyanotoxins, since some studies report that the use of contaminated water for agricultural irrigation can impair plant growth and lead to contamination of food products. Interestingly, at environmentally relevant concentrations, cylindrospermopsin (CYN) seems to cause no deleterious effects in plants, and it might even promote their yield. However, studies assessing CYN accumulation in the edible tissues at environmental concentrations are lacking. The objective of this work was to evaluate the effects of cyanobacterial biomass CYN producing or non-producing on lettuce plant growth, and that of CYN accumulation in edible tissues. This study consisted of growing lettuce plants, under controlled conditions, for 25 days in soil (1) with no extra nutrient addition (control) and supplementation with (2) cyanobacterial biomass that did not produce CYN, (3) cyanobacterial biomass that produced CYN (~10 µg of dissolved CYN), and (4) cyanobacterial biomass that produced CYN, treated by boiling for 5 min (~25 µg of dissolved CYN). At the end of the exposure, lettuce growth was assessed, as well as CYN accumulation in tissues and soil. The results showed that leaf growth was significantly increased (p < 0.05) in lettuce plants supplemented with cyanobacterial biomass, especially at condition (3), which was five-fold higher compared with the control group. Regarding CYN accumulation, for conditions (3) and (4), the toxin was detected in the tissues of plants, as well as in soil at the following decreasing order of concentrations: soil > roots > leaves. Interestingly, the concentration determined in lettuce leaves in condition (4) was three-fold lower when compared with the condition (3). Nevertheless, for both conditions, although CYN has been detected in lettuce leaves, the concentration in the edible part did not exceed the proposed provisional tolerable daily intake (TDI) of 0.03 µg/kg/BW. In conclusion, these results suggest that the use of cyanobacterial biomass as lettuce biofertilizer, even containing CYN at environmentally relevant concentrations, can positively influence plant growth and development without compromising the safety of edible tissues.
- Cylindrospermopsin and glyphosate accumulation in lettuce (Lactuca sativa) simultaneously exposed to both toxicants in hydroponic and soil systemsPublication . Sengupta, S.; Freitas, Marisa; Pinto, Edgar; Ferreira, I.; Oliveira, F.; Azevedo, J.; Prieto, A.; Diez-Quijada, L.; Jos, A.; Cameán, A.M.; Campos, A.; Vasconcelos, V.In nature, the simultaneous occurrence of multiple emergent contaminants such as cyanotoxins (e.g., cylindrospermopsin (CYN)) and herbicides (e.g., glyphosate (GLY)), is highly expectable and it can be anticipated, mainly in the aquatic and terrestrial environments. The use of contaminated water for irrigation can be hazardous to the agricultural sector and some studies have reported that, individually, these contaminants can be accumulated in the edible tissues exerting a negative influence on crop plants safety and ultimately in human health. Furthermore, recent studies have suggested that some cyanotoxins (e.g., microcystins) can change the membrane permeability of roots, resulting in changes in the accumulation rates of other contaminants in plants. Since edible plants are exposed to a wide variety of substances through irrigation water, there is increasing concern in the potential adverse effects of the interactions between those substances when present simultaneously, especially when this can have potential public health consequences. The aim of this study was to determine the accumulation of CYN in Lactuca sativa simultaneously exposed to GLY at environmentally relevant concentrations. Lettuce plants were exposed for 15 days to 50 μg/L or kg of CYN-containing crude extract (Chrysosporum ovalisporum culture - LEGE X001) and 750 μg/L or kg of GLY, in hydroponic and soil systems, respectively. The resins (HP20, SP700, and XAD18) were evaluated for MC-LR uptake kinetics, capacities, and extraction efficiencies and simple procedures were developed for determining MC-LR concentration in binding disc extracts by Adda-ELISA (U.S. EPA Method 546). The XAD18-DGT/Adda-ELISA method had a 7-d deployment time detection limit of ≈0.05 μg/L and capacity of > 250 μg/L of MC-LR in water samples which encompass U.S. EPA and WHO advisory concentrations for drinking and recreational waters. The XAD18DGT/Adda-ELISA method determined timeaveraged MC-LR concentrations in waters with wide ranging pH (4.9–8.3) and ionic strength (0.04–0.8 M) under well-stirred and quiescent conditions with 90–101% accuracy. In addition to high sensitivity and accuracy, the method is simple, inexpensive, and applicable for determining MC-LR and related MCs concentrations in waterbodies with wide ranging chemical characteristics and hydrodynamic conditions. 4.11.07 Cylindrospermopsin and Glyphosate Accumulation in Lettuce (Lactuca sativa) Simultaneously Exposed to Both Toxicants in Hydroponic and Soil Systems S. Sengupta, School of Bio Sciences and Technology Vellore Institute of Technology, Vellore - 632 014, Tamil Nadu, India; M.M. Freitas, School of Health, Polytechnic Institute of Porto; E. Pinto, I. Ferreira, LAQV/REQUIMTE, Departament of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge de Viterbo Ferreira 228, 4050313 Porto, Portugal; F. Oliveira, J. Azevedo, CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450–208 Porto; A. Prieto, L. Diez-Quijada, A. Jos, A.M. Cameán, Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n◦2, 41012 Seville, Spain / Toxicology; A. Campos, Interdisciplinary Centre of Marine and Environmental Sciences, CIIMAR; V. Vasconcelos, CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450–208 Porto In nature, the simultaneous occurrence of multiple emergent contaminants such as cyanotoxins (e.g., cylindrospermopsin (CYN)) and herbicides (e.g., glyphosate (GLY)), is highly expectable and it can be anticipated, mainly in the aquatic and terrestrial environments. The use of contaminated water for irrigation can be hazardous to the agricultural sector and some studies have reported that, individually, these contaminants can be accumulated in the edible tissues exerting a negative influence on crop plants safety and ultimately in human health. Furthermore, recent studies have suggested that some cyanotoxins (e.g., microcystins) can change the membrane permeability of roots, resulting in changes in the accumulation rates of other contaminants in plants. Since edible plants are exposed to a wide variety of substances through irrigation water, there is increasing concern in the potential adverse effects of the interactions between those substances when present simultaneously, especially when this can have potential public health consequences. The aim of this study was to determine the concentration of CYN and GLY in lettuce plants (roots and leaves) was determined by LC/MSMS. The results show that, at the described conditions, CYN was accumulated in roots (0.06-7.62 μg CYN/g Dw) and leaves (0.13-1.1 μg CYN/g Dw) of lettuce, especially when plants were exposed in hydroponic system. However, interestingly, when lettuce plants were exposed simultaneously to both toxicants the concentration of CYN assimilated by lettuce plants (roots and leaves) was respectively, 1.5fold and 1-3-2.2-fold lower than in the exposure to isolated CYN. Conversely, the plants exposed to the mixture in soil system, showed that the concentration of GLY incorporated by lettuce (roots and leaves) was higher than in the exposure to the isolated compound (0.04 - 0.21 µg GLY/g and < LOQ - 0.84 µg GLY/g, respectively). This finding highlights the potential for the enhancement of GLY accumulation in lettuce plants due to their cooccurrence with CYN, and it underlines the importance of further research regarding the mechanism involved.
- Cytotoxic activity of marine cyanobacteria against cancer cell linesPublication . Ribeiro, Maria João; Costa, Margarida; Leão, Pedro; Barros, Piedade; Vasconcelos, Vítor; Martins, RosárioMarine cyanobacteria possess an extensive capacity to produce compounds that were found to induce biological activities against cell lines and organisms. Many of the marine cyanobacteria compounds already described were found to induce toxicity against cancer cells.
- Cytotoxicity of picocyanobacteria strains of the genera Cyanobium on osteosarcoma cellsPublication . Martins, Rosário; Costa, Margarida; Garcia, Mónica; Barros, Piedade; Costa-Rodrigues, João; Vasconcelos, Vítor; Fernandes, MariaMarine cyanobacteria have been recognized as an important source of bioactive compounds. The cytotoxicity on cancer cell lines has been extensively explored and several cyanobacteria metabolites are already described as potential anticancer compounds or are considered useful templates for the design of new anticancer drugs. The majority of compounds have been isolated from filamentous or colonial cyanobacteria that growth in high densities along shores. In contrast, picoplanktonic forms have rarely been explored since, for these strains, there is a need for culture for biomass production. From our LEGE cyanobacteria culture collection we selected a panel of seven strains of the picocyanobacteria genera Cyanobium in order to explore it’s potential as anticancer agents. Strains were cultured under laboratory conditions. Freeze-dried biomass was extracted using methanol and dichloromethane to a crude extract and then fractionated using hexane, ethyl acetate and methanol. The cytotoxicity of crude extracts and fractions was evaluated in the osteosarcoma cell line MG63 by the reduction of the bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) and confirmed by the lactate dehydrogenase (LDH) assay. From the results, four of the seven Cyanobium strains were found to induce a significant decrease in cell viability. The highest percentage of inhibition of tumor cells growth was observed within the ethyl acetate, which is therefore, promising in terms of isolation of bioactive compounds
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