Browsing by Author "Vasconcelos, Vitor"
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- Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a PeroxiredoxinPublication . Frazão, Bárbara; Campos, Alexandre; Osório, Hugo; Thomas, Benjamin; Leandro, Sérgio; Teixeira, Alexandre; Vasconcelos, Vitor; Antunes, AgostinhoPelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS-PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.
- Antibiotic resistance in freshwater cyanobacteria and associated bacteriaPublication . Dias, Elsa; Dias, Daniela; Ferreira, Eugénia; Manageiro, Vera; Vasconcelos, Vitor; Pereira, Paulo; Caniça, ManuelaObjectives: Cyanobacteria are ubiquitous prokaryotes in aquatic ecosystems and although they can be exposed to antibiotics, their role on water resistome was never investigated. Thus, this work aimed to evaluate the antibiotic susceptibility patterns and resistance mechanisms of cyanobacteria and co-occurring bacteria in order to assess their contribution to the global pool of resistance determinants in freshwater. Methods: We investigated 4 cyanobacterial genera (Microcystis, Aphanizomenon, Anabaena and Planktothrix), previously isolated from freshwater reservoirs, and several bacteria isolated from those cyanobacterial cultures. Antibiotic susceptibility of cyanobacteria was evaluated by microdilution method, under specific culturing conditions, against beta-lactams, aminoglycosides, quinolones, sulfonamides and tetracyclines. Minimum inhibitory concentrations (MIC) were determined according to cyanobacterial cell dentisty (DO, 450nm) and microscopic examination of cultures integrity. Bacteria were identified by 16S sequencing and their susceptibility patterns were determined by disk diffusion, according to SFM 2012 non-specific breakpoints, against the same antibiotics. All strains were subjected to the search of class 1, 2 and 3 integrons and antibiotic resistance genes according to the phenotype. Results: Overall, we observed a great diversity of susceptibility to the tested antibiotics, among the distinct strains. Microcystis showed the lowest susceptibility regarding beta-lactams. Conversely, Microcystis was more susceptible to quinolones, while Planktothrix showed higher MIC values. Bacteria from cyanobacterial cultures were identified as Hydrogenophaga atypica, Limnobacter thioxidans, Rhizobium radiobacter, Sphingobium sp. and Brevundimonas lenta. Even though no known antibiotic resistance genes were yet identified, bacteria from different species and showing distinct phenotypes exhibited class 1 and 2 integrons. L. thioxidans, for example, revealed to be resistant to aminoglycosides and harbored a class 2 integron. Conclusions: Although no known antibiotic resistance genes were found in cyanobacteria and co-occurring bacteria, the presence of integrons and the susceptibility to antibiotics, suggest that they may play a role on freshwater resistome and eventually contribute to the dissemination of antibiotic resistance. These results may also be helpful to define guidelines and breakpoints to access cyanobacteria antibiotic susceptibility.
- Aplicabilidade da PCR em tempo real na amplificação de DNA cianobacteriano em amostras preservadas.Publication . Churro, Catarina; Pereira, Paulo; Valério, Elisabete; Vasconcelos, VitorO estudo e monitorização de florescências de cianobactérias envolve frequentemente o uso de amostras fixadas a fim de evitar a sua degradação. Com o aumento das técnicas moleculares disponiveis, a possibilidade de utilizar este recurso de DNA para explorar informação genética representa uma mais valia na investigação em cianobacterias e toxinas associadas. Neste trabalho, determinou-se a quantidade e qualidade do DNA cianobacteriano recuperado a partir de amostras ambientais e de culturas fixadas com (1) solução de Lugol, (2) formaldeído, (3) gluteraldeído e (4) solução de Transeau. A quantificação e capacidade de amplificação do DNA extraído foi avaliada por PCR em tempo real utilizando como fragmento alvo o gene rpoC1. A extração foi feita com fenol-cloróformio e a pureza do DNA foi determinada espectrofotometricamente pela razão DO260/DO280 e DO260/DO230. As amostras foram analisadas em 5 diluições seriadas de 1:10 para determinar o limite de detecção e a eficiência da reacção. A amplificação das várias diluições por PCR em tempo real foi comparada com a amplificação em PCR convencional. Nas amostras fixadas com solução de Lugol ou formaldeído, a quantidade de DNA obtido foi inferior à quantidade obtida na amostra controlo (sem fixação). Contudo obteve-se DNA de boa qualidade (DO260/DO280> 1,86 e DO260/DO230> 2,22). Na análise por PCR em tempo real o fragmento alvo foi amplificado exponencialmente, com réplicas consistentes e uma eficiência de 0,91 (r2=0,99; m=-3,55) para a solução de Lugol e eficiência de 1.02 (r2=0,98; m=-3,28) para o formaldeído. No entanto a quantificação do gene alvo nestas duas amostras foi significativamente inferior à do controlo o que indica que o gene alvo pode ser quantificado mas a sua concentração não pode ser extrapolada para concentração real da amostra não fixada. Todas as diluições das amostras referidas anteriormente foram amplificadas enquanto que por PCR convencional só se obteve produto nas três primeiras diluições, o que indica maior sensibilidade do PCR em tempo real para a amplificação de amostras fixadas. Para a fixação com a solução de Transeau e gluteraldeído obteve-se DNA de fraca qualidade (DO260/DO280 <1,6) o que influenciou a quantificação espectrofotométrica, assim como a análise do gene rpoC1 por PCR em tempo real, em que a amplificação não ocorreu correctamente e as réplicas foram inconsistentes.
- Applicability of the real-time PCR assay in the amplification of cyanobacterial DNA from preserved samplesPublication . Churro, Catarina; Valério, Elisabete; Pereira, Paulo; Vasconcelos, VitorThe study and monitoring of cyanobacterial blooms often involves the use of preserved samples to avoid cellular degradation. However, preserved samples may not be suitable for molecular biology studies because preservation methods can interfere with DNA quality/quantity. Real-time quantitative PCR analysis (qPCR) has been widely applied in molecular analysis and is considered a promising method for monitoring purposes. This study intended to evaluate the applicability of the real-time qPCR technique in samples that were subjected to different methods of preservation: (1) 15% Lugol’s iodine solution (2) 4% formaldehyde and (3) 25% glutaraldehyde. The ability to amplify and quantify DNA extracted from Planktothrix agardhii was assessed using the rpoC1 gene as the target fragment in both raw water samples and in vitro cultures. No reliable DNA amplification was obtained from glutaraldehyde-preserved samples. Successful amplification was obtained from Lugol’s and formaldehyde-preserved samples. In this case, however, the quantification that was achieved by real-time PCR cannot be used to infer cell numbers, because the Ct values that were obtained from the Lugol’s and formaldehydepreserved samples were significantly higher than the Ct values that were obtained from the unpreserved samples. Therefore real-time PCR can be used for the detection and identification of cyanobacteria in preserved samples but no reliable cell quantification can be performed using this method.
- Assessing the antibiotic susceptibility of freshwater Cyanobacteria spp.Publication . Dias, Elsa; Oliveira, Micaela; Jones-Dias, Daniela; Vasconcelos, Vitor; Ferreira, Eugénia; Manageiro, Vera; Caniça, ManuelaFreshwater is a vehicle for the emergence and dissemination of Antibiotic resistance. Cyanobacteria are ubiquitous in freshwater, where they are exposed to antibiotics and resistant organisms, but their role on water resistome was never evaluated. Data concerning the effects of antibiotics on cyanobacteria, obtained by distinct methodologies, is often contradictory. This emphasizes the importance of developing procedures to understand the trends of antibiotic susceptibility in cyanobacteria. In this study we aimed to evaluate the susceptibility of four cyanobacterial isolates from different genera (Microcystis aeruginosa, Aphanizomenon gracile, Chrisosporum bergii, Planktothix agradhii), and among them nine isolates from the same specie (M.aeruginosa) to distinct antibiotics (amoxicillin, ceftazidime, ceftriaxone, kanamycine, gentamicine, tetracycline, trimethoprim, nalidixicacid, norfloxacin). We used a method adapted from the bacteria standard broth microdilution. Cyanobacteria were exposed to serial dilution of each antibiotic (0.0015–1.6mg/L) in Z8 medium (20±1◦C; 14/10hL/D cycle; light intensity 16±421μEm−s−). Cell growth was followed overtime (OD450nm/microscopic examination) and the minimum inhibitory concentrations (MICs) were calculated for each antibiotic/ isolate. We found that β-lactams exhibited the lower MICs, aminoglycosides, tetracycline and norfloxacine presented intermediate MICs; none of the isolates were susceptible to trimethoprim and nalidixic acid. The reduced susceptibility of all tested cyanobacteria to some antibiotics suggests that they might be naturally non-susceptible to these compounds, or that they might became non-susceptible due to antibiotic contamination pressure, or to the transfer of genes from resistant bacteria present in the environment.
- Avaliação do efeito da microcistina-LR no crescimento, sistema antioxidante e indução de apoptose em Saccharomyces cerevisiaePublication . Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, VitorNeste trabalho pretendeu-se usar a levedura Saccharomyces cerevisiae como modelo para compreender melhor os mecanismos de toxicidade induzidos pela microcistina-LR. Este é o organismo eucariótico mais simples e estudado que tem sido amplamente utilizado como modelo no estudo de mecanismos de toxicidade, devido à facilidade de acesso à informação sobre o seu genoma (totalmente sequenciado e anotado), proteoma e processos bioquímicos correspondentes.
- Avaliação do efeito da microcistina-LR no crescimento, sistema antioxidante e indução de apoptose em Saccharomyces cerevisiaePublication . Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, VitorAs hepatotoxinas microcistinas são mundialmente frequentemente encontradas em corpos de água doce. Estas são conhecidas por causar hepatotoxicidade aguda em humanos / animais, promoção de tumores em animais e ao seu potencial carcinogénico. Em células de mamíferos, o mecanismo de toxicidade das microcistinas é atribuída a um processo que envolve várias vias, um deles relacionado com a inibição das fosfatases proteicas PP1 / PP2A e a produção de espécies reativas de oxigénio (ROS). No entanto, uma vez que ainda não estão completamente caracterizados os mecanismos moleculares de ação das microcistinas e os efeitos correspondentes, não é possível ainda fazer a identificação completa destes compostos tóxicos. Neste estudo foram avaliados os efeitos de várias concentrações de microcistina-LR no crescimento, níveis de ROS, resposta do sistema antioxidante e indução de apoptose da levedura Saccharomyces cerevisiae. Verificou-se que o crescimento microbiano não foi inibido na presença das várias concentrações de toxina. Contudo, após coloração das células com fluorocromos, verificou-se que a exposição das células à toxina induziu um aumento dos níveis intracelulares dos ROS. Este aumento provocou uma ativação do sistema antioxidante, especialmente na resposta da catalase. Além disso, observou-se uma inibição da SOD1, o que em conjunto com o tipo de ROS possivelmente presente, sugere que a espécie reativa de oxigénio maioritariamente induzida é peróxido de hidrogénio (H2O2). Observaram-se ainda sinais de apoptose após coloração das células com DAPI e após avaliação por citometria de fluxo, usando um kit de Anexina V-FITC. Os resultados obtidos neste estudo demonstram que a levedura Saccharomyces cerevisiae VL3 apresenta alguns dos principais efeitos tóxicos induzidos pela microcistina-LR em eucariotas superiores. Esta levedura, comprovou assim ser um simples e bom modelo eucariótico para estudar em mais detalhe os mecanismos moleculares de toxicidade induzidos pela microcistina-LR.
- Deciphering the role of cyanobacteria in water resistome: Hypothesis justifying the antibiotic resistance (phenotype and genotype) in Planktothrix genusPublication . Dias, Elsa; Oliveira, Micaela; Manageiro, Vera; Vasconcelos, Vitor; Caniça, ManuelaThe importance of environmental microorganisms in the emergence and dissemination of antibiotic resistance is an undeniable fact. However, cyanobacteria are not seen yet as putative players in the dynamic of environmental resistome, despite their ubiquity in water environments, where they are exposed to antibiotic pollution and in straight contact with native and pathogenic bacteria harboring antibiotic resistance genes (ARGs). In this work we evaluated the susceptibility of 8 strains of Planktothrix agardhii (from surface freshwaters reservoirs) and 8 strains of Planktothrix mougeotii (from a wastewater treatment plant) to several classes of antibiotics, using a microplate dilution method previously described by us. We also search for ARGs in those strains by molecular methods. None of the 16 tested strains were susceptible to trimethoprim, nalidixic acid and norfloxacin, from 0.0015–1.6 mg/L, but all were susceptible to streptomycin, gentamicin, kanamycin, ceftazidime and ceftriaxone. The minimum inhibitory concentrations (MICs) ranged between 0.05–0.8 mg/L for the aminoglycosides and 0.4–1.6 mg/L for the two β‑lactams. Major differences were found in the susceptibility to amoxicillin and tetracycline, with P. agardhii being susceptible (MIC of 0.05 mg/L and 0.4 mg/L, respectively) and P. mougeotii not susceptible. These distinct responses might be due to differences between species. However, the lower susceptibility of wastewater strains suggests that antibiotic resistance phenotype of cyanobacteria is related with their habitat. The failure to detect acquired genes conferring resistance to trimethoprim/quinolones, strongly supports the hypothesis that cyanobacteria are intrinsically resistant to these antibiotics. Interestingly, we detected a class-1-type integron and a sul1 gene in 3 strains of both P. agardhii and P. mougeotii, which supports the possibility of cyanobacteria to acquire and transfer antibiotic resistance determinants. In conclusion, the identification of ARGs and related integrons, as well as the reduced susceptibility to some antibiotics, suggests that cyanobacteria may play a role on environmental resistome.
- Effects of microcystin-LR on Saccharomyces cerevisiae growth, oxidative stress and apoptosisPublication . Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, VitorMicrocystins (MC) are cyanotoxins occurring globally, known for causing acute hepatotoxicity in humans/animals, tumor promotion in animals and potential carcinogenicity. The mechanism of MC toxicity is considered a multi-pathway process involving the inhibition of protein phosphatases PP1/PP2A and the production of reactive oxygen species (ROS). However, their mechanism of action is not fully characterized, thus hampering the complete hazard identification. In this study, we evaluated the effect of several microcystin-LR concentrations on the growth, ROS levels, antioxidant system response and apoptosis induction on Saccharomyces cerevisiae. Our results showed that the growth of S. cerevisiae was not inhibited when compared to control cells. However, the staining of cells with DHR123 and DHE revealed an intracellular increase of the ROS levels. This ROS increase resulted in an augment of catalase activity and inhibition of SOD. All these facts suggest that hydrogen peroxide was the main ROS induced by MCLR. Signs of apoptosis were also detected in the cells exposed to toxin. Our results show that S. cerevisiae VL3 displays MCLR toxicity effects known to occur in higher eukaryotes and confirmed that it can be a simple and good model to help further in the elucidation of MCLR molecular mechanisms of toxicity.
- Evaluation of methanol preservation for molecular and morphological studies in cyanobacteria using Planktothrix agardhiiPublication . Churro, Catarina; Valério, Elisabete; Vieira, Luís; Pereira, Paulo; Vasconcelos, VitorMolecular studies on cyanobacteria often involve filtering and freezing of samples leading to loss of cell morphological features. Methanol is often used in preservation of biological materials in association with other fixatives. This study evaluates the application of methanol in the preservation of DNA for molecular studies as well as for the preservation of cell morphology for morphometric analysis in filamentous cyanobacteria. In the present study, both culture and environmental bloom samples were preserved using a cold methanol dehydration series (50, 70, and 100 %) and stored at −20 °C for up to 2 years. The DNA quantity and quality, nucleotide sequence retrieval, and real-time PCR quantification were analyzed over time. Morphometric cell analysis was performed on preserved samples. Results show that the DNA extracted from samples preserved up to 6 months was successfully quantified by real-time PCR. After that period, the DNA quantity decreased with the preservation time. Nevertheless, we were able to detect/amplify the target fragment in samples preserved up to 2 years. The DNA sequence and cell morphology were also maintained during the preservation time. Thus, methanol preservation is an adequate method to preserve molecular information and morphological features after long storage periods.
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