Browsing by Author "Marani, Mariela M."
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- Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three-dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organismsPublication . Plácido, Alexandra; Coelho, Andreia; Abreu Nascimento, Lucas; Gomes Vasconcelos, Andreanne; Barroso, M. Fátima; Ramos-Jesus, Joilson; Costa, Vladimir; Lima, Francisco das Chagas Alves; Delerue-Matos, Cristina; Ramos, Ricardo Martins; Marani, Mariela M.; Leite, José Roberto de Souza de AlmeidaTransgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc.
- Layer-by-layer films containing peptides of the Cry1Ab16 toxin from Bacillus thuringiensis for potential biotechnological applicationsPublication . Plácido, Alexandra; Farias, Emanuel Airton de Oliveira; Marani, Mariela M.; Vasconcelos, Andreanne G.; Mafud, Ana C.; Mascarenhas, Yvonne P.; Eiras, Carla; Leite, José R.S.A.; Delerue-Matos, CristinaCry1Ab16 is a toxin of crystalline insecticidal proteins that has been widely used in genetically modified organisms (GMOs) to gain resistance to pests. For the first time, in this study, peptides derived fromthe immunogenic Cry1Ab16 toxin (from Bacillus thuringiensis) were immobilized as layer-by-layer (LbL) films. Given the concern about food and environmental safety, a peptide with immunogenic potential, PcL342–354C, was selected for characterization of the electrochemical, optical, and morphological properties. The results obtained by cyclic voltammetry (CV) showed that the peptide have an irreversible oxidation process in electrolyte of 0.1 mol·L−1 potassiumphosphate buffer (PBS) at pH7.2. Itwas also observed that the electrochemical response of the peptide is governed mainly by charge transfer. In an attempt to maximize the electrochemical signal of peptide, it was intercalated with natural (agar, alginate and chitosan) or synthetic polymers (polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate (PSS)). The presence of synthetic polymers on the film increased the electrochemical signal of PcL342–354C up to 100 times. Images by Atomic Force Microscopy (AFM) showed that the immobilized PcL342–354C formed self-assembled nanofibers with diameters ranging from 100 to 200 nm on the polymeric film. By UV–Visible spectroscopy (UV–Vis) it was observed that the ITO/PE/PSS/PcL342–354C film grows linearly up to the fifth layer, thereafter tending to saturation. X-ray diffraction confirmed the presence on the films of crystalline ITO and amorphous polypeptide phases. In general, the ITO/PEI/PSS/PcL342–354C film characterization proved that this systemis an excellent candidate for applications in electrochemical sensors and other biotechnological applications for GMOs and environmental indicators.
- Structure and function of a novel antioxidant peptide from the skin of tropical frogsPublication . Barbosa, Eder Alves; Oliveira, Ana; Plácido, Alexandra; Socodato, Renato; Portugal, Camila C.; Mafud, Ana Carolina; Ombredane, Alicia S.; Moreira, Daniel C.; Vale, Nuno; Bessa, Lucinda J.; Joanitti, Graziella A.; Alves, Cláudia; Gomes, Paula; Delerue-Matos, Cristina; Mascarenhas, Yvonne Primerano; Marani, Mariela M.; Relvas, João B.; Pintado, Manuela; Leite, José Roberto S.A.The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin-I, has a molecular weight [M+H]+ = 1543.69Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin-I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin-I has a novel primary sequence with low similarity compared with previously described amphibian's AOPs. Antioxidin-I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin-I presented a low cytotoxicity and suppressed menadione-induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia-induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide.
- Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coliPublication . Marani, Mariela M.; Perez, Luis O.; de Araujo, Alyne Rodrigues; Plácido, Alexandra; Sousa, Carla F.; Quelemes, Patrick Veras; Oliveira, Mayara; Gomes-Alves, Ana G.; Pueta, Mariana; Gameiro, Paula; Tomás, Ana M.; Delerue-Matos, Cristina; Eaton, Peter; Camperi, Silvia A.; Basso, Néstor G.; Leite, José Roberto de Souza de AlmeidaPatagonia's biodiversity has been explored from many points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7μM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian region's unexplored biodiversity as a source for new molecule discovery.
- Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coliPublication . Marani, Mariela M.; Perez, Luis O.; Araujo, Alyne Rodrigues de; Plácido, Alexandra; Sousa, Carla F.; Quelemes, Patrick Veras; Oliveira, Mayara; Gomes-Alves, Ana G.; Pueta, Mariana; Gameiro, Paula; Tomás, Ana M.; Delerue-Matos, Cristina; Eaton, Peter; Camperi, Silvia A.; Basso, Néstor G.; Leite, Jose Roberto de Souza de AlmeidaPatagonia's biodiversity has been explored frommany points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7 μM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian region's unexplored biodiversity as a source for new molecule discovery.