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Chronoamperometric magnetogenosensing for simultaneous detection of two Roundup Ready™ soybean lines: GTS 40-3-2 and MON89788

Publication . Plácido, Alexandra; Pereira, Clara; Barroso, M. Fátima; de-los-Santos-Álvarez, Noemí; Delerue-Matos, Cristina

Development of expeditious analytical methods for the detection of genetically modified organisms (GMOs) is increasingly necessary, not only to verify compliance with labelling, but also to help industry to efficiently control the reception of raw materials. On the basis of this, a disposable electrochemical magnetogenoassay is proposed for simultaneous detection of two Roundup Ready (RR) soybean lines GTS 40-3-2 and MON89788, using gold-coated magnetic nanoparticles (Fe3O4@Au) as nanosupport. To perform this magnetogenoassay, a sandwich-type hybridization assay was used with different enzymatic labelling systems (fluorescein isothiocyanate and digoxigenin) and dual screen-printed carbon electrodes (SPdCEs), which allowed the simultaneous readout of each target. A linear relationship ranging from 0.1 to 2.5 nM and from 0.1 to 1.0 nM was achieved for GTS 40-3-2 and MON89788 events, respectively, and both assays showed a similar detection limit of about 0.1 nM. Furthermore, a good performance in terms of precision and selectivity was achieved. The proposed approach is a step forward for event-specific multiplex detection.

2019-03Journal article

Open access

Acetylated cashew gum-based nanoparticles for the incorporation of alkaloid epiisopiloturine

Publication . Rodrigues, Jessica do Amaral; Araújo, Alyne Rodrigues de; Pitombeira, Nadia Aline; Plácido, Alexandra; Almeida, Miguel Peixoto de; Veras, Leiz Maria Costa; Delerue-Matos, Cristina; Lima, Filipe Camargo Dalmatti Alves; Neto, Augusto Batagin; Paula, Regina Célia Monteiro de; Feitosa, Judith Pessoa Andrade; Eaton, Peter; Leite, José Roberto Souza A.; Silva, Durcilene Alves da

The natural alkaloid epiisopiloturine has recently become the focus of study for various medicinal properties, particularly for its anti-inflammatory and antischistosomal effect. The incorporation of active molecules in natural polymeric matrices has garnered increasing interest during recent decades. A new derivative of cashew gum successfully obtained by gum acetylation has shown great potential as a carrier in controlled drug release systems. In this work, epiisopiloturine was encapsulated in acetylated cashew gum nanoparticles in order to increase solubility and allow slow release, whereas the morphology results were supported by computer simulations. The particles were produced under a variety of conditions, and thoroughly characterized using light scattering and microscopic techniques. The particles were spherical and highly stable in solution, and showed drug incorporation at high levels, up to 55% efficiency. Using a dialysis-based in vitro assay, these particles were shown to release the drug via a Fickian diffusion mechanism, leading to gradual drug release over approximately 6 h. These nanoparticles show potential for the use as drug delivery system, while studies on their potential anti-inflammatory action, as well as toxicity and efficacy assays would need to be performed in the future to confirm their suitability as drug delivery candidates.

2019-05Journal article

Open access

Synergistic and antibiofilm properties of ocellatin peptides against multidrug-resistant Pseudomonas aeruginosa

Publication . Bessa, Lucinda J; Eaton, Peter; Dematei, Anderson; Plácido, Alexandra; Vale, Nuno; Gomes, Paula; Delerue-Matos, Cristina; Leite, José Roberto Sá; Gameiro, Paula

Aim:To test ocellatin peptides (ocellatins-PT2-PT6) for antibacterial and antibiofilm activities and synergy with antibiotics against Pseudomonas aeruginosa. Materials & methods: Normal- and checkerboard-broth microdilution methods were used. Biofilm studies included microtiter plate-based assays and microscopic analysis by confocal laser scanning microscopy and atomic force microscopy. Results: Ocellatins were more active against multidrug-resistant isolates of P. aeruginosa than against susceptible strains. Ocellatin-PT3 showed synergy with ciprofloxacin and ceftazidime against multidrug-resistant isolates and was capable of preventing the proliferation of 48-h mature biofilms at concentrations ranging from 4 to 8× the MIC. Treated biofilms had low viability and were slightly more disaggregated. Conclusion: Ocellatin-PT3 may be promising as a template for the development of novel antimicrobial peptides against P. aeruginosa.

2018Journal article

Open access

Structure and function of a novel antioxidant peptide from the skin of tropical frogs

Publication . 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.

2018-02Journal article

Open access

Lycopene-rich extract from red guava ( Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway

Publication . Santos, Raimunda C. dos; Ombredane, Alicia S.; Souza, Jéssica Maria T.; Vasconcelos, Andreanne G.; Plácido, Alexandra; Amorim, Adriany das G.N.; Barbosa, Eder Alves; Lima, Filipe C.D.A.; Ropke, Cristina D.; Alves, Michel M.M.; Arcanjo, Daniel D.R.; Carvalho, Fernando A.A.; Delerue-Matos, Cristina; Joanitti, Graziella A.; Leite, José Roberto de S.A.

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M]+• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC50]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC50=1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC50≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway.

2018Journal article

Open access