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- Chitosan-based silver nanoparticles: A study of the antibacterial, antileishmanial and cytotoxic effectsPublication . Lima, Douglas dos Santos; Gullon, Beatriz; Cardelle-Cobas, Alejandra; Brito, Lucas M; Rodrigues, Klinger AF; Quelemes, Patrick V; Ramos-Jesus, Joilson; Arcanjo, Daniel DR; Plácido, Alexandra; Batziou, Krystallenia; Quaresma, Pedro; Eaton, Peter; Delerue-Matos, Cristina; Carvalho, Fernando Aecio A; Silva, Durcilene Alves da; Pintado, Manuela; Leite, Jose Roberto de SáSilver nanoparticles have been studied as an alternative for treatment of microbial infections and leishmaniasis, without promoting induction of microbial or parasite resistance. In this study, chitosan-based silver nanoparticles were synthesized from silver nitrate (AgNO3), sodium borohydride as a reducing agent, and the biopolymer chitosan as a capping agent. The chitosan-based silver nanoparticles were characterized by ultraviolet–visible, Fourier transform infrared, dynamic light scattering, zeta potential, atomic force microscopy, and transmission electron microscope. The antibacterial assay was performed by determination of the minimum inhibitory concentration. The antileishmanial and the cytotoxic effects induced by AgNO3, chitosan, and chitosan-based silver nanoparticles were analyzed by resazurin and MTT colorimetric assays, respectively. AgNO3, chitosan, and chitosan-based silver nanoparticles induced a marked activity against all bacterial strains and promastigote forms of Leishmania amazonensis at minimum inhibitory concentrations ranging from 1.69 to 3.38 µg Ag/mL. Interestingly, the chitosan-based silver nanoparticles presented less cytotoxicity than the AgNO3 alone and were more active against L. amazonensis than solely chitosan. Furthermore, the cytotoxic concentrations (CC50) of both chitosan and chitosan-based silver nanoparticles against macrophages were significantly higher than the IC50 against promastigotes. Thus, the chitosan-based silver nanoparticles represent a promising alternative for the treatment of microbial infections and leishmaniasis.
- Electrochemical genoassays on gold-coated magnetic nanoparticles to quantify genetically modified organisms (GMOs) in food and feed as GMO percentagePublication . Plácido, Alexandra; Pereira, Clara; Guedes, Alexandra; Barroso, M. Fátima; Miranda-Castro, Rebeca; Santos-Álvarez, Noemí de-los-; Delerue-Matos, CristinaThe integration of nanomaterials in the field of (bio)sensors has allowed developing strategies with improved analytical performance. In this work, ultrasmall core-shell Fe3O4@Au magnetic nanoparticles (MNPs) were used as the platform for the immobilization of event-specific Roundup Ready (RR) soybean and taxon-specific DNA sequences. Firstly, monodisperse Fe3O4 MNPs were synthesized by thermal decomposition and subsequently coated with a gold shell through reduction of Au(III) precursor on the surface of the MNPs in the presence of an organic capping agent. This nanosupport exhibited high colloidal stability, average particle size of 10.2 ± 1.3 nm, and spherical shape. The covalent immobilization of ssDNA probe onto the Au shell of the Fe3O4@Au MNPs was achieved through a self-assembled monolayer (SAM) created from mixtures of alkane thiols (6-mercapto-1-hexanol and mercaptohexanoic acid). The influence of the thiols ratio on the electrochemical performance of the resulting electrochemical genoassays was studied, and remarkably, the best analytical performance was achieved for a pure mercaptohexanoic acid SAM. Two quantification assays were designed; one targeting an RR sequence and a second targeting a reference soybean gene, both with a sandwich format for hybridization, signaling probes labelled with fluorescein isothiocyanate (FITC), enzymatic amplification and chronoamperometric detection at screen-printed carbon electrodes (SPCE). The magnetogenoassays exhibited linear ranges from 0.1 to 10.0 nM and from 0.1 to 5.0 nM with similar detection limits of 0.02 nM and 0.05 nM for the event-specific (RR) and the taxon-specific (lectin) targets, respectively. The usefulness of the approach was demonstrated by its application to detect genetically modified organisms (GMOs) in feed and food.
- Quaternized cashew gum: An anti-staphylococcal and biocompatible cationic polymer for biotechnological applicationsPublication . Quelemes, Patrick V.; Araújo, Alyne R. de; Plácido, Alexandra; Delerue-Matos, Cristina; Maciel, Jeanny S.; Bessa, Lucinda J.; Ombredane, Alicia S.; Joanitti, Graziella A.; Soares, Maria José dos S.; Eaton, Peter; Silva, Durcilene A. da; Leite, José Roberto S.A.Chemical modifications to cashew gum (CG) structure have been previously reported to obtain new physicochemical characteristics, however until now there were no reports of modifications by introduction of new functional groups to add cationic character. This study presents a quaternization route for CG using a quaternary ammonium reagent. The chemical features of the quaternized cashew gum derivatives (QCG) were analyzed by: FTIR, elemental analysis, degree of substitution, Zeta potential, 1H NMR and 1H-13C correlation (HSQC). QCG were evaluated for their anti-staphylococcal activity by determining minimum inhibitory and bactericidal concentrations against pathogenic Staphylococcus spp. and by imaging using atomic force microscopy. Moreover, the mammalian cell biocompatibility were also assessed through hemolytic and cell toxicity assays. QCG presented promising antimicrobial activity against methicillin-resistant S. aureus and biocompatibility on tested cells. These results show that QCG could be a promising tool in the development of biomaterials with an anti-septic action.
- Synergistic and antibiofilm properties of ocellatin peptides against multidrug-resistant Pseudomonas aeruginosaPublication . Bessa, Lucinda J; Eaton, Peter; Dematei, Anderson; Plácido, Alexandra; Vale, Nuno; Gomes, Paula; Delerue-Matos, Cristina; Leite, José Roberto Sá; Gameiro, PaulaAim: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.
- Lycopene-rich extract from red guava ( Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathwayPublication . 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.
- Chronoamperometric magnetogenosensing for simultaneous detection of two Roundup Ready™ soybean lines: GTS 40-3-2 and MON89788Publication . Plácido, Alexandra; Pereira, Clara; Barroso, M. Fátima; de-los-Santos-Álvarez, Noemí; Delerue-Matos, CristinaDevelopment 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.
- 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.
- Structure–Activity Relationship of Piplartine and Synthetic Analogues against Schistosoma mansoni and Cytotoxicity to Mammalian CellsPublication . Campelo, Yuri; Ombredane, Alicia; Vasconcelos, Andreanne; Albuquerque, Lucas; Moreira, Daniel; Plácido, Alexandra; Rocha, Jefferson; Fokoue, Harold Hilarion; Yamaguchi, Lydia; Mafud, Ana; Mascarenhas, Yvonne; Delerue-Matos, Cristina; Borges, Tatiana; Joanitti, Graziella; Arcanjo, Daniel; Kato, Massuo; Kuckelhaus, Selma; Silva, Marcos; Moraes, Josué; Leite, JoséSchistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure–function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5⁻10 µM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.