Browsing by Author "Ramos-Jesus, Joilson"
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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.
- Copper nanoparticles stabilized with cashew gum: Antimicrobial activity and cytotoxicity against 4T1 mouse mammary tumor cell linePublication . Amorim, Adriany; Mafud, Ana Carolina; Nogueira, Silvania; Ramos-Jesus, Joilson; Araújo, Alyne Rodrigues de; Plácido, Alexandra; Neta, Maria Brito; Alves, Michel Muálem Moraes; Carvalho, Fernando Aécio Amorim; Rufino Arcanjo, Daniel Dias; Braun, Sacha; López, Marta Sánchez-Paniagua; López-Ruiz, Beatriz; Delerue-Matos, Cristina; Mascarenhas, Yvonne; Silva, Durcilene; Eaton, Peter; Leite, José Roberto Souza AlmeidaCopper nanoparticles stabilized with cashew (CG-CuNPs) were synthesized by reduction reaction using ascorbic acid and sodium borohydride, using the cashew gum (CG) as a natural polymer stabilizer. Dynamic light scattering, atomic force microscopy, Fourier-transform infrared spectroscopy, UV-Vis spectrophotometry, and x-ray diffraction were used to characterize the nanoparticles (CG-CuNPs), and copper was quantified by electrochemical measurement. The UV-vis spectra of the CG-CuNPs confirmed the formation of nanoparticles by appearance of a surface plasmon band at 580 nm after 24 h of reaction. The Fourier-transform infrared spectrum of CG-CuNPs showed the peak at 1704 cm−1 from cashew gum, confirming the presence of the gum in the nanoparticles. The average size of CG-CuNPs by dynamic light scattering and atomic force microscopy was around 10 nm, indicating small, approximately spherical particles. Antimicrobial assays showed that CG-CuNPs had activity against Staphylococcus aureus ATCC 29213 with a minimal inhibitory concentration of 0.64 mM. The cytotoxicity assay on BALB/c murine macrophages showed lower cytotoxic effects for CG-CuNPs than CuSO4·5H2O. Viability cell assays for CG-CuNPs at (0.250 mM) inhibited by 70% the growth of 4T1 LUC (4T1 mouse mammary tumor cell line) and NIH 3T3 cells (murine fibroblast cells) over a 24-h period. Therefore, CG-CuNPs can be used as an antimicrobial agent with lower cytotoxic effects than the CuSO4·5H2O precursor.
- 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.
- Development of an electrochemical DNA-based biosensor for the detection of the cardiovascular pharmacogenetic-altering SNP CYP2C9*3Publication . Morais, Stephanie L.; Magalhães, Júlia M.C. S.; Domingues, Valentina F.; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaCardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C9*3 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C9*3 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C9*3 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C9*3 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015–1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.
- Development of electrochemical genosensors for the CYPC*2 gene polymorphism detectionPublication . Sousa, António J. S. F.; Costa, Inês; Banegas, Rodrigo S.; Morais, Stephanie L.; Magalhães, Júlia; Rodrigues, Valentina; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaPharmacogenetic studies search for heritable genetic polymorphisms that influence responses to drug therapy. Pharmacogenetics has many possible applications in cardiovascular pharmacotherapy including screening for polymorphisms to choose agents with the greatest potential for efficacy and least risk of toxicity. Pharmacogenetics also informs dose adaptations for specific drugs in patients with aberrant metabolism. Cardiovascular diseases (CVD) are considered one of the leading causes of death worldwide. To prevent cardiovascular complications and further loss of life oral anticoagulants (e.g., warfarin) are frequently prescribed to patients. Nevertheless, warfarin therapeutic agent presents narrow therapeutic windows with well-documented health risks. Some of these dose-responses are a result of specific single-nucleotide polymorphism (SNP) genetic variations present in a patient´s DNA. Among them, determined SNP in the cytochrome P4502C9 (CYP2C9), namely the CYP2C9*2, gene has been identified as dose-response altering SNP. Therefore, the need for a rapid, selective, low-cost and in real time detection device is crucial before prescribing any anticoagulant. In this work an analytical approach based on electrochemical genosensor technique is under development to create a low-cost genotyping platform able to genotype SNPs related with the therapeutic response of warfarin. Analyzing public databases, two specific 71 bp DNA probes, one with adenine (TA) and other with guanine (TG) SNP genetic variation were selected and designed. The design of this electrochemical genosensor consists of ssDNA immobilization onto gold surfaces that act as the SNPs complementary probes. The hybridization reaction is performed in a sandwich format of the complementary ssDNA, using an enzymatic scheme to amplify the electrochemical signal. The electrochemical signal was performed by using chronoamperometric technique.
- Identification of Eschweilenol C in derivative of Terminalia fagifolia Mart. and green synthesis of bioactive and biocompatible silver nanoparticlesPublication . Araujo, Alyne Rodrigues de; Ramos-Jesus, Joilson; Oliveira, Taiane Maria de; Carvalho, Andressa Maria A. de; Nunes, Paulo Humberto M.; Daboit, Tatiane Caroline; Carvalho, Ana P.; Barroso, M. Fátima; Almeida, Miguel Peixoto de; Plácido, Alexandra; Rodrigues, Artur; Portugal, Camila C.; Socodato, Renato; Relvas, João B.; Delerue-Matos, Cristina; Silva, Durcilene Alves da; Eaton, Peter; Leite, José Roberto de Souza de A.A green synthetic route was developed to prepare silver nanoparticles (AgNPs) in aqueous solution for biological applications. Eschweilenol C, a compound derivative ellagic acid was identified as the main constituent of the aqueous fraction of the ethanolic extract of Terminalia fagifolia Mart. by NMR analysis. In the green synthesis, the ethanolic extract of T. fagifolia and its aqueous fraction were used to promote silver reduction and nanoparticle stabilization. The synthesized AgNPs presented a spherical or polygonal morphology shape by TEM analysis and AgNPs showed high levels of antioxidant and considerable antibacterial and antifungal activities. Synthesized nanoparticles presented significant antioxidant activity by sequestration of DPPH and ABTS radicals, in addition to iron reduction (FRAP assay) and measurement of antioxidant capacity in ORAC units, in addition, AgNP synthesized with the aqueous fraction also demonstrated antioxidant potential in microglial cells. Gram-positive and Gram-negative bacteria were susceptible to growth inhibition by the nanoparticles, among which the AgNPs formed by the ethanolic extract was the most effective. The data obtained by AFM images suggested that AgNPs could lead to the lysis of bacteria and subsequent death. The antifungal assays showed high efficiency against yeasts and dermatophytes. This work represents the first description of antifungal activity by AgNPs against Fonsecaea pedrosoi, the etiologic agent of chromoblastomycosis. In relation to biocompatibility, the AgNPs induced lower haemolysis than AgNO3.
- VKORC1 gene polymorphism as cardiovascular biomarker: Detection by electrochemical genosensorsPublication . Costa, Inês; J. S. F. Sousa, António; Banegas, Rodrigo S.; Morais, Stephanie L.; Magalhães, Júlia; Rodrigues, Valentina; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaWarfarin is an anticoagulant generally used to prevent cardiovascular diseases. Since of the low therapeutic index of warfarin and frequent complications of prevention or treatment, significant differences in individual doses of warfarin are needed to achieve prophylactic and therapeutic ranges. Recent studies have been reporting that genetic variants of vitamin K epoxide reductase complex (VKORC1) influence the response to warfarin and doses [9]. So, the genetic and pharmacogenetic information of the major cardiovascular diseases plays an important role in the identification of the cardiovascular risk factors and in the diagnosis and treatment of these conditions. This work addresses the development of a disposable electrochemical genosensor able of detecting single nucleotide polymorphism (SNP) in the VKORC1 gene. Analysing public databases, two specific 52 bp DNA probes, one with adenine (TA) and another with guanine (TG) SNP genetic variation were selected and selected and designed. The genosensor methodology implied the immobilization of a mixed self-assembled monolayer (SAM) linear VKORC1 DNA-capture probe and mercaptohexanol (MCH) onto screen-printed gold electrodes (SPGE). To improve the genosensor´s selectivity and avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the VKORC1 allele was designed using a complementary fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. Preliminary studies indicate that differences in the electrochemical answers were obtained depending of the hybridization reaction format. In fact, higher electrochemical intensities were measured when the hybridization reaction was performed with a complementary DNA (without SNPs). These results suggested that the sensor is able to discriminate between the complementary DNA and single base mismatch targets having a great potential for the DNA polymorphism analysis.