Saulo Garrido - Academia.edu (original) (raw)

Papers by Saulo Garrido

Research paper thumbnail of Pharmaceutical nanotechnology: Antimicrobial peptides as potential new drugs against WHO list of critical, high, and medium priority bacteria

European Journal of Medicinal Chemistry

Research paper thumbnail of B1CTcu5 analogs as promising antimicrobial peptides, replacing the sequence cysteine

Proceedings of 7th International Electronic Conference on Medicinal Chemistry, 2021

Research paper thumbnail of Influence of the Testing Gage Length in the Strength, Young’s Modulus and Weibull Modulus of Carbon Fibres

Carbon Fibres are the most used reinforcement for advanced composites. due to their superior elec... more Carbon Fibres are the most used reinforcement for advanced composites. due to their superior electrical and mechanical properties and light weight, which in turn is translated into a high strength/density ratio composite. Their usefulness is intrisically related to their microstructure, i.e. , amount of functional groups, degree of heat treatment and porosity. The amount of functional groups has a strong influence on the composite properties while heat treatment and porosity are responsible for carbon fibre’s strength and modulus. During processing a number of cracks, voids and fibrillar misalignments, are created either during the PAN precursor processing and during heat treatment to final convertion to carbon. Such inhomogeneities give rise to an appreciable scatter in carbon fibres strength. Their brittle failure behaviour and scatter in strength are characteristic features of any other ceramics material. The most used statistical tool that deals with this characteristic variabil...

Research paper thumbnail of AN. Avaliação da Atividade Antimicrobiana do Peptídeo LeuAB-2: Um Promissor Conservante de Alimentos

Journal of Basic and Applied Pharmaceutical Sciencies, 2016

Introducao: A contaminacao de alimentos e uma das principais preocupacoes da industria alimentici... more Introducao: A contaminacao de alimentos e uma das principais preocupacoes da industria alimenticia, com consequente diminuicao da seguranca e qualidade nutricional. Abre-se entao, a necessidade do desenvolvimento de metodologias alternativas de conservacao, capaz de disponibilizar para a populacao alimentos de qualidade cada vez melhor e mais seguros sob o ponto de vista microbiologico e toxicologico. Dentre os diversos tipos de microrganismos patogenico, que podem ser transmitidos pelos alimentos, destaca-se o genero Salmonella, cuja importância epidemiologica das doencas veiculadas por alimentos, decorre da alta prevalencia do mesmo. Desta forma, e de vital importância, a busca por substâncias naturais que exibam atividades antimicrobianas especificas e, acima de tudo, que as exercam atraves de mecanismos de acao alternativos aos conservantes quimicos disponiveis. Entre estas substâncias, os peptideos antimicrobianos (PAMs) surgem como uma alternativa interessante, ja que atuam me...

Research paper thumbnail of Peptides from CcdB protein as novel inhibitors of DNA gyrase

Research paper thumbnail of CB. Desenvolvimento e caracterização de lipossomas de diferentes composições lipídicas contendo o peptídeo antifúngico Histatina-5

Journal of Basic and Applied Pharmaceutical Sciencies, 2016

Introducao: A candidiase e uma infeccao da mucosa oral e vaginal do organismo humano, provocado p... more Introducao: A candidiase e uma infeccao da mucosa oral e vaginal do organismo humano, provocado pelo microrganismo Candida albicans. A patogenicidade das especies de Candida esta relacionada com a formacao de biofilmes, que agem como um revestimento impermeavel e protetor, e torna o microrganismo resistente a medicamentos convencionais e ao sistema imune do hospedeiro. Neste caso, os antifungicos mais comumente utilizados, como polienos (nistatina e anfotericina B) ou azois (itraconazol, miconazol, fluconazol) nao apresentam acao eficaz. Por esse motivo, a busca por novas opcoes de tratamento esta em constante desenvolvimento principalmente na area da biotecnologia. Um exemplo disso sao os peptideos biologicamente ativos e antifungicos da familia das Histatinas, entre eles a Histatina-5. Trata-se de um peptideo naturalmente encontrado na saliva humana que possui como alvo de acao a mitocondria das celulas de C. albicans. No entanto, Histatina-5 sofre a acao de proteases e de outras ...

Research paper thumbnail of Síntese e caracterização do análogo peptídico derivado do sistema toxina/antitoxina CcdB/CcdA

Journal of Basic and Applied Pharmaceutical Sciencies, 2018

Introducao: Sistemas toxina/antitoxina (TA) sao amplamente encontrados nos plasmideos e cromossom... more Introducao: Sistemas toxina/antitoxina (TA) sao amplamente encontrados nos plasmideos e cromossomos de bacterias e Archaea. Sao pequenas proteinas expressadas por esses micro-organismos que possuem importante papel em varios eventos fisiologicos como a manutencao plasmidial, formacao de celulas persistentes, resistencia ao estresse e protecao contra bacteriofagos. O sistema TA codificado pelo plasmideo F e composto pela toxina CcdB (11,7kDa), uma proteina que mata a celula atraves de um mecanismo que envolve a enzima DNA girase em Escherichia coli, e a antitoxina CcdA (8,3kDa), que previne a acao da toxina formando um complexo estavel com a mesma. A sintese de peptideos de menor tamanho analogos a essas proteinas e que mantenham as propriedades originais das mesmas, e uma ferramenta importante para o estudo estrutural e funcional desses sistemas. Objetivo: Neste trabalho foi sintetizado, purificado e caracterizado o analogo peptidico derivado do sistema toxina/antitoxina. Este fragm...

Research paper thumbnail of Assessment of the Bioactive Potential of Cheese Whey Protein Hydrolysates Using Immobilized Alcalase

Food and Bioprocess Technology, 2020

The hydrolysis of bovine cheese whey (BCW) proteins was performed by the Alcalase immobilized on ... more The hydrolysis of bovine cheese whey (BCW) proteins was performed by the Alcalase immobilized on the support glyoxyl-corncob-powder (AGCCP). The whey protein hydrolysates (WPH) obtained with AGCPP were fractionated by RP-HPLC according to the hydrophilicity of the peptides (F1, hydrophilic; F2, intermediate hydrophilicity; and F3, hydrophobic). The fractions were analyzed by MALDI-TOF, and molecular weight peptides (< 1500 m/z) were identified. Total WPH obtained by AGCCP presented high capacity reduction of the ABTS radical (57.82%) and high chelating activity of iron II (76.2%). The obtained peptides showed high antimicrobial activity (87.75-100%) against the species Escherichia coli (ATCC 43895) and Listeria monocytogenes (ATCC 7644). Only F2 was effective against the Candida albicans strain ATCC 18804 tested (MIC = 10 mg/ mL). The results of this study show that the hydrolysis of BCW proteins using the AGCCP derivative represents a very interesting and viable way for obtention of bioactive peptides from cheese whey.

Research paper thumbnail of Hidrólise das proteínas do soro do queijo utilizando a alcalase imobilizada em pó de sabugo de milho

Brazilian Journal of Development, 2020

A hidrólise das proteínas do soro de queijo foi feita a 50º C, pH=9, 100 rpm por 24 h, utilizando... more A hidrólise das proteínas do soro de queijo foi feita a 50º C, pH=9, 100 rpm por 24 h, utilizando o derivado alcalase glioxil pó de sabugo de milho (AGSM) e os resultados comparados com o derivado alcalase glioxil agarose (AGA) e enzima livre (AL). AL apresentou menor KM = 0,243mM e maior grau de hidrólise (DH)=59,63% com relação aos derivados, provavelmente devido aos efeitos difusionais da imobilização, dificultando o acesso do substrato a enzima. AGSM apresentou melhor estabilidade térmica (62 e 15,5 vezes maior do que a AL e AGA respectivamente). A SDS-PAGE demonstrou a ocorrência de hidrólise utilizando a AGSM, além do DH=26,59% e 3,29U/mg. Confirmando esta hidrólise, o perfil cromatográfico mostrou um aumento no número de picos em diferentes tempos de retenção. Resultados semelhantes foram encontrados para AGA, portanto, o SM representa uma fonte inovativa, de baixo custo, para imobilização da alcalase e obtenção de hidrolisados proteicos. Palavras-chave: Alcalase, soro de queijo, hidrolisados proteicos, ligação covalente multipontual, pó de sabugo de milho.

Research paper thumbnail of A Novel Antifungal System With Potential for Prolonged Delivery of Histatin 5 to Limit Growth of Candida albicans

Frontiers in Microbiology, 2019

Currently 75-88% of fungal infections are caused by Candida species, and Candida albicans is the ... more Currently 75-88% of fungal infections are caused by Candida species, and Candida albicans is the main microorganism that causes these infections, especially oral candidiasis. An option for treatment involves the use of the antifungal peptide Histatin 5 (Hst 5), which is naturally found in human saliva but undergoes rapid degradation when present in the oral cavity, its site of action. For this reason, it is important to develop a way of applying this peptide to the oral lesions, which promotes the gradual release of the peptide. In the present study, we have evaluated the development of liposomes of different lipid compositions, loaded with the peptide as a way to promote its release slowly and gradually, preserving its antifungal potential. For this, the peptide 0WHistatin 5, an analog of the peptide Hst 5, was synthesized, which contains the amino acid tryptophan in its sequence. The solid phase synthesis method was used, followed by cleavage and purification. The liposomes were produced by thin film hydration technique in three different lipid compositions, F1, F2, and F3 and were submitted to an extrusion and sonication process to standardize the size and study the best technique for their production. The liposomes were characterized by dynamic light scattering, and tests were performed to determine the encapsulation efficiency, release kinetics, stability, and evaluation of antifungal activity. The extruded liposomes presented average size in the range of 100 nm, while sonicated liposomes presented a smaller size in the range of 80 nm. The encapsulation efficiency was higher for the sonicated liposomes, being 34.5% for F1. The sonicated F3 presented better stability when stored for 60 days at 4°C. The liposomes showed the ability to release the peptide for the total time of 96 h, with the first peak after 5 h, and a further increase of the released after 30 h. Time-kill assay showed that the liposomes were able to control yeast growth for 72 h. The data suggest that the liposomes loaded with 0WHistatin 5 maintained the action of the peptide and were able to limit the growth of C. albicans, being a suitable system for use in the treatment of oral candidiasis.

Research paper thumbnail of Addendum: Bassan, J.C.; et al. Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein. Materials 2016, 9(5), 357

Research paper thumbnail of Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein

Materials, 2016

In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic... more In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic support (corn cob powder-CCP) with the goal of obtaining peptides with bioactive potential from cheese whey. The pretreated support was activated with glyoxyl groups, glutaraldehyde and IDA-glyoxyl. The immobilization yields of the derivatives were higher than 83%, and the retention of catalytic activity was higher than 74%. The trypsin-glyoxyl-CCP derivative was thermally stable at 65˝C, a value that was 1090-fold higher than that obtained with the free enzyme. The trypsin-IDA-glyoxyl-CCP and trypsin-glutaraldehyde-CCP derivatives had thermal stabilities that were 883-and five-fold higher, respectively, then those obtained with the free enzyme. In the batch experiments, trypsin-IDA-glyoxyl-CCP retained 91% of its activity and had a degree of hydrolysis of 12.49%, while the values for trypsin-glyoxyl-CCP were 87% and 15.46%, respectively. The stabilized derivative trypsin-glyoxyl-CCP was also tested in an upflow packed-bed reactor. The hydrodynamic characterization of this reactor was a plug flow pattern, and the kinetics of this system provided a relative activity of 3.04˘0.01 U¨g´1 and an average degree of hydrolysis of 23%, which were suitable for the production of potentially bioactive peptides.

Research paper thumbnail of Cationic Pd(II) complexes acting as topoisomerase II inhibitors: Synthesis, characterization, DNA interaction and cytotoxicity

Journal of Inorganic Biochemistry, 2016

Research paper thumbnail of Toxin-antitoxin systems and its biotechnological applications

African Journal of Biotechnology, 2014

Toxin-antitoxin (TA) systems are important genetic modules composed by two elements: a toxin, tha... more Toxin-antitoxin (TA) systems are important genetic modules composed by two elements: a toxin, that is always a protein, and an antitoxin, that can be a RNA or a protein and neutralizes the toxic effect of toxin. These systems are widespread in bacteria and archaea, found on plasmids and chromosomes. According to the nature of the antitoxin and its mode of interaction with the toxin, TA systems are grouped into five types. In general, the antitoxin is less stable than the toxin and is rapidly degraded in special conditions, leaving the toxin free to act on its cellular targets. TA modules are important in several events in cell physiology such as plasmid maintenance, formation of persister cells, stress resistance, protection from bacteriophages and regulation of biofilm formation, acting on crucial cellular processes including translation, replication, cytoskeleton formation and membrane integrity. TA systems components have proven to be very useful in biotechnology, being used to enhance cloning selection and protein expression in living bacterial cells. Furthermore, they are also considered as promising targets for the development of antibacterial drugs and can be used in gene therapy. Here, we reported current aspects and the application of TA modules in biotechnology research.

Research paper thumbnail of DNA binding, topoisomerase inhibition and cytotoxicity of palladium(II) complexes with 1,10-phenanthroline and thioureas

Inorganica Chimica Acta, 2016

Metallointercalators represent a promising alternative in cancer chemotherapy. We present herein ... more Metallointercalators represent a promising alternative in cancer chemotherapy. We present herein DNA binding, topoisomerase inhibition and cytotoxic studies on a series of complexes of general formulae [Pd (phen)(tu ⁄) 2 ] 2+ incorporating the intercalator 1,10-phenanthroline and thiourea ligands (L = thiourea 1, N-methylthiourea 2, N,N 0-dimethylthiourea 3). DNA-unwinding results showed that the complexes can induce the unwinding of the plasmid DNA. The binding constants (K b) for the interaction of the complexes with SS-DNA were determined by UV spectroscopy. Competitive experiments with ethidium bromide (EB) were investigated by fluorescence spectroscopy and show that all the complexes were able to displace EB from the DNA-EB complex. The results suggest that they may interact with DNA by intercalation. Compounds were tested against human oral carcinoma cell line (KB), human breast cancer cell line (MCF7) and cisplatin-resistant human breast cancer cell line (MCF7-R) and showed good cytotoxic activity towards MCF7-R. Compounds 2 and 3 were also able to cause topo II inhibition.

Research paper thumbnail of Peptide KSL-W-Loaded Mucoadhesive Liquid Crystalline Vehicle as an Alternative Treatment for Multispecies Oral Biofilm

Molecules, 2015

Decapeptide KSL-W shows antibacterial activities and can be used in the oral cavity, however, it ... more Decapeptide KSL-W shows antibacterial activities and can be used in the oral cavity, however, it is easily degraded in aqueous solution and eliminated. Therefore, we aimed to develop liquid crystalline systems (F1 and F2) for KSL-W buccal administration to treat multispecies oral biofilms. The systems were prepared with oleic acid, polyoxypropylene (5) polyoxyethylene (20) cetyl alcohol (PPG-5-CETETH-20), and a 1% poloxamer 407 dispersion as the oil phase (OP), surfactant (S), and aqueous phase (AP), respectively. We characterized them using polarized light microscopy (PLM), small-angle X-ray scattering (SAXS), rheology, and in vitro bioadhesion, and performed in vitro biological analysis. PLM showed isotropy (F1) or anisotropy with lamellar mesophases (F2), confirmed by peak ratio quantification using SAXS. Rheological tests demonstrated that F1 exhibited Newtonian behavior but not F2, which showed a structured AP concentration-dependent system. Bioadhesion studies revealed an AP concentration-dependent increase in the system's bioadhesiveness (F2 = 15.50˘1.00 mN¨s) to bovine teeth blocks. Antimicrobial testing revealed 100% inhibition of multispecies oral biofilm growth after KSL-W administration, which was incorporated in the F2 aqueous phase at a concentration of 1 mg/mL. Our results suggest that this system could serve as a potential vehicle for buccal administration of antibiofilm peptides.

Research paper thumbnail of Hidrólise enzimática de proteínas lácteas para obtenção de peptídeos bioativos utilizando alcalase comercial

Anais do Simpósio Nacional de Bioprocessos, 2015

Research paper thumbnail of In Vitro Identification of Histatin 5 Salivary Complexes

PloS one, 2015

With recent progress in the analysis of the salivary proteome, the number of salivary proteins id... more With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein-protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed be...

Research paper thumbnail of Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion

PLOS ONE, 2015

Milk whey proteins are well known for their high biological value and versatile functional proper... more Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.

Research paper thumbnail of Histatin 5 inhibits adhesion of C. albicans to Reconstructed Human Oral Epithelium

Frontiers in Microbiology, 2015

Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surf... more Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. The formation of a saliva-derived protein pellicle on the mucosa surface can provide protection against C. albicans on oral epithelial cells, and narrow information is available on the mucosal pellicle composition. Histatins are one of the most abundant salivary proteins and presents antifungal and antibacterial activities against many species of the oral microbiota, however, its presence has never been studied in oral mucosa pellicle. The objective of this study was to evaluate the potential of histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37 • C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37 • C at 5% CO 2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. HOET pre-incubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization on epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis.

Research paper thumbnail of Pharmaceutical nanotechnology: Antimicrobial peptides as potential new drugs against WHO list of critical, high, and medium priority bacteria

European Journal of Medicinal Chemistry

Research paper thumbnail of B1CTcu5 analogs as promising antimicrobial peptides, replacing the sequence cysteine

Proceedings of 7th International Electronic Conference on Medicinal Chemistry, 2021

Research paper thumbnail of Influence of the Testing Gage Length in the Strength, Young’s Modulus and Weibull Modulus of Carbon Fibres

Carbon Fibres are the most used reinforcement for advanced composites. due to their superior elec... more Carbon Fibres are the most used reinforcement for advanced composites. due to their superior electrical and mechanical properties and light weight, which in turn is translated into a high strength/density ratio composite. Their usefulness is intrisically related to their microstructure, i.e. , amount of functional groups, degree of heat treatment and porosity. The amount of functional groups has a strong influence on the composite properties while heat treatment and porosity are responsible for carbon fibre’s strength and modulus. During processing a number of cracks, voids and fibrillar misalignments, are created either during the PAN precursor processing and during heat treatment to final convertion to carbon. Such inhomogeneities give rise to an appreciable scatter in carbon fibres strength. Their brittle failure behaviour and scatter in strength are characteristic features of any other ceramics material. The most used statistical tool that deals with this characteristic variabil...

Research paper thumbnail of AN. Avaliação da Atividade Antimicrobiana do Peptídeo LeuAB-2: Um Promissor Conservante de Alimentos

Journal of Basic and Applied Pharmaceutical Sciencies, 2016

Introducao: A contaminacao de alimentos e uma das principais preocupacoes da industria alimentici... more Introducao: A contaminacao de alimentos e uma das principais preocupacoes da industria alimenticia, com consequente diminuicao da seguranca e qualidade nutricional. Abre-se entao, a necessidade do desenvolvimento de metodologias alternativas de conservacao, capaz de disponibilizar para a populacao alimentos de qualidade cada vez melhor e mais seguros sob o ponto de vista microbiologico e toxicologico. Dentre os diversos tipos de microrganismos patogenico, que podem ser transmitidos pelos alimentos, destaca-se o genero Salmonella, cuja importância epidemiologica das doencas veiculadas por alimentos, decorre da alta prevalencia do mesmo. Desta forma, e de vital importância, a busca por substâncias naturais que exibam atividades antimicrobianas especificas e, acima de tudo, que as exercam atraves de mecanismos de acao alternativos aos conservantes quimicos disponiveis. Entre estas substâncias, os peptideos antimicrobianos (PAMs) surgem como uma alternativa interessante, ja que atuam me...

Research paper thumbnail of Peptides from CcdB protein as novel inhibitors of DNA gyrase

Research paper thumbnail of CB. Desenvolvimento e caracterização de lipossomas de diferentes composições lipídicas contendo o peptídeo antifúngico Histatina-5

Journal of Basic and Applied Pharmaceutical Sciencies, 2016

Introducao: A candidiase e uma infeccao da mucosa oral e vaginal do organismo humano, provocado p... more Introducao: A candidiase e uma infeccao da mucosa oral e vaginal do organismo humano, provocado pelo microrganismo Candida albicans. A patogenicidade das especies de Candida esta relacionada com a formacao de biofilmes, que agem como um revestimento impermeavel e protetor, e torna o microrganismo resistente a medicamentos convencionais e ao sistema imune do hospedeiro. Neste caso, os antifungicos mais comumente utilizados, como polienos (nistatina e anfotericina B) ou azois (itraconazol, miconazol, fluconazol) nao apresentam acao eficaz. Por esse motivo, a busca por novas opcoes de tratamento esta em constante desenvolvimento principalmente na area da biotecnologia. Um exemplo disso sao os peptideos biologicamente ativos e antifungicos da familia das Histatinas, entre eles a Histatina-5. Trata-se de um peptideo naturalmente encontrado na saliva humana que possui como alvo de acao a mitocondria das celulas de C. albicans. No entanto, Histatina-5 sofre a acao de proteases e de outras ...

Research paper thumbnail of Síntese e caracterização do análogo peptídico derivado do sistema toxina/antitoxina CcdB/CcdA

Journal of Basic and Applied Pharmaceutical Sciencies, 2018

Introducao: Sistemas toxina/antitoxina (TA) sao amplamente encontrados nos plasmideos e cromossom... more Introducao: Sistemas toxina/antitoxina (TA) sao amplamente encontrados nos plasmideos e cromossomos de bacterias e Archaea. Sao pequenas proteinas expressadas por esses micro-organismos que possuem importante papel em varios eventos fisiologicos como a manutencao plasmidial, formacao de celulas persistentes, resistencia ao estresse e protecao contra bacteriofagos. O sistema TA codificado pelo plasmideo F e composto pela toxina CcdB (11,7kDa), uma proteina que mata a celula atraves de um mecanismo que envolve a enzima DNA girase em Escherichia coli, e a antitoxina CcdA (8,3kDa), que previne a acao da toxina formando um complexo estavel com a mesma. A sintese de peptideos de menor tamanho analogos a essas proteinas e que mantenham as propriedades originais das mesmas, e uma ferramenta importante para o estudo estrutural e funcional desses sistemas. Objetivo: Neste trabalho foi sintetizado, purificado e caracterizado o analogo peptidico derivado do sistema toxina/antitoxina. Este fragm...

Research paper thumbnail of Assessment of the Bioactive Potential of Cheese Whey Protein Hydrolysates Using Immobilized Alcalase

Food and Bioprocess Technology, 2020

The hydrolysis of bovine cheese whey (BCW) proteins was performed by the Alcalase immobilized on ... more The hydrolysis of bovine cheese whey (BCW) proteins was performed by the Alcalase immobilized on the support glyoxyl-corncob-powder (AGCCP). The whey protein hydrolysates (WPH) obtained with AGCPP were fractionated by RP-HPLC according to the hydrophilicity of the peptides (F1, hydrophilic; F2, intermediate hydrophilicity; and F3, hydrophobic). The fractions were analyzed by MALDI-TOF, and molecular weight peptides (< 1500 m/z) were identified. Total WPH obtained by AGCCP presented high capacity reduction of the ABTS radical (57.82%) and high chelating activity of iron II (76.2%). The obtained peptides showed high antimicrobial activity (87.75-100%) against the species Escherichia coli (ATCC 43895) and Listeria monocytogenes (ATCC 7644). Only F2 was effective against the Candida albicans strain ATCC 18804 tested (MIC = 10 mg/ mL). The results of this study show that the hydrolysis of BCW proteins using the AGCCP derivative represents a very interesting and viable way for obtention of bioactive peptides from cheese whey.

Research paper thumbnail of Hidrólise das proteínas do soro do queijo utilizando a alcalase imobilizada em pó de sabugo de milho

Brazilian Journal of Development, 2020

A hidrólise das proteínas do soro de queijo foi feita a 50º C, pH=9, 100 rpm por 24 h, utilizando... more A hidrólise das proteínas do soro de queijo foi feita a 50º C, pH=9, 100 rpm por 24 h, utilizando o derivado alcalase glioxil pó de sabugo de milho (AGSM) e os resultados comparados com o derivado alcalase glioxil agarose (AGA) e enzima livre (AL). AL apresentou menor KM = 0,243mM e maior grau de hidrólise (DH)=59,63% com relação aos derivados, provavelmente devido aos efeitos difusionais da imobilização, dificultando o acesso do substrato a enzima. AGSM apresentou melhor estabilidade térmica (62 e 15,5 vezes maior do que a AL e AGA respectivamente). A SDS-PAGE demonstrou a ocorrência de hidrólise utilizando a AGSM, além do DH=26,59% e 3,29U/mg. Confirmando esta hidrólise, o perfil cromatográfico mostrou um aumento no número de picos em diferentes tempos de retenção. Resultados semelhantes foram encontrados para AGA, portanto, o SM representa uma fonte inovativa, de baixo custo, para imobilização da alcalase e obtenção de hidrolisados proteicos. Palavras-chave: Alcalase, soro de queijo, hidrolisados proteicos, ligação covalente multipontual, pó de sabugo de milho.

Research paper thumbnail of A Novel Antifungal System With Potential for Prolonged Delivery of Histatin 5 to Limit Growth of Candida albicans

Frontiers in Microbiology, 2019

Currently 75-88% of fungal infections are caused by Candida species, and Candida albicans is the ... more Currently 75-88% of fungal infections are caused by Candida species, and Candida albicans is the main microorganism that causes these infections, especially oral candidiasis. An option for treatment involves the use of the antifungal peptide Histatin 5 (Hst 5), which is naturally found in human saliva but undergoes rapid degradation when present in the oral cavity, its site of action. For this reason, it is important to develop a way of applying this peptide to the oral lesions, which promotes the gradual release of the peptide. In the present study, we have evaluated the development of liposomes of different lipid compositions, loaded with the peptide as a way to promote its release slowly and gradually, preserving its antifungal potential. For this, the peptide 0WHistatin 5, an analog of the peptide Hst 5, was synthesized, which contains the amino acid tryptophan in its sequence. The solid phase synthesis method was used, followed by cleavage and purification. The liposomes were produced by thin film hydration technique in three different lipid compositions, F1, F2, and F3 and were submitted to an extrusion and sonication process to standardize the size and study the best technique for their production. The liposomes were characterized by dynamic light scattering, and tests were performed to determine the encapsulation efficiency, release kinetics, stability, and evaluation of antifungal activity. The extruded liposomes presented average size in the range of 100 nm, while sonicated liposomes presented a smaller size in the range of 80 nm. The encapsulation efficiency was higher for the sonicated liposomes, being 34.5% for F1. The sonicated F3 presented better stability when stored for 60 days at 4°C. The liposomes showed the ability to release the peptide for the total time of 96 h, with the first peak after 5 h, and a further increase of the released after 30 h. Time-kill assay showed that the liposomes were able to control yeast growth for 72 h. The data suggest that the liposomes loaded with 0WHistatin 5 maintained the action of the peptide and were able to limit the growth of C. albicans, being a suitable system for use in the treatment of oral candidiasis.

Research paper thumbnail of Addendum: Bassan, J.C.; et al. Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein. Materials 2016, 9(5), 357

Research paper thumbnail of Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein

Materials, 2016

In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic... more In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic support (corn cob powder-CCP) with the goal of obtaining peptides with bioactive potential from cheese whey. The pretreated support was activated with glyoxyl groups, glutaraldehyde and IDA-glyoxyl. The immobilization yields of the derivatives were higher than 83%, and the retention of catalytic activity was higher than 74%. The trypsin-glyoxyl-CCP derivative was thermally stable at 65˝C, a value that was 1090-fold higher than that obtained with the free enzyme. The trypsin-IDA-glyoxyl-CCP and trypsin-glutaraldehyde-CCP derivatives had thermal stabilities that were 883-and five-fold higher, respectively, then those obtained with the free enzyme. In the batch experiments, trypsin-IDA-glyoxyl-CCP retained 91% of its activity and had a degree of hydrolysis of 12.49%, while the values for trypsin-glyoxyl-CCP were 87% and 15.46%, respectively. The stabilized derivative trypsin-glyoxyl-CCP was also tested in an upflow packed-bed reactor. The hydrodynamic characterization of this reactor was a plug flow pattern, and the kinetics of this system provided a relative activity of 3.04˘0.01 U¨g´1 and an average degree of hydrolysis of 23%, which were suitable for the production of potentially bioactive peptides.

Research paper thumbnail of Cationic Pd(II) complexes acting as topoisomerase II inhibitors: Synthesis, characterization, DNA interaction and cytotoxicity

Journal of Inorganic Biochemistry, 2016

Research paper thumbnail of Toxin-antitoxin systems and its biotechnological applications

African Journal of Biotechnology, 2014

Toxin-antitoxin (TA) systems are important genetic modules composed by two elements: a toxin, tha... more Toxin-antitoxin (TA) systems are important genetic modules composed by two elements: a toxin, that is always a protein, and an antitoxin, that can be a RNA or a protein and neutralizes the toxic effect of toxin. These systems are widespread in bacteria and archaea, found on plasmids and chromosomes. According to the nature of the antitoxin and its mode of interaction with the toxin, TA systems are grouped into five types. In general, the antitoxin is less stable than the toxin and is rapidly degraded in special conditions, leaving the toxin free to act on its cellular targets. TA modules are important in several events in cell physiology such as plasmid maintenance, formation of persister cells, stress resistance, protection from bacteriophages and regulation of biofilm formation, acting on crucial cellular processes including translation, replication, cytoskeleton formation and membrane integrity. TA systems components have proven to be very useful in biotechnology, being used to enhance cloning selection and protein expression in living bacterial cells. Furthermore, they are also considered as promising targets for the development of antibacterial drugs and can be used in gene therapy. Here, we reported current aspects and the application of TA modules in biotechnology research.

Research paper thumbnail of DNA binding, topoisomerase inhibition and cytotoxicity of palladium(II) complexes with 1,10-phenanthroline and thioureas

Inorganica Chimica Acta, 2016

Metallointercalators represent a promising alternative in cancer chemotherapy. We present herein ... more Metallointercalators represent a promising alternative in cancer chemotherapy. We present herein DNA binding, topoisomerase inhibition and cytotoxic studies on a series of complexes of general formulae [Pd (phen)(tu ⁄) 2 ] 2+ incorporating the intercalator 1,10-phenanthroline and thiourea ligands (L = thiourea 1, N-methylthiourea 2, N,N 0-dimethylthiourea 3). DNA-unwinding results showed that the complexes can induce the unwinding of the plasmid DNA. The binding constants (K b) for the interaction of the complexes with SS-DNA were determined by UV spectroscopy. Competitive experiments with ethidium bromide (EB) were investigated by fluorescence spectroscopy and show that all the complexes were able to displace EB from the DNA-EB complex. The results suggest that they may interact with DNA by intercalation. Compounds were tested against human oral carcinoma cell line (KB), human breast cancer cell line (MCF7) and cisplatin-resistant human breast cancer cell line (MCF7-R) and showed good cytotoxic activity towards MCF7-R. Compounds 2 and 3 were also able to cause topo II inhibition.

Research paper thumbnail of Peptide KSL-W-Loaded Mucoadhesive Liquid Crystalline Vehicle as an Alternative Treatment for Multispecies Oral Biofilm

Molecules, 2015

Decapeptide KSL-W shows antibacterial activities and can be used in the oral cavity, however, it ... more Decapeptide KSL-W shows antibacterial activities and can be used in the oral cavity, however, it is easily degraded in aqueous solution and eliminated. Therefore, we aimed to develop liquid crystalline systems (F1 and F2) for KSL-W buccal administration to treat multispecies oral biofilms. The systems were prepared with oleic acid, polyoxypropylene (5) polyoxyethylene (20) cetyl alcohol (PPG-5-CETETH-20), and a 1% poloxamer 407 dispersion as the oil phase (OP), surfactant (S), and aqueous phase (AP), respectively. We characterized them using polarized light microscopy (PLM), small-angle X-ray scattering (SAXS), rheology, and in vitro bioadhesion, and performed in vitro biological analysis. PLM showed isotropy (F1) or anisotropy with lamellar mesophases (F2), confirmed by peak ratio quantification using SAXS. Rheological tests demonstrated that F1 exhibited Newtonian behavior but not F2, which showed a structured AP concentration-dependent system. Bioadhesion studies revealed an AP concentration-dependent increase in the system's bioadhesiveness (F2 = 15.50˘1.00 mN¨s) to bovine teeth blocks. Antimicrobial testing revealed 100% inhibition of multispecies oral biofilm growth after KSL-W administration, which was incorporated in the F2 aqueous phase at a concentration of 1 mg/mL. Our results suggest that this system could serve as a potential vehicle for buccal administration of antibiofilm peptides.

Research paper thumbnail of Hidrólise enzimática de proteínas lácteas para obtenção de peptídeos bioativos utilizando alcalase comercial

Anais do Simpósio Nacional de Bioprocessos, 2015

Research paper thumbnail of In Vitro Identification of Histatin 5 Salivary Complexes

PloS one, 2015

With recent progress in the analysis of the salivary proteome, the number of salivary proteins id... more With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein-protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed be...

Research paper thumbnail of Buffalo Cheese Whey Proteins, Identification of a 24 kDa Protein and Characterization of Their Hydrolysates: In Vitro Gastrointestinal Digestion

PLOS ONE, 2015

Milk whey proteins are well known for their high biological value and versatile functional proper... more Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase-A. When the TNBS method was used the obtained hydrolysates presented DH of 55 and 62% for H1 and H2, respectively. Otherwise for the OPA method the DH was 27 and 43% for H1 and H2, respectively. The total antioxidant activities of the H1 and H2 samples with and without previous enzymatic hydrolysis, determined by DPPH using diphenyl-p-picrylhydrazyl radical, was 4.9 and 12 mM of Trolox equivalents (TE) for H2 and H2Dint, respectively. The increased concentrations for H1 and H2 samples were approximately 99% and 75%, respectively. The in vitro gastrointestinal digestion efficiency for the samples that were first hydrolyzed was higher compared with samples not submitted to previous hydrolysis. After in vitro gastrointestinal digestion, several amino acids were released in higher concentrations, and most of which were essential amino acids. These results suggest that buffalo cheese whey is a better source of bioavailable amino acids than bovine cheese whey.

Research paper thumbnail of Histatin 5 inhibits adhesion of C. albicans to Reconstructed Human Oral Epithelium

Frontiers in Microbiology, 2015

Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surf... more Candida albicans is the most pathogenic fungal species, commonly colonizing on human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. The formation of a saliva-derived protein pellicle on the mucosa surface can provide protection against C. albicans on oral epithelial cells, and narrow information is available on the mucosal pellicle composition. Histatins are one of the most abundant salivary proteins and presents antifungal and antibacterial activities against many species of the oral microbiota, however, its presence has never been studied in oral mucosa pellicle. The objective of this study was to evaluate the potential of histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37 • C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37 • C at 5% CO 2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. HOET pre-incubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization on epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis.