RESUMO
The present study addressed the protective effects against oxidative stress (OS) of a cocoa powder extract (CPEX) on the protein expression profile of S. cerevisiae. A proteomic analysis was performed after culture preincubation with CPEX either without stress (-OS) or under stress conditions (+OS) (5 mM of H2O2). LC-MS/MS identified 33 differentially expressed proteins (-OS: 14, +OS: 19) that were included By Gene Ontology analysis in biological processes: biosynthesis of amino acids, carbohydrate metabolism and reactive oxygen species metabolic process. In a gene-knockout strains study, eight proteins were identified as putative candidates for being involved in the protective mechanism of cocoa polyphenols against OS induced by H2O2. CPEX was able to exert its antioxidant activity in yeast mainly through the regulation of: (a) amino acids metabolism proteins by modulating the production of molecules with known antioxidant roles; (b) stress-responsive protein Yhb1, but we were unable to fully understand its down-regulation; (c) protein Prb1, which can act by clipping Histone H3 N-terminal tails that are related to cellular resistance to DNA damaging agents.
Assuntos
Cacau/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ontologia Genética , Mutação/genética , Substâncias Protetoras/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Pulsed electric fields (PEF) technology has been used as a sustainable method for extracting antioxidant bioactive compounds from different food matrices. In the present study, the optimal conditions of PEF extraction for mushrooms (2.5 kV/cm, 50 kJ/kg, 6 h) were applied to Lentinula edodes, Agaricus brunnescens, and Pleurotus ostreatus to evaluate the total antioxidant capacity of the extracts, followed by the Triple TOF-LC-MS-MS analysis of the phenolic profile compared to A. bisporus by high-performance liquid chromatography coupled to mass spectrophotometry. In addition, the microporation effect of the technology on the mushroom surface was evaluated using scanning electron microscopy. A comparison was made with a maceration extraction (aqueous stirring for 6 h). The results showed that PEF-assisted extraction enhanced the recovery of antioxidant compounds such as 3,5-dicaffeoylquinic and cinnamic acid with contents up to 236.85 µg/100 g dry weight and 2043.26 µg/100 g dry weight from A. bisporus, respectively. However, mixed results were obtained for certain phenolic compounds, including vanillic acid from L. edodes, ellagic acid from P. ostreatus, and thymol from all mushrooms. These results indicate that the application of PEF technology is effective for the extraction of antioxidant compounds in fungal matrices by creating micropores in cell membranes that allow great recovery in matrices with high content of bioactive compounds.
RESUMO
Saccharomyces cerevisiae has been used as a model organism to study the capacity of cocoa and red grape extracts to trigger an antioxidant response. A methodology adapted to microtiter plates has been developed to monitor yeast growth after culture preincubation with food ingredients and exposure to oxidative stress by hydrogen peroxide and menadione. This methodology proved effective in measuring the ability of cocoa and red grape extracts to promote an antioxidant response in yeast, and also the prospect of conducting dose-response studies. Additionally, the method has proven useful to perform studies with mutant strains lacking genes that may be related to the mechanism of action underlying the antioxidant properties. Thus, in a single assay, it is possible to elucidate the sensitivity of strains to oxidative stress, the ability of an ingredient to promote an antioxidant response, and the possible implication of certain genes. Results of assays using strain hst3Δ showed that the antioxidant protection provided by exposure to cocoa and red grape extracts was not present in the strain lacking gene HST3 when H2 O2 and menadione were used as oxidizing agents. This effect was previously reported for cocoa extract only, with H2 O2 as stressor. Moreover, the results showed that the mutant strain hst3Δ is more resistant to menadione and H2 O2 in the absence of preincubation with cocoa and red grape extract, hinting at the possible implication of sirtuin Hst3 in the antioxidant cellular response.
Assuntos
Antioxidantes/farmacologia , Cacau/química , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Vitis/química , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: To compare the application of non-invasive ventilation (NIV) versus continuous positive airway pressure (CPAP) in the treatment of patients with cardiogenic pulmonary edema (CPE) admitted to an intensive care unit (ICU). METHODS: In a prospective, randomized, controlled study performed in an ICU, patients with CPE were assigned to NIV (n=56) or CPAP (n=54). Primary outcome was intubation rate. Secondary outcomes included duration of ventilation, length of ICU and hospital stay, improvement of gas exchange, complications, ICU and hospital mortality, and 28-day mortality. The outcomes were analyzed in hypercapnic patients (PaCO2>45mmHg) with no underlying chronic lung disease. RESULTS: Both devices led to similar clinical and gas exchange improvement; however, in the first 60min of treatment a higher PaO2/FiO2 ratio was observed in the NIV group (205±112 in NIV vs. 150±84 in CPAP, P=.02). The rate of intubation was similar in both groups (9% in NIV vs. 9% in CPAP, P=1.0). There were no differences in duration of ventilation, ICU and length of hospital stay. There were no significant differences in ICU, hospital and 28-d mortality between groups. In the hypercapnic group, there were no differences between NIV and CPAP. CONCLUSIONS: Either NIV or CPAP are recommended in patients with CPE in the ICU. Outcomes in the hypercapnic group with no chronic lung disease were similar using NIV or CPAP.
Assuntos
Pressão Positiva Contínua nas Vias Aéreas , Unidades de Terapia Intensiva , Ventilação não Invasiva , Edema Pulmonar/terapia , Idoso , Idoso de 80 Anos ou mais , Dióxido de Carbono/sangue , Feminino , Mortalidade Hospitalar , Humanos , Hipercapnia/etiologia , Intubação Intratraqueal/estatística & dados numéricos , Estimativa de Kaplan-Meier , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Oxigênio/sangue , Pressão Parcial , Estudos Prospectivos , Edema Pulmonar/sangue , Edema Pulmonar/complicações , Troca Gasosa Pulmonar , Resultado do TratamentoRESUMO
The involvement of Toll-like receptor 2 (TLR2) and TLR4 in triggering signal transduction pathways leading to prostaglandin E(2) (PGE(2)) production in response to Candida albicans has been studied in cells from wild-type, TLR2-/- and TLR4-/- knockout mice. In vitro PGE(2) production by macrophages challenged with zymosan, yeast or hypha cells was strongly inhibited in TLR2-deficient cells, but not in TLR4-/- cells, as compared to macrophages from wild-type mice. PGE(2) production was dependent on de novo cyclooxygenase-2 (Cox2) synthesis, since unchallenged cells failed to produce PGE(2) and specific Cox2 inhibition during challenge totally blocked PGE(2) production. Similar results were obtained following in vitro challenge of splenocytes from mice intravenously infected with the low-virulent C. albicans PCA2 strain. This indicates that TLR2 is the major receptor that mediates PGE(2) production in response to C. albicans, probably by upregulating Cox2 expression.
Assuntos
Candida albicans/imunologia , Dinoprostona/biossíntese , Macrófagos Peritoneais/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Células Cultivadas , Ciclo-Oxigenase 2 , Feminino , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Knockout , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like , Receptor 4 Toll-LikeAssuntos
Anemia Hemolítica Autoimune , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Betacoronavirus , COVID-19 , Criança , Humanos , SARS-CoV-2RESUMO
In this work, we studied the role of toll-like receptor-2 (TLR2) in murine defenses against Candida albicans. TLR2-deficient mice experimentally infected intraperitoneally (i.p.) or intravenously (i.v.) in vivo had very significant impaired survival compared with that of control mice. In vitro production of TNF-alpha and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2-/- mice in response to yeasts and hyphae of C. albicans were significantly lower (80% and 40%, respectively; P <0.05) than production by macrophages from wild-type mice. This impaired production of TNF-alpha and MIP-2 probably contributed to the 41% decreased recruitment of neutrophils to the peritoneal cavity of i.p. infected TLR2-/- mice. In contrast, in vitro phagocytosis of yeasts and production of reactive oxygen intermediates (ROI) were not affected in macrophages from TLR2-/- animals. Our data indicate that TLR2 plays a major role in the response of macrophages to C. albicans, triggering cytokine and chemokine expression, and it is essential for in vivo protection against infection.
Assuntos
Candida albicans/patogenicidade , Candidíase/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Candida albicans/imunologia , Contagem de Células , Células Cultivadas , Quimiocina CXCL2 , Quimiocinas/biossíntese , Modelos Animais de Doenças , Citometria de Fluxo , Hifas/imunologia , Imunidade Inata , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptor 2 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Previous work by our group showed that Toll-like receptor 2 (TLR2) is essential for activation of innate immunity, playing a major role in the response of macrophages to Candida albicans, triggering cytokine and chemokine expression, and therefore TLR2 -/- mice are more susceptible to systemic primary candidiasis. In this work, we used a murine model of systemic C. albicans infection, in which resistance to reinfection with virulent wild-type cells is induced by prior exposure of mice to a low-virulence agerminative strain of C. albicans (primary sublethal infection), to study the influence of TLR2 gene deletion on (i) the ability to develop an acquired resistance upon vaccination; (ii) the development of the acquired humoral response; and (iii) the production of Th1 cytokines IFN-gamma, IL-12 and TNF-alpha. Our results indicate that, although TLR2 -/- mice have a very impaired production of Th1 cytokines compared with control mice, they are equally capable of mounting a specific humoral response to the fungus and developing a vaccine-induced resistance.
Assuntos
Anticorpos Antifúngicos/sangue , Candida albicans/imunologia , Candidíase/imunologia , Citocinas/biossíntese , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Imunidade Inata , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interleucina-12/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 2 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The fungal species Candida albicans is an opportunistic pathogen, which causes serious infections in humans, particularly in immunocompromised patients. Depending on the underlying host defect, C. albicans causes a variety of infections, ranging from superficial mucocutaneous candidiasis to life-threatening disseminated infections. Both the limited spectrum of antifungal drugs currently in clinical use and the emergence of resistances make necessary the development of new effective antifungal drugs with minimal side effects; however, such a research is limited by the small number of specific target sites identified to date. The cell wall is a fungal specific dynamic structure essential to almost every aspect of the biology and pathogenicity of C. albicans. Its structure confers physical protection and shape to fungal cells, and as the most external part of the fungus, the cell wall mediates the interaction with the host, including adhesion to host tissues and modulation of the host anti-Candida immune response. Consequently, the fungal cell wall can be considered as a suitable target for development of new antifungal compounds. Therefore two distinct types of potential cell wall-related targets can be envisaged, according to their mode of action in inhibiting infection: (i) inhibition of cell wall biogenesis, which may impair cell wall integrity and thus cell viability, and (ii) modification of host-fungus interactions by inhibiting or blocking putative virulence factors, which may impair host colonization and progress of the infectious process. Antibodies specific to cell wall antigens may protect against infection by a variety of mechanisms and may evolve into save antifungal agents.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candida/metabolismo , Candida/ultraestrutura , Candidíase/imunologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Farmacorresistência Fúngica , Humanos , ImunoterapiaRESUMO
We have studied the role of myeloid differentiation factor 88 (MyD88), the universal Toll-like receptor (TLR) adaptor protein, in murine defenses against Candida albicans. MyD88-deficient mice, experimentally infected in vivo, had a very significant impaired survival, and a higher tissue fungal burden when compared with control mice. The recruitment of neutrophils to the site of infection was also significantly diminished in MyD88-\- mice. In vitro production of proinflammatory cytokines such as TNF-alpha, IFN-gamma and IL-12p70, by antigen-stimulated splenocytes from mice intravenously infected with the low-virulence C. albicans PCA2 strain, could not be detected in MyD88-\- mice. This default of production of Th1 cytokines in MyD88-deficient mice correlated with a greatly diminished frequency of IFN-gamma-producing CD4 + T lymphocytes. Also, the frequency of IFN-gamma-producing CD8 + T lymphocytes was lower in MyD88-\- mice than in control mice. Although C. albicans-specific antibody titers in PCA2-infected mice appeared more quickly in MyD88-\- mice than in control mice, the MyD88-\- group was not able to maintain the Candida-specific IgM nor IgG titers at the third week of infection. The complexity of antigens recognized by sera from MyD88-\- mice was quite similar to that from infected control mice. Taken together, these data show that MyD88-\- mice are extremely susceptible to C. albicans infections, suggesting that MyD88-dependent signaling pathways are essential for both the innate and adaptive immune responses to C. albicans.
Assuntos
Antígenos de Diferenciação/fisiologia , Candida albicans/imunologia , Citocinas/biossíntese , Receptores Imunológicos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Candida albicans/fisiologia , Candidíase/imunologia , Rim/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , Neutrófilos/imunologia , Baço/citologia , Baço/metabolismo , Células Th1/imunologiaRESUMO
Background: To compare the application of non-invasive ventilation (NIV) versus continuous positive airway pressure (CPAP) in the treatment of patients with cardiogenic pulmonary edema (CPE) admitted to an intensive care unit (ICU). Methods: In a prospective, randomized, controlled study performed in an ICU, patients with CPE were assigned to NIV (n=56) or CPAP (n=54). Primary outcome was intubation rate. Secondary outcomes included duration of ventilation, length of ICU and hospital stay, improvement of gas exchange, complications, ICU and hospital mortality, and 28-day mortality. The outcomes were analyzed in hypercapnic patients (PaCO2 > 45 mmHg) with no underlying chronic lung disease. Results: Both devices led to similar clinical and gas exchange improvement; however, in the first 60 min of treatment a higher PaO2/FiO2 ratio was observed in the NIV group (205±112 in NIV vs. 150±84 in CPAP, P=.02). The rate of intubation was similar in both groups (9% in NIV vs. 9% in CPAP, P=1.0). There were no differences in duration of ventilation, ICU and length of hospital stay. There were no significant differences in ICU, hospital and 28-d mortality between groups. In the hypercapnic group, there were no differences between NIV and CPAP. Conclusions: Either NIV or CPAP are recommended in patients with CPE in the ICU. Outcomes in the hypercapnic group with no chronic lung disease were similar using NIV or CPAP
Introducción: Comparar la efectividad de la ventilación no invasiva (VNI) frente a la presión positiva continúa en la vía aérea (CPAP) en pacientes ingresados por edema agudo de pulmón (EAP) cardiogénico en una unidad de cuidados intensivos (UCI). Métodos: Ensayo clínico donde 56 pacientes fueron asignados a VNI y 54 pacientes a CPAP. El objetivo primario fue la tasa de intubación. Los objetivos secundarios fueron: duración de ventilación, estancia en UCI y en el hospital, mejoría gasométrica, complicaciones y mortalidad en UCI, hospitalaria y a los 28 días. Los objetivos fueron analizados en pacientes hipercápnicos (PaCO2 >45mmHg) sin patologia pulmonar. Resultados: Ambos dispositivos obtuvieron similar mejoría clínica y del intercambio gaseoso, sin embargo, la VNI mostró un aumento más rápido de la oxigenación (medido por el cociente PaO2/FiO2) en los primeros 60 minutos de aplicación (205 ± 112 en VNI vs. 150 ± 84 en CPAP, p= 0,02). La tasa de intubación fue similar en ambos grupos (9% en VNI vs. 9% en CPAP, p= 1,0). No hubo diferencias en la duración de la ventilación, ni en la estancia en UCI ni hospitalaria. Tampoco hubo diferencias significativas en la mortalidad en UCI, hospitalaria y a los 28 días entre ambos grupos. En el subgrupo de pacientes hipercápnicos tampoco se observaron diferencias significativas en los objetivos analizados. Conclusiones: La VNI como la CPAP se pueden emplear en pacientes con EAP en la UCI. En pacientes hipercápnicos sin patología pulmonar no se observa beneficio de la VNI sobre la CPAP
Assuntos
Humanos , Edema Pulmonar/terapia , Ventilação não Invasiva/métodos , Respiração com Pressão Positiva/métodos , Insuficiência Respiratória/terapia , Cuidados Críticos/métodos , Estudos Prospectivos , Hipercapnia/terapiaRESUMO
We have constructed a conditional null mutant Candida albicans strain for the UBI3 gene which encodes a ubiquitin fusion protein involved in ribosome biogenesis. A one-step gene disruption procedure, using the plasmid pCaDis, was designed to place the second copy of the UBI3 gene under the control of the tightly regulated MET3 promoter in a C. albicans heterozygous strain (UBI3/Deltaubi3::hisG), previously isolated in the first step of the ura-blaster protocol. Analysis of the conditional null mutant in repressing and inducing conditions indicates that UBI3 is an essential gene whose expression is required for growth of C. albicans.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/genética , Proteínas de Plantas/fisiologia , Proteínas Ribossômicas/fisiologia , Ribossomos/fisiologia , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation of colony morphology variants; and (iii) induction of hyphal and pseudohypal development. These results indicate that polyubiquitin in C. albicans is involved in the negative control of switching, as well as in maintaining the yeast cell morphology, probably by silencing mechanisms triggering the hyphal and pseudohyphal development in the absence of environmental inducers.