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1.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669133

RESUMO

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Menispermaceae/química , Extratos Vegetais/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Glucose/biossíntese , Células Hep G2 , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Injeções Intraperitoneais , Masculino , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Estreptozocina/administração & dosagem , Células Tumorais Cultivadas , Água/química
2.
ACS Appl Mater Interfaces ; 13(7): 8082-8094, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33570927

RESUMO

Antibiotic-resistant bacteria are a significant and growing threat to human health. Recently, two-dimensional (2D) nanomaterials have shown antimicrobial activity and have the potential to be used as new approaches to treating antibiotic resistant bacteria. In this Research Article, we exfoliate transition metal dichalcogenide (TMDC) nanosheets using synthetic single-stranded DNA (ssDNA) sequences, and demonstrate the broad-spectrum antibacterial activity of MoSe2 encapsulated by the T20 ssDNA sequence in eliminating several multidrug-resistant (MDR) bacteria. The MoSe2/T20 is able to eradicate Gram-positive Escherichia coli and Gram-positive Staphylococcus aureus at much lower concentrations than graphene-based nanomaterials. Eradication of MDR strains of methicillin-resistant S. aureus (MRSA), Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii are shown to occur at at 75 µg mL-1 concentration of MoSe2/T20, and E. coli at 150 µg mL-1. Molecular dynamics simulations show that the thymine bases in the T20 sequence lie flat on the MoSe2 surface and can, thus, form a very good conformal coating and allow the MoSe2 to act as a sharp nanoknife. Electron microscopy shows the MoSe2 nanosheets cutting through the cell membranes, resulting in significant cellular damage and the formation of interior voids. Further assays show the change in membrane potential and reactive oxygen species (ROS) formation as mechanisms of antimicrobial activity of MoSe2/T20. The cellular death pathways are also examined by mRNA expression. This work shows that biocompatible TMDCs, specifically MoSe2/T20, is a potent antimicrobial agent against MDR bacteria and has potential for clinical settings.


Assuntos
Antibacterianos/farmacologia , Calcogênios/farmacologia , DNA de Cadeia Simples/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Metais Pesados/farmacologia , Células A549 , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/química , Cápsulas/química , Cápsulas/farmacologia , Calcogênios/química , DNA de Cadeia Simples/síntese química , Enterococcus faecalis/efeitos dos fármacos , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Metais Pesados/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Pseudomonas aeruginosa/efeitos dos fármacos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Propriedades de Superfície
3.
Food Funct ; 12(5): 2171-2188, 2021 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-33566044

RESUMO

The present study was designed to explore the beneficial mitochondrial effects and anti-oxidative activities of plant sterol ester of α-linolenic acid (PS-ALA) through AMP-activated protein kinase (AMPK) signaling in the treatment of nonalcoholic fatty liver disease (NAFLD) using in vivo and in vitro models. The mitochondrial function was evaluated and the oxidative stress index was measured. The protein expression was analyzed by immunohistochemical, immunofluorescence, and western blotting methods. The results showed that PS-ALA significantly suppressed NAFLD and alleviated steatosis in HepG2 cells induced by oleic acid (OA). In addition, PS-ALA promoted mitochondrial biogenesis, enhanced mitochondrial fatty acid oxidation capacity, improved mitochondrial dynamics, and restored mitochondrial membrane potential. Moreover, PS-ALA reduced reactive oxygen species production both in the liver tissue of HFD-fed mice and OA-loaded HepG2 cells. At the molecular level, PS-ALA accelerated the phosphorylation of AMPK and increased the protein expression of peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) and nuclear NF-E2-related factor 2 (Nrf2). Furthermore, the stimulating effects of PS-ALA on the PGC-1α/Nrf1/Tfam pathway and Nrf2/HO-1 pathway as well as its mitochondrial biogenesis promotion effects and anti-oxidative activities were abrogated by the AMPK inhibitor in OA-treated HepG2 cells. In conclusion, the protective effects of PS-ALA on NAFLD appear to be associated with improving mitochondrial function and oxidative stress via activating AMPK signaling.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ésteres/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fitosteróis/uso terapêutico , Ácido alfa-Linoleico/química , Animais , Dieta Hiperlipídica , Ativação Enzimática/efeitos dos fármacos , Ésteres/química , Células Hep G2 , Humanos , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Mitocôndrias Hepáticas/ultraestrutura , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Fosforilação , Fitosteróis/química , Espécies Reativas de Oxigênio/análise , Transdução de Sinais/efeitos dos fármacos
4.
ACS Appl Mater Interfaces ; 13(7): 7977-7986, 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33586952

RESUMO

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as "nanoreactors" for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision are lacking. We report the construction of a light-responsive encapsulin nanoreactor for "on demand" production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-singlet oxygen generator (miniSOG), a biological photosensitizer that is activated by blue light to generate ROS, primarily singlet oxygen (1O2). We established that the nanocompartments stably encased miniSOG and in response to blue light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, we showed that ROS generated by the nanoreactor triggered photosensitized oxidation reactions which exerted a toxic effect on tumor cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROS-driven processes in biomedicine and biotechnology.


Assuntos
Antineoplásicos/farmacologia , Engenharia Biomédica , Corantes Fluorescentes/farmacologia , Luz , Neoplasias Pulmonares/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Células A549 , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Flavoproteínas/química , Flavoproteínas/metabolismo , Corantes Fluorescentes/química , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Nanocompostos/química , Tamanho da Partícula , Fármacos Fotossensibilizantes/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/análise , Oxigênio Singlete/metabolismo , Espectrometria de Fluorescência , Propriedades de Superfície
5.
ACS Appl Mater Interfaces ; 13(3): 4450-4462, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33443399

RESUMO

Biosensors that can automatically and continuously track fluctuations in biomarker levels over time are essential for real-time sensing in biomedical and environmental applications. Although many electrochemical sensors have been developed to quickly and sensitively monitor biomarkers, their sensing stability in complex biofluids is disturbed by unavoidable nonspecific adhesion of proteins or bacteria. Recently, various substrate surface modification techniques have been developed to resist biofouling, yet functionalization of electrodes in sensors to be anti-biofouling is rarely achieved. Here, we report an integrated three-electrode system (ITES) modified with a "liquid-like" polydimethylsiloxane (PDMS) brush that can continuously and stably monitor reactive oxygen species (ROS) in complex fluids. Based on the slippery "liquid-like" coating, the modified ITES surface could prevent the adhesion of various liquids as well as the adhesion of proteins and bacteria. The "liquid-like" coating does not significantly affect the sensitivity of the electrode in detecting ROS, while the sensing performance could remain stable and free of bacterial attack even after 3 days of incubation with bacteria. In addition, the PDMS brush-modified ITES (PMITES) could continuously record ROS levels in bacterial-rich fluids with excellent stability over 24 h due to the reduced bacterial contamination on the electrode surface. This technique offers new opportunities for continuous and real-time monitoring of biomarkers that will facilitate the development of advanced sensors for biomedical and environmental applications.


Assuntos
Incrustação Biológica/prevenção & controle , Técnicas Biossensoriais/instrumentação , Dimetilpolisiloxanos/química , Técnicas Eletroquímicas/instrumentação , Eletrodos , Desenho de Equipamento , Células HeLa , Humanos , Oxirredução , Espécies Reativas de Oxigênio/análise
6.
Biosensors (Basel) ; 11(2)2021 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-33498809

RESUMO

Reactive oxygen species (ROS) have been found in plants, mammals, and natural environmental processes. The presence of ROS in mammals has been linked to the development of severe diseases, such as diabetes, cancer, tumors, and several neurodegenerative conditions. The most common ROS involved in human health are superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH). Organic and inorganic molecules have been integrated with various methods to detect and monitor ROS for understanding the effect of their presence and concentration on diseases caused by oxidative stress. Among several techniques, fluorescence and electrochemical methods have been recently developed and employed for the detection of ROS. This literature review intends to critically discuss the development of these techniques to date, as well as their application for in vitro and in vivo ROS detection regarding free-radical-related diseases. Moreover, important insights into and further steps for using fluorescence and electrochemical methods in the detection of ROS are presented.


Assuntos
Técnicas Eletroquímicas , Espécies Reativas de Oxigênio/análise , Animais , Fluorescência , Humanos , Peróxido de Hidrogênio , Estresse Oxidativo , Superóxidos
7.
Yakugaku Zasshi ; 141(1): 111-124, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33390438

RESUMO

Phototoxicity is a toxic response elicited by topically applied or systemically administered photoreactive chemicals after exposure to light and can be broadly categorized into photoirritation, photoallergy, photogenotoxicity, and photocarcinogenicity. The need in the 21st century for accurate evaluation of photosafety has led to the publication of a number of guidelines from government agencies in Europe and the U.S.A. as well as the Organisation for Economic Co-operation and Development (OECD). In this review, we first discuss the mechanisms of phototoxicity and how they can be evaluated. We then discuss the state of the art and challenges now faced in photosafety evaluation of pharmaceuticals and cosmetics. Additionally, we describe the latest developments in OECD test guidelines (TG) for assessing photosafety, including revisions to the in vitro 3T3 neutral red uptake (NRU) phototoxicity test (TG 432) and the newly adopted reactive oxigen species (ROS) assay (TG 495). We will emphasize the importance of selecting the most appropriate means of evaluation with reference to the latest guidelines and other legal criteria for conducting photosafety evaluation.


Assuntos
Dermatite Fototóxica/diagnóstico , Dermatite Fototóxica/etiologia , Luz/efeitos adversos , Vermelho Neutro/toxicidade , Células 3T3 , Animais , Células Cultivadas , Humanos , Camundongos , Espécies Reativas de Oxigênio/análise , Segurança , Testes de Toxicidade/métodos
8.
Eur J Med Chem ; 213: 113162, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33493826

RESUMO

Based on the previous research results of our research group, to further improve the anti-inflammatory activity of hesperetin, we substituted triazole at the 7-OH branch of hesperetin. We also evaluated the anti-inflammatory activity of 39 new hesperetin derivatives. All compounds showed inhibitory effects on nitric oxide (NO) and inflammatory factors in lipopolysaccharide-induced RAW264.7 cells. Compound d5 showed a strong inhibitory effect on NO (half maximal inhibitory concentration = 2.34 ± 0.7 µM) and tumor necrosis factor-α, interleukin (IL)-1ß, and (IL-6). Structure-activity relationships indicate that 7-O-triazole is buried in a medium-sized hydrophobic cavity that binds to the receptor. Compound d5 can also reduce the reactive oxygen species production and significantly inhibit the expression of inducible NO synthase and cyclooxygenase-2 through the nuclear factor-κB signaling pathway. In vivo results indicate that d5 can reduce liver inflammation in mice with acute liver injury (ALI) induced by CCI4. In conclusion, d5 may be a candidate drug for treating inflammation associated with ALI.


Assuntos
Desenho de Fármacos , Hesperidina/farmacologia , Inflamação/tratamento farmacológico , Fígado/efeitos dos fármacos , Triazóis/farmacologia , Animais , Tetracloreto de Carbono , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Hesperidina/síntese química , Hesperidina/química , Inflamação/metabolismo , Inflamação/patologia , Fígado/lesões , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Células RAW 264.7 , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
9.
J Mater Chem B ; 9(4): 1009-1017, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33427275

RESUMO

The design and synthesis of near-infrared (NIR) emissive fluorophores for the imaging of organelles and photodynamic therapy have received enormous attention. Hence, the development of NIR emissive fluorophores for high-fidelity lysosome targeting, two-photon fluorescence imaging, and the inducing of photo-triggered cancer-cell apoptosis is highly desirable. In this study, a novel lysosome-targeting two-photon fluorescent photosensitizer (TTRh-CN) is prepared and comprehensively investigated. TTRh-CN demonstrates near-infrared (NIR) emission, good biocompatibility, and superior photostability, and it can act as a two-photon fluorescent agent for the clear visualization of living cells and the vascular system within tissue, with deep-tissue penetration abilities. Furthermore, TTRh-CN can efficiently produce ROS in conjunction with lysosomes in situ upon light irradiation, which can damage lysosomes, up-regulate LC3 and Beclin1, increase BAX release, and induce cell apoptosis. The efficacy of TTRh-CN as a photosensitizer is explored in vivo. All these results confirm that TTRh-CN can serve as a potential platform for the two-photon fluorescence imaging of cells/tissue and for organelle-specific photodynamic therapy.


Assuntos
Antineoplásicos/farmacologia , Materiais Biocompatíveis/farmacologia , Imagem Óptica , Fotoquimioterapia , Fótons , Fármacos Fotossensibilizantes/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Teoria da Densidade Funcional , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Raios Infravermelhos , Lisossomos/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Estrutura Molecular , Tamanho da Partícula , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/química , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Propriedades de Superfície , Células Tumorais Cultivadas
10.
Chemosphere ; 262: 128305, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33182158

RESUMO

The fine particulate matter (PM2.5) was collected at academic campus of Indian Institute of Technology, Delhi, India from January-December 2017. The PM2.5 samples were analysed for carcinogenic (Cd, Cr, As, Ni, and Pb) and non-carcinogenic (V, Cu, Zn, Fe) trace metals and their elicited effects on carcinoma epithelial cell line A549. Toxicological testing was done with ELISA kit. Same analyses were repeated for standard reference material (NIST-1648a) represents urban particulate matter. The student-t test and spearman correlation were used for data analysis. The seasonality in PM2.5 mass concentration and chemical composition showed effect on biological outcomes. The PM2.5 in post-monsoon and winter had higher amount of trace metals compared to mass collected in pre-monsoon and monsoon. Following the trend in PM mass concentration significantly (p < 0.5) lower cell viability was observed in post-monsoon and winter compared to other two seasons. NIST UPM 1648(a) samples always had higher cytotoxicity compared to ambient PM2.5 Delhi sample. Strong association of Chromium, Nickel, Cadmium, and Zinc was observed with cell viability and reactive oxygen species (ROS) production. In winter IL-6, IL-8 production were 2.8 and 3 times higher than values observed in post-monsoon and 53 and 9 times higher than control. In winter season trace metals As, Cu, Fe, in pre-monsoon Cr, Ni, As, Pb, V, and Fe, in post-monsoon Cd and V strongly correlated with ROS generation. ROS production in winter and pre-monsoon seasons found to be 2.6 and 1.3 times higher than extremely polluted post-monsoon season which had 2 to 3 times higher PM2.5 concentration compared to winter and pre-monsoon. The result clearly indicated that the presence of Fe in winter and pre-monsoon seasons catalysed the ROS production, probably OH˙ radical caused high cytokines production which influenced the cell viability reduction, while in post-monsoon PM majorly composed of Pb, As, Fe and Cu and affected by photochemical smog formation showed significant association between ROS production with cell viability. Overall, in Delhi most toxic seasons for respiratory system are winter and post-monsoon and safest season is monsoon.


Assuntos
Poluentes Atmosféricos/toxicidade , Monitoramento Ambiental/métodos , Células Epiteliais/efeitos dos fármacos , Metais Pesados/toxicidade , Material Particulado/toxicidade , Espécies Reativas de Oxigênio/análise , Células A549 , Poluentes Atmosféricos/análise , Sobrevivência Celular/efeitos dos fármacos , Citocinas/análise , Células Epiteliais/imunologia , Células Epiteliais/patologia , Humanos , Índia , Metais Pesados/análise , Material Particulado/análise , Estações do Ano
11.
Methods Mol Biol ; 2202: 215-229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857358

RESUMO

Photodynamic therapy employs nontoxic dyes called photosensitizers (PS) that are excited by visible light of the correct wavelength to produce a variety of reactive oxygen species (ROS) by an interaction between the long-lived PS triplet states with ambient oxygen. The most important type of ROS in photodynamic therapy (PDT) is singlet oxygen, which is produced by a Type II energy transfer process. On the other hand, superoxide, hydrogen peroxide, and hydroxyl radicals can be produced by a Type I electron transfer process. This chapter describes a set of fluorescent probes that can be used to tease apart these different ROS produced when various PS are illuminated in solution. Singlet oxygen sensor green (SOSG) is used for singlet oxygen, 4-hydroxyphenyl-fluorescein (HPF) for hydroxyl radicals, Amplex Red for hydrogen peroxide, and nitroblue-tetrazolium or XTT for superoxide.


Assuntos
Corantes Fluorescentes/química , Fotoquimioterapia/métodos , Espécies Reativas de Oxigênio/análise , Transporte de Elétrons , Transferência de Energia , Luz , Fármacos Fotossensibilizantes , Espécies Reativas de Oxigênio/metabolismo
12.
Methods Mol Biol ; 2202: 43-50, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857344

RESUMO

Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.


Assuntos
Hipóxia Celular/fisiologia , Microscopia de Fluorescência/métodos , Superóxidos/análise , Animais , Linhagem Celular , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/metabolismo , Humanos , Hipóxia/metabolismo , Microscopia/métodos , Mitocôndrias/metabolismo , Oxirredução , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo
13.
Methods Mol Biol ; 2202: 81-91, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857348

RESUMO

The budding yeast Saccharomyces cerevisiae is a facultative organism that is able to utilize both anaerobic and aerobic metabolism, depending on the composition of carbon source in the growth medium. When glucose is abundant, yeast catabolizes it to ethanol and other by-products by anaerobic fermentation through the glycolysis pathway. Following glucose exhaustion, cells switch to oxygenic respiration (a.k.a. "diauxic shift"), which allows catabolizing ethanol and the other carbon compounds via the TCA cycle and oxidative phosphorylation in the mitochondria. The diauxic shift is accompanied by elevated reactive oxygen species (ROS) levels and is characterized by activation of ROS defense mechanisms. Traditional measurement of the diauxic shift is done through measuring optical density of cultures grown in a batch at intermediate time points and generating a typical growth curve or by estimating the reduction of glucose and accumulation of ethanol in growth media over time. In this manuscript, we describe a method for determining changes in ROS levels upon yeast growth, using carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA). H2-DCFDA is a widely used fluorescent dye for measuring intracellular ROS levels. H2-DCFDA enables a direct measurement of ROS in yeast cells at intermediate time points. The outcome of H2-DCFDA fluorescent readout measurements correlates with the growth curve information, hence providing a clear understanding of the diauxic shift.


Assuntos
Microscopia de Fluorescência/métodos , Espécies Reativas de Oxigênio/análise , Carbono/metabolismo , Respiração Celular/fisiologia , Etanol/metabolismo , Fermentação , Fluorescência , Regulação Fúngica da Expressão Gênica/genética , Glucose/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Methods Mol Biol ; 2202: 93-102, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857349

RESUMO

Reactive oxygen species (ROS) could have a negative impact on sperm cellular function and viability. This chapter describes a protocol for oxidative stress evaluation using dichlorofluorescein (DCF) which can specifically reveal intracellular reactive oxygen species. The protocol described here has been used in human and teleost species sperm samples. The method can be used with two approaches: (1) flow cytometry, for quantification of DCF+ cells, or (2) confocal microscopy, for the localization of ROS within the cells.


Assuntos
Microscopia Confocal/métodos , Espécies Reativas de Oxigênio/análise , Espermatozoides/metabolismo , Animais , Peixes , Citometria de Fluxo/métodos , Fluoresceínas/química , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/fisiologia
15.
Methods Mol Biol ; 2202: 103-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857350

RESUMO

Reactive oxygen species (ROS) are continuously produced in semen and are essential for important spermatozoa functions that allow fertilization. However, an excessive amount of ROS is associated with poor sperm quality, which can compromise male fertility potential. This chemiluminescence assay is based on the production of light through the reaction between luminol and ROS. The emitted light is converted to an electrical signal by a luminometer, and the ROS levels in the sample are calculated as relative light units (RLU) per second per million spermatozoa per milliliter (RLU/s/million sperm/mL).


Assuntos
Medições Luminescentes/métodos , Espécies Reativas de Oxigênio/análise , Sêmen/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Luminescência , Luminol/metabolismo , Masculino , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Sêmen/fisiologia , Espermatozoides/metabolismo
16.
Methods Mol Biol ; 2202: 111-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857351

RESUMO

Different experimental conditions can be used to detect the presence of reactive oxygen species (ROS) in the photodynamic inactivation of microorganisms. Here, we describe the effect of the media and the addition of ROS scavengers to obtain insight about the oxidative processes that take place during the photokilling of bacteria. In addition, 9,10-dimethylanthracene was used to sense the generation of singlet molecular oxygen, O2(1Δg), in microbial cells. Thus, the contribution of type I or type II pathways in the photocytotoxicity action can be rapidly detected and compared between different photosensitizers.


Assuntos
Espécies Reativas de Oxigênio/análise , Oxigênio Singlete/análise , Oxigênio Singlete/metabolismo , Antracenos/química , Bactérias/metabolismo , Meios de Cultura/química , Luz , Oxirredução/efeitos dos fármacos , Oxigênio/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
Methods Mol Biol ; 2202: 125-135, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857352

RESUMO

Reactive oxygen species (ROS) production within biofilms is studied with a simple and easy setup based on fluorescence microscopy. Herein, a biofilm is exposed to different ROS inducers: a bactericidal antibiotic (ciprofloxacin) and a BODIPY-based photosensitizer (I2B-OAc). Real-time ROS induction in the core of the biofilms is monitored utilizing two fluorescent reporters-AMDA and H2DCFDA-the first one with selectivity toward singlet oxygen (1O2) and the latest for other ROS (O2•-, H2O2, and OH•-). A point-by-point methodology is reported, starting with the sample preparation all the way through the microscope setup and, finally, processing of the images.


Assuntos
Microscopia de Fluorescência/métodos , Espécies Reativas de Oxigênio/análise , Oxigênio Singlete/análise , Antibacterianos/farmacologia , Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Compostos de Boro/farmacologia , Ciprofloxacino/farmacologia , Peróxido de Hidrogênio , Oxirredução/efeitos dos fármacos , Oxigênio/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/metabolismo
18.
Methods Mol Biol ; 2202: 137-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857353

RESUMO

Electron paramagnetic resonance (EPR) spectroscopy is an established method for the measurement of free radicals. Solar radiation is essential for human life as it stimulates vitamin D synthesis and well-being. However, an excessive dose of solar radiation leads to the formation of free radicals. Here, we describe an EPR method for measuring the amount of radicals induced by UVA irradiation in excised skin. For the first time, a wavelength stable UVA LED (365 nm) was used. The method allows the quantitative determination of radicals in skin before, during, and after UVA irradiation. A dose-dependent radical production could be demonstrated, independent of the yielded power.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Espécies Reativas de Oxigênio/análise , Pele/metabolismo , Óxidos N-Cíclicos/química , Radicais Livres/química , Humanos , Marcadores de Spin , Raios Ultravioleta
19.
Methods Mol Biol ; 2202: 189-197, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857356

RESUMO

The most common way to demonstrate the reactive oxygen species (ROS)-mediated pathways in photodynamic therapy (PDT) and in sonodynamic therapy (SDT) is the use of specific ROS inhibitors. We present a general method to establish the relative efficiency of different sonosensitizers which produce the same ROS. To demonstrate it, we use peroxides as sonosensitizers which produce singlet molecular oxygen. The method is easily generalized by all types of ROS.


Assuntos
Estimulação Acústica/métodos , Fotoquimioterapia/métodos , Espécies Reativas de Oxigênio/análise , Apoptose , Humanos , Oxigênio Singlete/análise , Oxigênio Singlete/metabolismo , Som
20.
Methods Mol Biol ; 2202: 199-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857357

RESUMO

The assessment of reactive oxygen species has increasing importance in biomedical sciences, due to their biological role in signaling pathways and induction of cell damage at low and high concentrations, respectively. Detection of lipid peroxidation with sensing probes such as some BODIPY dyes has now wide application in studies using fluorescent microplate readers, flow cytometry, and fluorescence microscopy. Two phenylbutadiene derivatives of BODIPY are commonly used as peroxidation probes, non-oxidized probes and oxidized products giving red and green fluorescence, respectively. Peculiar features of lipoperoxidation and BODIPY dye properties make this assessment a rather complex process, not exempt of doubts and troubles. Color changes and fluorescence fading that are not due to lipid peroxidation must be taken into account to avoid misleading results. As a characteristic feature of lipoperoxidation is the propagation of peroxyl radicals, pitfalls and advantages of a delayed detection by BODIPY probes should be considered.


Assuntos
Peroxidação de Lipídeos/fisiologia , Microscopia de Fluorescência/métodos , Espécies Reativas de Oxigênio/metabolismo , Compostos de Boro/farmacologia , Butadienos/farmacologia , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Peróxidos/análise , Peróxidos/metabolismo , Espécies Reativas de Oxigênio/análise , Espectrometria de Fluorescência/métodos
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