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1.
Nat Commun ; 11(1): 3881, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753572

RESUMO

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.


Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais/instrumentação , Cálcio/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Imagem Óptica/instrumentação , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Propanolaminas/farmacologia
2.
Chemosphere ; 260: 127591, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32758773

RESUMO

This study examines the organization and morphology of Bacillus globigii (BG) spores, a common surrogate for Bacillus anthracis, which were seeded and then recovered at various times from several points within a conventional, pilot-scale activated sludge system. Recovered BG spores were enumerated, microscopically examined, and tested for resistance to chemical (i.e. 5% H2O2 for 8 min), thermal (80 °C for 30 min), and ultraviolet light (8 W, 254 nm UV for 1 min) inactivation. Spores exposed to activated sludge germinated, sporulated, and exhibited unique multilayer clustering patterns and statistically significant changes (p < 0.005) in dimensional morphology. Spores collected in the later experimental stages (i.e., during weeks 6 and 7) were significantly more resistant (p ≤ 0.05) to inactivation than those collected on the first day of testing. These results have direct consequences for sludge treatment requirements at wastewater treatment plants that receive spore-containing waste streams.


Assuntos
Bacillus/fisiologia , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Esgotos/microbiologia , Esporos Bacterianos/isolamento & purificação , Raios Ultravioleta , Purificação da Água/métodos , Microscopia de Força Atômica , Microscopia de Contraste de Fase , Projetos Piloto , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/efeitos da radiação , Esporos Bacterianos/ultraestrutura
3.
PLoS Pathog ; 16(8): e1008740, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822429

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation-coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe3+ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress.


Assuntos
Proteínas de Bactérias/genética , Edição de RNA , Xanthomonas/genética , Xanthomonas/metabolismo , Adenosina/genética , Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/farmacologia , Inosina/genética , Inosina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Doenças das Plantas/microbiologia , Virulência/efeitos dos fármacos , Xanthomonas/efeitos dos fármacos , Xanthomonas/patogenicidade
5.
PLoS One ; 15(7): e0236405, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702060

RESUMO

Regulatory small RNAs play an essential role in maintaining cell homeostasis in bacteria in response to environmental stresses such as iron starvation. Prokaryotes generally encode a large number of RNA regulators, yet their identification and characterisation is still in its infancy for most bacterial species. Burkholderia cenocepacia is an opportunistic pathogen with high innate antimicrobial resistance, which can cause the often fatal cepacia syndrome in individuals with cystic fibrosis. In this study we characterise a small RNA which is involved in the response to iron starvation, a condition that pathogenic bacteria are likely to encounter in the host. BrrF is a small RNA highly upregulated in Burkholderia cenocepacia under conditions of iron depletion and with a genome context consistent with Fur regulation. Its computationally predicted targets include iron-containing enzymes of the tricarboxylic acid (TCA) cycle such as aconitase and succinate dehydrogenase, as well as iron-containing enzymes responsible for the oxidative stress response, such as superoxide dismutase and catalase. Phenotypic and gene expression analysis of BrrF deletion and overexpression mutants show that the regulation of these genes is BrrF-dependent. Expression of acnA, fumA, sdhA and sdhC was downregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. TCA cycle genes not predicted as target for BrrF were not affected in the same manner by iron depletion. Likewise, expression of sodB and katB was dowregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. BrrF overexpression reduced aconitase and superoxide dismutase activities and increased sensitivity to hydrogen peroxide. All phenotypes and gene expression changes of the BrrF deletion mutant could be complemented by overexpressing BrrF in trans. Overall, BrrF acts as a regulator of central metabolism and oxidative stress response, possibly as an iron-sparing measure to maintain iron homeostasis under conditions of iron starvation.


Assuntos
Proteínas de Bactérias/genética , Burkholderia cenocepacia/genética , Ferro/metabolismo , Pequeno RNA não Traduzido/genética , Aconitato Hidratase/metabolismo , Burkholderia cenocepacia/metabolismo , Catalase/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Ferro/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/genética
6.
Proc Natl Acad Sci U S A ; 117(28): 16313-16323, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601209

RESUMO

Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.


Assuntos
Peroxirredoxinas/química , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
7.
PLoS One ; 15(7): e0236389, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730276

RESUMO

Streptococcus pneumoniae (Pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Despite recent successes due to pneumococcal vaccination and antibiotic use, Pneumococcus remains a significant medical problem. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase (LPO), thiocyanate anion and hydrogen peroxide (H2O2). LPO oxidizes thiocyanate using H2O2 into the final product hypothiocyanite that has antimicrobial effects against a wide range of microorganisms. However, hypothiocyanite's effect on Pneumococcus has never been studied. Our aim was to determine whether hypothiocyanite can kill S. pneumoniae. Bactericidal activity was measured in a cell-free in vitro system by determining the number of surviving pneumococci via colony forming units on agar plates, while bacteriostatic activity was assessed by measuring optical density of bacteria in liquid cultures. Our results indicate that hypothiocyanite generated by LPO exerted robust killing of both encapsulated and nonencapsulated pneumococcal strains. Killing of S. pneumoniae by a commercially available hypothiocyanite-generating product was even more pronounced than that achieved with laboratory reagents. Catalase, an H2O2 scavenger, inhibited killing of pneumococcal by hypothiocyanite under all circumstances. Furthermore, the presence of the bacterial capsule or lytA-dependent autolysis had no effect on hypothiocyanite-mediated killing of pneumococci. On the contrary, a pneumococcal mutant deficient in pyruvate oxidase (main bacterial H2O2 source) had enhanced susceptibility to hypothiocyanite compared to its wild-type strain. Overall, results shown here indicate that numerous pneumococcal strains are susceptible to LPO-generated hypothiocyanite.


Assuntos
Lactoperoxidase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Tiocianatos/farmacologia , Anti-Infecciosos/farmacologia , Autólise , Cápsulas Bacterianas/efeitos dos fármacos , Catalase/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxirredução , Piruvato Oxidase/deficiência , Piruvato Oxidase/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/crescimento & desenvolvimento
9.
Chemosphere ; 257: 127241, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32526468

RESUMO

The role of endogenous hydrogen sulphide (H2S) in silicon-induced improvement in boron toxicity (BT) tolerance in pepper plants was studied. Two-week old seedlings were subjected to control (0.05 mM B) or 2.0 mM BT in a nutrient solution. These two treatments were combined with 2.0 mM Si. BT caused considerable reduction in biomass, chlorophyll a &b, photosystem II maximum quantum efficiency (Fv/Fm), glutathione and ascorbate in the pepper seedlings. However, it enhanced malondialdehyde (MDA) and hydrogen peroxide, electrolyte leakage, proline, H2S, and activities of catalase, superoxide dismutase, peroxidase, and L-DES. Silicon stimulated growth, proline content and activities of various antioxidant biomolecules and enzymes, leaf Ca2+, K+ and N, endogenous H2S and L-DES activity, but reduced H2O2 and MDA contents, membrane leakage and leaf B. Silicon-induced B tolerance was further enhanced by 0.2 mM NaHS, a H2S donor. A scavenger of H2S, hypotaurine (0.1 mM HT), was supplied together with Si and Si + NaHS to assess the involvement of H2S in Si-induced BT tolerance of pepper plants. Hypotaurine inverted the positive role of Si on the antioxidant defence system by reducing endogenous H2S, but NaHS supply along with Si + HT reversed the negative effects of HT, showing that H2S participated in Si-induced BT tolerance of pepper plants.


Assuntos
Boro/toxicidade , Sulfeto de Hidrogênio/química , Poluentes do Solo/toxicidade , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Catalase/metabolismo , Clorofila A , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Malondialdeído , Estresse Oxidativo/efeitos dos fármacos , Plântula/efeitos dos fármacos , Silício/química , Sulfetos , Superóxido Dismutase/metabolismo
10.
Toxicol Lett ; 332: 27-35, 2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-32585298

RESUMO

Reactive oxygen species (ROS) within the cell are rapidly detoxified by antioxidants such as glutathione. Depletion of glutathione will therefore increase levels of intracellular ROS, which can lead to oxidative DNA damage and the induction of apoptosis. The working hypothesis was that Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) would be more sensitive in response to GSH depletion due to their deficiency in the removal of the oxidative DNA modification, 8-oxo-7,8-dihydroguanine (8-oxoG). Following GSH depletion, an increase in intracellular ROS and a subsequent induction of apoptosis was measured in mOgg1-/- MEFs; as expected. Unexpectedly, an elevated basal level of ROS was identified in mOgg1-/- MEFs compared to wild type MEFs; which we suggest is partly due to the differential expression of key anti-oxidant genes. The elevated basal ROS levels in mOgg1-/- MEFs were not accompanied by a deficiency in ATP production or a large increase in 8-oxoG levels. Although 8-oxoG levels did increase following GSH depletion in mOgg1-/- MEFs; this increase was significantly lower than observed following treatment with a non-toxic dose of hydrogen peroxide. Reconstitution of Ogg1 into mOgg1-/- MEFs resulted in an increased viability following glutathione depletion, however this rescue did not differ between a repair-proficient and a repair-impaired variant of Ogg1. The data indicates that induction of apoptosis in response to oxidative stress in mOgg1-/- MEFs is independent of DNA damage and OGG1-initiated DNA repair.


Assuntos
Apoptose/efeitos dos fármacos , Dano ao DNA , DNA Glicosilases/genética , Fibroblastos/efeitos dos fármacos , Glutationa/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Butionina Sulfoximina/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-32502715

RESUMO

WD40 proteins play crucial roles in response to abiotic stress. By screening the genome sequences of Salvia miltiorrhiza Bunge, 225 SmWD40 genes were identified and divided into 9 subfamilies (I-IX). Physiological, biochemical, gene structure, conserved protein motif and GO annotation analyses were performed on SmWD40 family members. The SmWD40-170 was found in 110 SmWD40 genes that contain drought response elements, SmWD40-170 was one of these genes whose response in terms of expression under drought was significant. The expression of SmWD40-170 was also up-regulated by ABA and H2O2. Through observed the stomatal phenotype of SmWD40-170 transgenic lines, the stomatal closure was abolished under dehydration, ABA and H2O2 treatment in SmWD40-170 knockdown lines. Abscisic acid (ABA), as the key phytohormone, elevates reactive oxygen species (ROS) levels under drought stress. The ABA-ROS interaction mediated the generation of H2O2 and the activation of anion channel in guard cells. The osmolality alteration of guard cells further accelerated the stomatal closure. As a second messenger, nitric oxide (NO) regulated ABA signaling, the NO stimulated protein kinase activity inhibited the K+ influx which result in stomatal closure. These NO-relevant events were essential for ABA-induced stomatal closure. The reduction of NO production was also observed in the guard cells of SmWD40-170 knockdown lines. The abolished of stomatal closure attributed to the SmWD40-170 deficiency induced the reduction of NO content. In general, the SmWD40-170 is a critical drought response gene in SmWD40 gene family and regulates ABA- and H2O2-induced stomatal movement by affecting the synthesis of NO.


Assuntos
Secas , Genes de Plantas , Estômatos de Plantas/fisiologia , Salvia miltiorrhiza/fisiologia , Repetições WD40 , Ácido Abscísico/farmacologia , Peróxido de Hidrogênio/farmacologia , Óxido Nítrico , Potássio , Salvia miltiorrhiza/genética , Estresse Fisiológico
12.
Nucleic Acids Res ; 48(12): 6799-6810, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32484546

RESUMO

Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.


Assuntos
Mitocôndrias/genética , Nitrosação/genética , Estresse Nitrosativo/genética , Treonina-tRNA Ligase/genética , Aminoacil-tRNA Sintetases/genética , Aminoacilação/genética , Animais , Domínio Catalítico/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Cinética , Camundongos , Mitocôndrias/enzimologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Treonina-tRNA Ligase/química
13.
Phytochemistry ; 176: 112414, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32446133

RESUMO

Five undescribed phenolics named pithecellobiumin C-G, along with thirteen known ones were isolated from the twigs and leaves of Archidendron clypearia (Jack) I.C.Nielsen. Their structures were elucidated based on comprehensive spectroscopic analyses, combined with computer-assisted structure elucidation software (ACD/Structure Elucidator) and gauge-independent atomic orbitals (GIAO) NMR chemical shift calculations. The absolute configurations were determined by comparison of experimental and calculated specific rotation and ECD curves. These compounds were tested for their neuroprotective activities against H2O2-induced injury in human neuroblastoma SH-SY5Y cells by MTT assay. Pithecellobiumin C-E exhibited noticeable neuroprotective effect. Further pharmacological study demonstrated that they could prevent cell death through inhibiting the apoptosis induction. Flow cytometry assays also proved that these compounds could attenuate reactive oxygen species (ROS) level and mitochondrial dysfunction in SH-SY5Y cells.


Assuntos
Fabaceae , Fármacos Neuroprotetores/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio
14.
Food Chem ; 327: 127059, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32447138

RESUMO

The aim of this study was to purify and identify antioxidant peptides from watermelon seed protein hydrolysates (WSPHs-I: Mw < 1 kDa) and further evaluate their cytoprotective effects against H2O2-induced oxidative stress in HepG2 cells. After purification by Sephadex G-15 and semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC), five peptides, RDPEER (P1), KELEEK (P2), DAAGRLQE (P3), LDDDGRL (P4), and GFAGDDAPRA (P5) were sequenced by LC-MS/MS and synthesized with solid-phase synthesis method. These peptides showed desirable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity (IC50: 0.216 ± 0.01-0.435 ± 0.03), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity (IC50: 0.54 ± 0.02-1.23 ± 0.03), and oxygen radical absorbance capacity (ORAC) (82.36 ± 1.2-130.67 ± 2.2 µM TE/mg). Among them, peptide P1 exhibited the strongest antioxidant capacity. Moreover, the results suggested that peptide P1 may protect HepG2 cells from H2O2-induced oxidative damage by significantly inhibiting reactive oxygen species (ROS), [Ca2+]i, malondialdehyde (MDA) levels and increasing antioxidative enzyme activities.


Assuntos
Citrullus/química , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/química , Hidrolisados de Proteína/química , Sequência de Aminoácidos , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Cromatografia Líquida de Alta Pressão , Citrullus/metabolismo , Células Hep G2 , Humanos , Oxirredução , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Proteínas de Plantas/metabolismo , Substâncias Protetoras/química , Substâncias Protetoras/isolamento & purificação , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sementes/química , Sementes/metabolismo , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem
15.
Arch Microbiol ; 202(7): 1873-1880, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32448965

RESUMO

The aim is to evaluate the prooxidant and antimicrobial effects of Fe3O4 and TiO2 nanoparticles and thalicarpine by luminescent and standard microbiological assays. Their effect on the kinetics of free-radical oxidation reactions (at pH 7.4 and pH 8.5) is studied in the following model systems, using activated chemiluminescence: chemical, with Fenton's reagent (H2O2-FeSO4)-for the generation of hydroxyl radicals (.OH); chemical, with oxidant hydrogen peroxide (H2O2); chemical (NAD.H-PhMS), for the generation of superoxide radicals (O2.-). Fe3O4 nanoparticles exhibit highly pronounced antioxidant properties; TiO2 nanoparticles exhibit mild to moderate prooxidant properties at neutral and alkaline conditions. Those properties are tested by the chemiluminescent method for the first time. Thalicarpine and its combination with TiO2 nanoparticles exhibit pronounced antioxidant activities at pH 8.5 which are lost and transformed into well-presented prooxidant effects at pH 7.4. That is a result-supported proof on the observed typical properties of thalicarpine and TiO2, namely antibacterial, organic-preserving and anti-pathogenic activities. The antimicrobial effect is tested on Gram-positive and Gram-negative bacteria: two strains of Escherichia coli, Bacillus cereus 1095 and Staphylococcus aureus. All bacteria are destroyed after the application of TiO2, but not Fe3O4 nanoparticles, showing their antibacterial effect. Thalicarpine, in combination with TiO2, showed even synergetic antibacterial effect.


Assuntos
Aporfinas/farmacologia , Bactérias/efeitos dos fármacos , Nanopartículas , Titânio/farmacologia , Anti-Infecciosos/farmacologia , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Ferro/química , Oxidantes/farmacologia , Oxirredução , Espécies Reativas de Oxigênio
16.
PLoS One ; 15(5): e0227522, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374759

RESUMO

Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.


Assuntos
Proliferação de Células/genética , Canais Iônicos/genética , Proteínas de Membrana/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Sistemas CRISPR-Cas/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Camundongos , NADPH Oxidases/genética , RNA Interferente Pequeno/genética , Neoplasias de Mama Triplo Negativas/patologia
17.
Exp Biol Med (Maywood) ; 245(11): 933-939, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32397762

RESUMO

IMPACT STATEMENT: There is a critical shortage of personal protective equipment (PPE) around the globe. This article describes the safe collection, storage, and decontamination of N95 respirators using hydrogen peroxide vapor (HPV). This article is unique because it describes the HPV process in an operating room, and is therefore, a deployable method for many healthcare settings. Results presented here offer creative solutions to the current PPE shortage.


Assuntos
Betacoronavirus/efeitos dos fármacos , Descontaminação/métodos , Peróxido de Hidrogênio/farmacologia , Máscaras/virologia , Dispositivos de Proteção Respiratória/virologia , Infecções por Coronavirus/prevenção & controle , Humanos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle
18.
Food Chem ; 328: 127135, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32473490

RESUMO

Watermelon seed, a watermelon processing industry by-product, is a good protein source for the preparation of antioxidant peptides due to its high protein content, low cost, special amino acid composition. Antioxidant hydrolysates obtained from watermelon seed protein (WSP) after slit divergent ultrasound (SDU) treatment were studied. The stepwise multiple linear regression model verified that the reducing power of watermelon seed protein hydrolysates (WSPHs) is positively related with -SH and ß-turn content of WSP (R2 = 0.931, p < 0.01). Using the degree of hydrolysis (DH) and reducing power as indicators, the WSPHs was prepared under the optimal conditions (ultrasound frequency: 20/28 kHz, time: 60 min, power density: 100 W/L) and divided into three components by ultrafiltration membrane (1 and 5 kDa). Compared with WSPHs and other fractions, WSPHs-I (Mw < 1 kDa) not only significantly protected HepG2 cells from H2O2-induced damage, but also greatly alleviated the liver injury caused by d-galactose in male SD rats.


Assuntos
Antioxidantes/farmacologia , Citrullus/química , Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacologia , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Enzimas/metabolismo , Galactose/toxicidade , Células Hep G2 , Humanos , Peróxido de Hidrogênio/farmacologia , Hidrólise , Modelos Lineares , Masculino , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Ratos Sprague-Dawley , Sementes/química , Ultrassom/instrumentação , Ultrassom/métodos
19.
J Infect Chemother ; 26(8): 831-837, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32414689

RESUMO

Biofilm is a complex structure consisting of microorganisms such as bacteria, fungi and an extracellular matrix (ECM). Biofilms are involved in most microbial infections and show persistent resistance to antibiotic treatment and immune response. Both Aspergillus fumigatus and Streptococcus pneumoniae are colonizers that can form biofilms in the respiratory tract. These pathogens have been simultaneously isolated from the same patient, but their interaction is poorly understood. We observed morphological changes in single- and mixed-species biofilms prepared for confocal laser scanning microscopy and scanning electron microscopy (SEM). Pneumococci suppressed the development of a fungal biofilm, and it even disrupted a preformed fungal biofilm. When a preformed fungal biofilm was treated with pneumococci, the mycelial network was fragmented, and only bacteria could develop. SEM revealed that the fragmented mycelium was further disrupted into fine filaments as treatment time progressed, and that the ECM of the preformed fungal biofilm had disappeared. The pneumococcal culture supernatant contained mycelial fragmentation activity that was heat-sensitive. The culture supernatant of a mutant pneumococcal strain deficient in pneumolysin (Δply) also exhibited the mycelial fragmentation activity. Enolase and lactate oxidase, which are involved in glycolysis and hydrogen peroxide production, were identified in the culture supernatant of the Δply mutant. Neither the wild type nor the mutant strain could fragment the mycelium in the presence of catalase. These data suggest that hydrogen peroxide could fragment the mycelium and would terminate the co-existence of A. fumigatus and S. pneumoniae in biofilm.


Assuntos
Aspergillus fumigatus/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Streptococcus pneumoniae/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Microscopia Eletrônica de Varredura , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/fisiologia , Estreptolisinas/metabolismo
20.
Chem Biol Interact ; 324: 109086, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275923

RESUMO

Oxidative stress-induced apoptosis of retinal ganglion cells (RGCs) contributes to the development and progression of glaucoma. Sestrin2 (Sesn2), a stress-inducible protein, has a potent antioxidant capacity that can provide cytoprotection against various noxious stimuli. However, whether Sesn2 is involved in protecting RGCs from oxidative stress remains unexplored. The purpose of this study was to evaluate the role of Sesn2 in regulating hydrogen peroxide (H2O2)-induced oxidative stress of RGCs. Here, we showed that Sesn2 expression was induced in RGCs following H2O2 exposure. Sesn2 depletion markedly exacerbated H2O2-induced apoptosis and reactive oxygen species (ROS) generation in RGCs. Notably, upregulation of Sesn2 significantly decreased H2O2-induced apoptosis and ROS generation. Moreover, Sesn2 overexpression increased the nuclear translocation of nuclear factor erythroid-derived 2-like 2 (Nrf2), elevated Nrf2/antioxidant response element (ARE)-mediated transcriptional activity and upregulated the expression of Nrf2 target genes in H2O2-stimulated RGCs. Interestingly, we found that Sesn2 promoted Nrf2/ARE activation through downregulation of kelch-like ECH-associated protein 1 (Keap1). Restoration of Keap1 or inhibition of Nrf2 significantly reversed the Sesn2-mediated protective effect in H2O2-stimulated RGCs. In conclusion, these results elucidated that Sesn2 confers a protective effect in RGCs against H2O2-induced oxidative stress by reinforcing Nrf2/ARE activation via downregulation of Keap1. Our study suggests that the Sesn2/Keap1/Nrf2 axis may play an important role in retinal degeneration in glaucoma.


Assuntos
Apoptose/fisiologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo/fisiologia , Células Ganglionares da Retina/metabolismo , Animais , Elementos de Resposta Antioxidante/fisiologia , Apoptose/efeitos dos fármacos , Regulação para Baixo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Células Ganglionares da Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima
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