Articoli di riviste: "RoGFP2"

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Albrecht, Simone C., Mirko C. Sobotta, Daniela Bausewein, Isabel Aller, Rüdiger Hell, Tobias P. Dick e Andreas J. Meyer. "Redesign of Genetically Encoded Biosensors for Monitoring Mitochondrial Redox Status in a Broad Range of Model Eukaryotes". Journal of Biomolecular Screening 19, n. 3 (16 agosto 2013): 379–86. http://dx.doi.org/10.1177/1087057113499634.

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The development of genetically encoded redox biosensors has paved the way toward chemically specific, quantitative, dynamic, and compartment-specific redox measurements in cells and organisms. In particular, redox-sensitive green fluorescent proteins (roGFPs) have attracted major interest as tools to monitor biological redox changes in real time and in vivo. Most recently, the engineering of a redox relay that combines glutaredoxin (Grx) with roGFP2 as a translational fusion (Grx1-roGFP2) led to a biosensor for the glutathione redox potential ( EGSH). The expression of this probe in mitochondria is of particular interest as mitochondria are the major source of oxidants, and their redox status is closely connected to cell fate decisions. While Grx1-roGFP2 can be expressed in mammalian mitochondria, it fails to enter mitochondria in various nonmammalian model organisms. Here we report that inversion of domain order from Grx1-roGFP2 to roGFP2-Grx1 yields a biosensor with perfect mitochondrial targeting while fully maintaining its biosensor capabilities. The redesigned probe thus allows extending in vivo observations of mitochondrial redox homeostasis to important nonmammalian model organisms, particularly plants and insects.

2

Liu, Ting-Hang, Mohammad A. Yaghmour, Miin-Huey Lee, Thomas M. Gradziel, Johan H. J. Leveau e Richard M. Bostock. "An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces". Phytopathology® 110, n. 2 (febbraio 2020): 297–308. http://dx.doi.org/10.1094/phyto-07-19-0237-r.

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The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant–microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 μM for oxidation by H2O2 and menadione, respectively, and 10 μM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

3

Xu, Xiuling, Katharina von Löhneysen, Katrin Soldau, Deborah Noack, Andrew Vu e Jeffrey S. Friedman. "A novel approach for in vivo measurement of mouse red cell redox status". Blood 118, n. 13 (29 settembre 2011): 3694–97. http://dx.doi.org/10.1182/blood-2011-03-342113.

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Abstract Maintenance of a reducing redox balance is a critical physiologic function of red cells (RBC) that can be perturbed in variety of RBC pathologies. Here we describe a new approach to evaluate in vivo RBC redox status using a redox sensitive GFP (roGFP2) sensor under control of a β-globin mini-promoter, directing expression specifically to erythroid cells. RoGFP2 expressing RBCs demonstrate ratiometric and reversible shifts in fluorescence on exposure to oxidants and reductants. We demonstrate that roGFP2 expressing RBC can be used to monitor thiol redox status during in vitro phenylhydrazine treatment and over the course of in vivo RBC aging, where a shift to a more oxidized state is observed in older cells. Thus, roGFP2 transgenic mice are a new and versatile tool that can be used to probe how RBC redox status responds in the context of drug therapy, physiologic stressors and pathologic states.

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Xu, Xiuling, Katharina von Loehneysen, Deborah Noack, Andrew Vu e Jeff S. Friedman. "A Novel Approach for In Vivo Measurement of Red Cell Redox Status". Blood 116, n. 21 (19 novembre 2010): 2036. http://dx.doi.org/10.1182/blood.v116.21.2036.2036.

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Abstract Abstract 2036 Maintenance of a reducing redox balance is a critical physiologic function of red cell metabolic machinery. Perturbation of this balance, whether inherited or acquired, is found in a variety of red cell pathologies. Methods for evaluation of red cell redox status include direct approaches such as determining glutathione (GSH, GSSG) levels, and indirect approaches such as measuring fluorescence of oxidation sensitive dyes. Here we describe an alternative method for evaluation of red cell redox status that can be used in vivo and in real-time assays. Engineered variants of GFP possessing two solvent accessible cysteine residues function as molecular redox sensors with distinct fluorescence characteristics. Excitation spectrum shifts upon the oxidation of cysteine residues forming a disulfide. A higher ratio of fluorescence when comparing excitation at 405nm versus 488nm indicates rising levels of oxidized GFP and a shift in cellular redox status. To validate redox GFPs in erythroid cells, we first performed in vitro assays with MEL cells over-expressing several related GFP sensors (ro-GFPs), selecting the brightest molecule (roGFP2) for further study. The sensor function of roGFP2 in MEL cells was verified by stimulation with exogenous oxidant (1mM H202) or reductant (10 mM DTT) as shown in the figure below. In order to create a physiologic in vivo model for study of red cell redox status, transgenic mice expressing roGFP2 specifically in the erythroid lineage were generated. roGFP2 expressing red cells demonstrate the expected shift in fluorescence upon exposure to H202 or t-butyl peroxide in a short-term assay. In vivo, we have measured red cell lifespan (using biotin-labeling) in roGFP2 transgenic animals to follow redox status of red cells as a function of cell age. Expression of roGFP2 has no effect on red cell survival. Interestingly, when comparing old red cells (age > 50days) with younger cells (age < 50days), a shift in GFP fluorescence ratio indicating that a higher fraction of the sensor is oxidized in the aged cells was observed. This observation is consistent with the hypothesis that metabolic changes, in particular a decline in ability to reduce oxidative damage, contribute to red cell senescence. We are generating several murine strains with defined red cell defects also expressing roGFP2 in order to assess the role of changes in intra-erythrocyte redox status in a range of pathologic conditions. In vitro and in vivo assays using roGFP2 transgenic cells/mice are in process to determine the potential utility of this system as a screen for hematoxicity of drugs and other compounds. Figure 1 Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Figure 1. Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Disclosures: No relevant conflicts of interest to declare.

5

de Cubas, Laura, Valeriy V. Pak, Vsevolod V. Belousov, José Ayté e Elena Hidalgo. "The Mitochondria-to-Cytosol H2O2 Gradient Is Caused by Peroxiredoxin-Dependent Cytosolic Scavenging". Antioxidants 10, n. 5 (6 maggio 2021): 731. http://dx.doi.org/10.3390/antiox10050731.

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Fluorescent protein-based reporters used to measure intracellular H2O2 were developed to overcome the limitations of small permeable dyes. The two major families of genetically encoded redox reporters are the reduction-oxidation sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins and HyPer and derivatives. We have used the most sensitive probes of each family, roGFP2-Tpx1.C169S and HyPer7, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular H2O2, the former is two-five times more sensitive than HyPer7, and roGFP2-Tpx1.C169S is partially oxidized in the cytosol of wild-type cells while HyPer7 is fully reduced. We have successfully expressed HyPer7 in the mitochondrial matrix, and it is ~40% oxidized, suggesting higher steady-state levels of peroxides, in the low micromolar range, than in the cytosol. Cytosolic HyPer7 can detect negligible H2O2 in the cytosol from mitochondrial origin unless the main H2O2 scavenger, the cytosolic peroxiredoxin Tpx1, is absent, while mitochondrial HyPer7 is oxidized to the same extent in wild-type and ∆tpx1 cells. We conclude that there is a bidirectional flux of H2O2 across the matrix and the cytosol, but Tpx1 rapidly and efficiently scavenges mitochondrial-generated peroxides and stops their steady-state cytosolic levels rising.

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Xu, Xiuling, e Jeff S. Friedman. "In Vivo Monitoring of Red Cell Redox Status to Screen for Potential Hematotoxicity of Anti-Malarial Drugs". Blood 118, n. 21 (18 novembre 2011): 2099. http://dx.doi.org/10.1182/blood.v118.21.2099.2099.

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Abstract Abstract 2099 Redox equilibrium is an important determinant of malaria pathology and host defensive response to malaria parasites. Many anti-malarial drugs are reported to increase oxidative stress in red cells (RBC). Drug-induced hemolysis, particularly in G6PD deficient individuals, limits the utility of approved 8-aminoquinolones such as primaquine. The search for derivatives of primaquine that maintain efficacy without RBC toxicity is hampered by lack of a predictive assay for hemolytic potential. In order to monitor in vivo RBC redox changes in response to anti-malaria drugs, we have established a transgenic mouse line specifically expressing a redox sensitive GFP (roGFP2) in RBC1. RoGFP2 is an engineered EGFP with 2 cysteines introduced at amino acid positions 147 and 2042. When oxidized, the 2 cysteines form a disulfide bond, resulting in a protein conformational change that alters the spectral properties of the GFP. By following the ratio of fluorescence emission at 520nm after excitation at 405 vs 488nm, the intracellular redox potential in live cells can be determined. Here we apply this novel mouse model to follow in vivo RBC redox status upon exposure of transgenic mice to a series of 8-AQÕs and control compounds including known hemolytic agents such as dapsone. Unlike in vitro studies, these whole animal experiments incorporate metabolic transformation of inert parent compounds, pharmacokinetics and a time-course that closely models clinical hemolytic reactions in susceptible individuals exposed to the same drugs. roGFP2 transgenic mice (N=4 per group) were given test compounds (primaquine, chloroquine, dapsone and 1161B*) by gavage 2X/day at a total dose of 50mg/Kg over a period of 5 days. Peripheral RBC were analyzed using a LSRII FACS (BD) to determine the 405/488 ratio, and thus follow the redox status as shown in the figure below. Chloroquine treatment did not cause detectable change in roGFP2 signal, in agreement with previous data that chloroquine does not induce ROS production or hematotoxicity. Treatment with 1161B induced a steady increase in roGFP2 ratio that was first evident at 3 days of treatment, while primaquine treatment has little effect. Dapsone serves as a positive control in this assay, as a known hemolytic agent requiring metabolic activation to produce a redox cycling metabolite.3 Treatment with dapsone produced a time dependent shift to a more oxidized state of roGFP2 that was first evident after 2 days of drug administration. Consistent with these results, CBCÕs obtained one week after start of treatment showed moderate and mild hemolysis in those mice receiving dapsone and 1161B, respectively. In summary, roGFP2 transgenic mice provide a promising platform for assessment of hemolytic potential of drugs—here demonstrated using dapsone and 8-aminoquinolones. While the mice used in this study were GFP transgenic on a wild-type background, studies are in progress to assess whether strains with specific RBC defects (such as G6PD hypomorphs, PRDX2 KO andSOD1 KO) are more sensitive to hemolytic insults. Preliminary studies indicate that PRDX2 and SOD1 KO mice are more sensitive to primaquine, but not chloroquine, according to the roGFP2 ratio changes. G6PD deficient animals may prove particularly useful for discriminating compounds such as primaquine that are safe for normal individuals, but cause hemolysis in the context of enzyme deficiency. *1161B is the B enantiomer of an 8-aminoquinolone derivative with better efficacy and reduced hematoxicity4,5 in mice, when compared to the racemic mixture of the same compound. Disclosures: No relevant conflicts of interest to declare.

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Fernández-Puente, Escarlata, e Jesús Palomero. "Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells". International Journal of Molecular Sciences 22, n. 19 (8 ottobre 2021): 10876. http://dx.doi.org/10.3390/ijms221910876.

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Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.

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García-Quirós, Estefanía, Juan de Dios Alché, Barbara Karpinska e Christine H. Foyer. "Glutathione redox state plays a key role in flower development and pollen vigour". Journal of Experimental Botany 71, n. 2 (26 settembre 2019): 730–41. http://dx.doi.org/10.1093/jxb/erz376.

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roGFP2 measurements demonstrate that ungerminated Arabidopsis pollen grains are highly oxidized. Glutathione-deficient flowers show increased cellular oxidation with poor pollen germination/tube growth, suggesting that the reduced state underpins growth functions.

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Morgan, Bruce, Mirko C. Sobotta e Tobias P. Dick. "Measuring EGSH and H2O2 with roGFP2-based redox probes". Free Radical Biology and Medicine 51, n. 11 (dicembre 2011): 1943–51. http://dx.doi.org/10.1016/j.freeradbiomed.2011.08.035.

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Costa, Cláudio F., Celien Lismont, Serhii Chornyi, Hongli Li, Mohamed A. F. Hussein, Hans R. Waterham e Marc Fransen. "Functional Analysis of GSTK1 in Peroxisomal Redox Homeostasis in HEK-293 Cells". Antioxidants 12, n. 6 (7 giugno 2023): 1236. http://dx.doi.org/10.3390/antiox12061236.

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Peroxisomes serve as important centers for cellular redox metabolism and communication. However, fundamental gaps remain in our understanding of how the peroxisomal redox equilibrium is maintained. In particular, very little is known about the function of the nonenzymatic antioxidant glutathione in the peroxisome interior and how the glutathione antioxidant system balances with peroxisomal protein thiols. So far, only one human peroxisomal glutathione-consuming enzyme has been identified: glutathione S-transferase 1 kappa (GSTK1). To study the role of this enzyme in peroxisomal glutathione regulation and function, a GSTK1-deficient HEK-293 cell line was generated and fluorescent redox sensors were used to monitor the intraperoxisomal GSSG/GSH and NAD+/NADH redox couples and NADPH levels. We provide evidence that ablation of GSTK1 does not change the basal intraperoxisomal redox state but significantly extends the recovery period of the peroxisomal glutathione redox sensor po-roGFP2 upon treatment of the cells with thiol-specific oxidants. Given that this delay (i) can be rescued by reintroduction of GSTK1, but not its S16A active site mutant, and (ii) is not observed with a glutaredoxin-tagged version of po-roGFP2, our findings demonstrate that GSTK1 contains GSH-dependent disulfide bond oxidoreductase activity.

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de Cubas, Laura, Jorge Mallor, Víctor Herrera-Fernández, José Ayté, Rubén Vicente e Elena Hidalgo. "Expression of the H2O2 Biosensor roGFP-Tpx1.C160S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H2O2 Levels, Transmembrane Gradients, and Response to Metals". Antioxidants 12, n. 3 (13 marzo 2023): 706. http://dx.doi.org/10.3390/antiox12030706.

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Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40–50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.

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Perez, Davis, Peter D. Dahlberg, Annina M. Sartor, Jiarui Wang, Julia Borden e William E. Moerner. "roGFP2 as an environmental sensor for cryogenic correlative light and electron microscopy". Biophysical Journal 121, n. 3 (febbraio 2022): 128a. http://dx.doi.org/10.1016/j.bpj.2021.11.2069.

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Lang, Lukas, Fabian Geissel, Britta Husemann e Marcel Deponte. "Kinetic and thermodynamic characterization of the roGFP2-Grx redox sensor at molecular level". Free Radical Biology and Medicine 208 (novembre 2023): S126. http://dx.doi.org/10.1016/j.freeradbiomed.2023.10.287.

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Krönauer, Christina, e Thomas Lahaye. "The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro". PLOS ONE 16, n. 8 (19 agosto 2021): e0256217. http://dx.doi.org/10.1371/journal.pone.0256217.

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The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

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Arias-Barreiro, Carlos R., Keisuke Okazaki, Apostolos Koutsaftis, Salmaan H. Inayat-Hussain, Akio Tani, Maki Katsuhara, Kazuhide Kimbara e Izumi C. Mori. "A Bacterial Biosensor for Oxidative Stress Using the Constitutively Expressed Redox-Sensitive Protein roGFP2". Sensors 10, n. 7 (24 giugno 2010): 6290–306. http://dx.doi.org/10.3390/s100706290.

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Müller, Alexandra, Jannis F. Schneider, Adriana Degrossoli, Nataliya Lupilova, Tobias P. Dick e Lars I. Leichert. "Systematic in vitro assessment of responses of roGFP2-based probes to physiologically relevant oxidant species". Free Radical Biology and Medicine 106 (maggio 2017): 329–38. http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.044.

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Müller, Alexandra, Jannis F. Schneider, Adriana Degrossoli, Nataliya Lupilova, Tobias P. Dick e Lars I. Leichert. "Fluorescence spectroscopy of roGFP2-based redox probes responding to various physiologically relevant oxidant species in vitro". Data in Brief 11 (aprile 2017): 617–27. http://dx.doi.org/10.1016/j.dib.2017.03.015.

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Esposito, Sonia, Alessandra Masala, Simona Sanna, Mauro Rassu, Viengsavanh Pimxayvong, Ciro Iaccarino e Claudia Crosio. "Redox-sensitive GFP to monitor oxidative stress in neurodegenerative diseases". Reviews in the Neurosciences 28, n. 2 (1 febbraio 2017): 133–44. http://dx.doi.org/10.1515/revneuro-2016-0041.

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AbstractRedox processes are key events in the degenerative cascade of many adult-onset neurodegenerative diseases (NDs), but the biological relevance of a single redox change is often dependent on the redox couple involved and on its subcellular origin. The biosensors based on engineered fluorescent proteins (redox-sensitive GFP [roGFP]) offer a unique opportunity to monitor redox changes in both physiological and pathological contexts in living animals and plants. Here, we review the use of roGFPs to monitor oxidative stress in different three adult-onset NDs: Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Despite the many differences spanning from incidence to onset, the hypotheses on biological processes underlying both sporadic and familiar ND forms in humans outline a model in which noncompeting mechanisms are likely to converge in various unsuccessful patterns to mediate the selective degeneration of a specific neuronal population. roGFPs, targeted to different cell compartments, are successfully used as specific markers of cell toxicity, induced by expression of causative genes linked to a determined ND. We also report the use of roGFP to monitor oxidative stress induced by the expression of the ALS-causative gene SOD1.

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Schuh, Anna Katharina, Mahsa Rahbari, Kim C. Heimsch, Franziska Mohring, Stanislaw J. Gabryszewski, Stine Weder, Kathrin Buchholz, Stefan Rahlfs, David A. Fidock e Katja Becker. "Stable Integration and Comparison of hGrx1-roGFP2 and sfroGFP2 Redox Probes in the Malaria Parasite Plasmodium falciparum". ACS Infectious Diseases 4, n. 11 (21 agosto 2018): 1601–12. http://dx.doi.org/10.1021/acsinfecdis.8b00140.

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Van Loi, Vu, e Haike Antelmann. "Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus". MethodsX 7 (2020): 100900. http://dx.doi.org/10.1016/j.mex.2020.100900.

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Ooi, Lia, Lee Heng e Izumi Mori. "A High-Throughput Oxidative Stress Biosensor Based on Escherichia coli roGFP2 Cells Immobilized in a k-Carrageenan Matrix". Sensors 15, n. 2 (22 gennaio 2015): 2354–68. http://dx.doi.org/10.3390/s150202354.

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Tung, Quach Ngoc, Vu Van Loi, Tobias Busche, Andreas Nerlich, Maren Mieth, Johanna Milse, Jörn Kalinowski, Andreas C. Hocke e Haike Antelmann. "Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum". Redox Biology 20 (gennaio 2019): 514–25. http://dx.doi.org/10.1016/j.redox.2018.11.012.

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Mourenza, Álvaro, José A. Gil, Luís M. Mateos e Michal Letek. "A Novel Screening Strategy Reveals ROS-Generating Antimicrobials That Act Synergistically against the Intracellular Veterinary Pathogen Rhodococcus equi". Antioxidants 9, n. 2 (28 gennaio 2020): 114. http://dx.doi.org/10.3390/antiox9020114.

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Abstract (sommario):

: Rhodococcus equi is a facultative intracellular pathogen that causes infections in foals and many other animals such as pigs, cattle, sheep, and goats. Antibiotic resistance is rapidly rising in horse farms, which makes ineffective current antibiotic treatments based on a combination of macrolides and rifampicin. Therefore, new therapeutic strategies are urgently needed to treat R. equi infections caused by antimicrobial resistant strains. Here, we employed a R. equi mycoredoxin-null mutant strain highly susceptible to oxidative stress to screen for novel ROS-generating antibiotics. Then, we used the well-characterized Mrx1-roGFP2 biosensor to confirm the redox stress generated by the most promising antimicrobial agents identified in our screening. Our results suggest that different combinations of antibacterial compounds that elicit oxidative stress are promising anti-infective strategies against R. equi. In particular, the combination of macrolides with ROS-generating antimicrobial compounds such as norfloxacin act synergistically to produce a potent antibacterial effect against R. equi. Therefore, our screening approach could be applied to identify novel ROS-inspired therapeutic strategies against intracellular pathogens.

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Wittig, Rainer, Verena Richter, Stephanie Wittig-Blaich, Petra Weber, Wolfgang S. L. Strauss, Thomas Bruns, Tobias P. Dick e Herbert Schneckenburger. "Biosensor-Expressing Spheroid Cultures for Imaging of Drug-Induced Effects in Three Dimensions". Journal of Biomolecular Screening 18, n. 6 (11 marzo 2013): 736–43. http://dx.doi.org/10.1177/1087057113480525.

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In the past, the majority of antitumor compound-screening approaches had been performed in two-dimensional (2D) cell cultures. Although easy to standardize, this method provides results of limited significance because cells are surrounded by an artificial microenvironment, are not exposed to hypoxia gradients, and lack cell-cell contacts. These nonphysiological conditions directly affect relevant parameters such as the resistance to anticancer drugs. Multicellular tumor spheroids more closely resemble the in vivo situation in avascularized tumors. To monitor cellular reactions within this three-dimensional model system, we stably transfected a spheroid-forming glioblastoma cell line with Grx1-roGFP2, a green fluorescent protein (GFP)–based glutathione-specific redox sensor that detects alterations in the glutathione redox potential. Functionality and temporal dynamics of the sensor were verified with redox-active substances in 2D cell culture. Based on structured illumination microscopy using nonphototoxic light doses, ratio imaging was then applied to monitor the response of the glutathione system to exogenous hydrogen peroxide in optical sections of a tumor spheroid. Our approach provides a proof of concept for biosensor-based imaging in 3D cell cultures.

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Ivashchenko, Oksana, Paul P. Van Veldhoven, Chantal Brees, Ye-Shih Ho, Stanley R. Terlecky e Marc Fransen. "Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk". Molecular Biology of the Cell 22, n. 9 (maggio 2011): 1440–51. http://dx.doi.org/10.1091/mbc.e10-11-0919.

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Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation–reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.

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Hartmann, Fabian Stefan Franz, Lina Clermont, Quach Ngoc Tung, Haike Antelmann e Gerd Michael Seibold. "The Industrial Organism Corynebacterium glutamicum Requires Mycothiol as Antioxidant to Resist Against Oxidative Stress in Bioreactor Cultivations". Antioxidants 9, n. 10 (9 ottobre 2020): 969. http://dx.doi.org/10.3390/antiox9100969.

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In aerobic environments, bacteria are exposed to reactive oxygen species (ROS). To avoid an excess of ROS, microorganisms are equipped with powerful enzymatic and non-enzymatic antioxidants. Corynebacterium glutamicum, a widely used industrial platform organism, uses mycothiol (MSH) as major low molecular weight (LMW) thiol and non-enzymatic antioxidant. In aerobic bioreactor cultivations, C. glutamicum becomes exposed to oxygen concentrations surpassing the air saturation, which are supposed to constitute a challenge for the intracellular MSH redox balance. In this study, the role of MSH was investigated at different oxygen levels (pO2) in bioreactor cultivations in C. glutamicum. Despite the presence of other highly efficient antioxidant systems, such as catalase, the MSH deficient ΔmshC mutant was impaired in growth in bioreactor experiments performed at pO2 values of 30%. At a pO2 level of 20%, this growth defect was abolished, indicating a high susceptibility of the MSH-deficient mutant towards elevated oxygen concentrations. Bioreactor experiments with C. glutamicum expressing the Mrx1-roGFP2 redox biosensor revealed a strong oxidative shift in the MSH redox potential (EMSH) at pO2 values above 20%. This indicates that the LMW thiol MSH is an essential antioxidant to maintain the robustness and industrial performance of C. glutamicum during aerobic fermentation processes.

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Trümper, Verena, Ilka Wittig, Juliana Heidler, Florian Richter, Bernhard Brüne e Andreas von Knethen. "Redox Regulation of PPARγ in Polarized Macrophages". PPAR Research 2020 (1 luglio 2020): 1–16. http://dx.doi.org/10.1155/2020/8253831.

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The peroxisome proliferator-activated receptor (PPARγ) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-γ- (IFNγ-) and interleukin-4- (IL4-) polarized macrophages (MΦ) initiates posttranslational modifications of PPARγ, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of MΦ, while the redox status of PPARγ was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPARγ were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFNγ, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPARγ-dependent reporter gene transactivation supporting a functional shift of PPARγ associated with the MΦ phenotype. These data suggest distinct mechanisms for regulating PPARγ function based on the redox state of MΦ.

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Christ, Loïck, Jérémy Couturier e Nicolas Rouhier. "Relationships between the Reversible Oxidation of the Single Cysteine Residue and the Physiological Function of the Mitochondrial Glutaredoxin S15 from Arabidopsis thaliana". Antioxidants 12, n. 1 (31 dicembre 2022): 102. http://dx.doi.org/10.3390/antiox12010102.

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Glutaredoxins (GRXs) are widespread proteins catalyzing deglutathionylation or glutathionylation reactions or serving for iron-sulfur (Fe-S) protein maturation. Previous studies highlighted a role of the Arabidopsis thaliana mitochondrial class II GRXS15 in Fe-S cluster assembly, whereas only a weak glutathione-dependent oxidation activity was detected with the non-physiological roGFP2 substrate in vitro. Still, the protein must exist in a reduced form for both redox and Fe-S cluster binding functions. Therefore, this study aimed at examining the redox properties of AtGRXS15. The acidic pKa of the sole cysteine present in AtGRXS15 indicates that it should be almost totally under a thiolate form at mitochondrial pH and thus possibly subject to oxidation. Oxidizing treatments revealed that this cysteine reacts with H2O2 or with oxidized glutathione forms. This leads to the formation of disulfide-bridge dimers and glutathionylated monomers which have redox midpoint potentials of −304 mV and −280 mV, respectively. Both oxidized forms are reduced by glutathione and mitochondrial thioredoxins. In conclusion, it appears that AtGRXS15 is prone to oxidation, forming reversible oxidation forms that may be seen either as a catalytic intermediate of the oxidoreductase activity and/or as a protective mechanism preventing irreversible oxidation and allowing Fe-S cluster binding upon reduction.

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Csukovich, Georg, Janina Huainig, Selina Troester, Barbara Pratscher e Iwan Anton Burgener. "The Intricacies of Inflammatory Bowel Disease: A Preliminary Study of Redox Biology in Intestinal Organoids". Organoids 2, n. 3 (3 settembre 2023): 156–64. http://dx.doi.org/10.3390/organoids2030012.

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We evaluated the redox status, precisely glutathione levels, which have a major impact in cellular detoxification and antioxidant defence in IBD-derived and healthy intestinal organoids. Therefore, we wanted to explore the differences in terms of their redox balance and mitochondrial fitness. To this end, we introduced a Grx1-roGFP2 construct into the organoids by lentiviral transduction before performing a stress assay by treating the organoids with hydrogen peroxide and examined the GSH/GSSG ratio using confocal imaging. Using ratio imaging, we could detect statistically significant differences between healthy and IBD-derived samples. To gain more insight, we also performed a GSH/GSSG assay, which directly measured glutathione levels. This analysis revealed that both organoid lines had higher levels of oxidized glutathione due to the stress treatment demonstrated by a lower GSH/GSSG ratio compared to the untreated control. Nevertheless, the results showed no significant difference between healthy and IBD-derived organoids. We further challenged organoids with hydrogen peroxide after incubation with MitoTracker® to see if mitochondrial fitness might be different in IBD-derived organoids. However, these results were also very comparable. In summary, our preliminary findings indicate that both organoid lines demonstrate a well-functioning system in terms of analysis but show no clear difference between healthy and IBD-derived samples.

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Nietzel, Thomas, Marlene Elsässer, Cristina Ruberti, Janina Steinbeck, José Manuel Ugalde, Philippe Fuchs, Stephan Wagner et al. "The fluorescent protein sensor roGFP2‐Orp1 monitorsin vivoH2O2and thiol redox integration and elucidates intracellular H2O2dynamics during elicitor‐induced oxidative burst in Arabidopsis". New Phytologist 221, n. 3 (27 novembre 2018): 1649–64. http://dx.doi.org/10.1111/nph.15550.

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Karpinska, Barbara, Sarah Owdah Alomrani e Christine H. Foyer. "Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling". Philosophical Transactions of the Royal Society B: Biological Sciences 372, n. 1730 (14 agosto 2017): 20160392. http://dx.doi.org/10.1098/rstb.2016.0392.

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Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction–oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1 , WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

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Velappan, Yazhini, Ambra de Simone, Santiago Signorelli, John A. Considine, Christine H. Foyer e Michael J. Considine. "Hydrogen Cyanamide Causes Reversible G2/M Cell Cycle Arrest Accompanied by Oxidation of the Nucleus and Cytosol". Antioxidants 12, n. 7 (23 giugno 2023): 1330. http://dx.doi.org/10.3390/antiox12071330.

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Hydrogen cyanamide (HC) has been widely used in horticulture to trigger bud burst following dormancy. Its use has been banned in some countries due to human health concerns, however the search for effective safe alternatives is delayed by lack of knowledge of the mechanism of HC action. Earlier studies demonstrate that HC stimulates the production of reactive oxygen species (ROS) and alters the rate of cell division. However, the relationships between HC effects on ROS, redox (reduction/oxidation) homeostasis and cell division are unknown. This study used Arabidopsis thaliana ((L.) Heynh.) seedlings expressing the redox reporter roGFP2 to measure the oxidation states of the nuclei and cytosol in response to HC treatment. The Cytrap dual cell cycle phase marker system and flow cytometry were used to study associated changes in cell proliferation. HC (1.5 mM) reversibly inhibited root growth during a 24 h treatment. Higher concentrations were not reversible. HC did not synchronise the cell cycle, in contrast to hydroxyurea. Rather, HC caused a gradual accumulation of cells in the G2/M phase and decline of G1/S phase cells, 16 to 24 h post-treatment. This was accompanied by increased oxidation of both the nuclei and cytosol. Taken together, these findings show that HC impairs proliferation of embryonic root meristem cells in a reversible manner through restriction of G2/M transition accompanied by increased cellular oxidation.

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Mourenza, Álvaro, Natalia Bravo-Santano, Inés Pradal, Jose A. Gil, Luis M. Mateos e Michal Letek. "Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi". Antioxidants 8, n. 11 (15 novembre 2019): 558. http://dx.doi.org/10.3390/antiox8110558.

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Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectives.

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Yadav, Shambhu, Bindia Chawla, Mohammad Anwar Khursheed, Rajesh Ramachandran e Anand Kumar Bachhawat. "The glutathione degrading enzyme, Chac1, is required for calcium signaling in developing zebrafish: redox as an upstream activator of calcium". Biochemical Journal 476, n. 13 (2 luglio 2019): 1857–73. http://dx.doi.org/10.1042/bcj20190077.

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Abstract Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the γ-glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a glutathione-degrading enzyme that acts only on reduced glutathione. The zebrafish chac1 expression was seen early in development, and in the latter stages, in the developing muscles, brain and heart. The chac1 knockdown was embryonic lethal, and the developmental defects were seen primarily in the myotome, brain and heart where chac1 was maximally expressed. The phenotypes could be rescued by the WT Chac1 but not by the catalytically inactive Chac1 that was incapable of degrading glutathione. The ability of chac1 to alter the intracellular glutathione redox potential in the live animals was examined using Grx1-roGFP2. The chac1 morphants lacked the increased degree of cellular oxidation seen in the WT zebrafish. As calcium is also known to be critical for the developing myotomes, brain and heart, we further investigated if the chac1 knockdown phenotypes were a consequence of the lack of calcium signals. We observed using GCaMP6s, that calcium transients normally seen in the developing embryos were strongly attenuated in these knockdowns. The study thus identifies Chac1 and the consequent change in intracellular glutathione redox potential as important upstream activators of calcium signaling during development.

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Perelmuter, Karen, Inés Tiscornia, Marcelo A. Comini e Mariela Bollati-Fogolín. "Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance". Sensors 22, n. 4 (9 febbraio 2022): 1324. http://dx.doi.org/10.3390/s22041324.

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Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

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Scheller, Daniel, Franziska Becker, Andrea Wimbert, Dominik Meggers, Stephan Pienkoß, Christian Twittenhoff, Lisa R. Knoke, Lars I. Leichert e Franz Narberhaus. "The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis". PLOS Genetics 19, n. 7 (10 luglio 2023): e1010669. http://dx.doi.org/10.1371/journal.pgen.1010669.

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Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5’-untranslated region (5’-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5’-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

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Ramazani, Yasaman, Noël Knops, Sante Princiero Berlingerio, Oyindamola Christiana Adebayo, Celien Lismont, Dirk J. Kuypers, Elena Levtchenko, Lambert P. van den Heuvel e Marc Fransen. "Therapeutic concentrations of calcineurin inhibitors do not deregulate glutathione redox balance in human renal proximal tubule cells". PLOS ONE 16, n. 4 (30 aprile 2021): e0250996. http://dx.doi.org/10.1371/journal.pone.0250996.

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The calcineurin inhibitors (CNI) cyclosporine A and tacrolimus comprise the basis of immunosuppressive regimes in all solid organ transplantation. However, long-term or high exposure to CNI leads to histological and functional renal damage (CNI-associated nephrotoxicity). In the kidney, proximal tubule cells are the only cells that metabolize CNI and these cells are believed to play a central role in the origin of the toxicity for this class of drugs, although the underlying mechanisms are not clear. Several studies have reported oxidative stress as an important mediator of CNI-associated nephrotoxicity in response to CNI exposure in different available proximal tubule cell models. However, former models often made use of supra-therapeutic levels of tissue drug exposure. In addition, they were not shown to express the relevant enzymes (e.g., CYP3A5) and transporters (e.g., P-glycoprotein) for the metabolism of CNI in human proximal tubule cells. Moreover, the used methods for detecting ROS were potentially prone to false positive results. In this study, we used a novel proximal tubule cell model established from human allograft biopsies that demonstrated functional expression of relevant enzymes and transporters for the disposition of CNI. We exposed these cells to CNI concentrations as found in tissue of stable solid organ transplant recipients with therapeutic blood concentrations. We measured the glutathione redox balance in this cell model by using organelle-targeted variants of roGFP2, a highly sensitive green fluorescent reporter protein that dynamically equilibrates with the glutathione redox couple through the action of endogenous glutaredoxins. Our findings provide evidence that CNI, at concentrations commonly found in allograft biopsies, do not alter the glutathione redox balance in mitochondria, peroxisomes, and the cytosol. However, at supra-therapeutic concentrations, cyclosporine A but not tacrolimus increases the ratio of oxidized/reduced glutathione in the mitochondria, suggestive of imbalances in the redox environment.

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Bend, John Richard, Xue Yan (Iris) Xue Yan Xia, Daofeng Chen, Abudi Awaysheh, Andrea Lo, Michael John Rieder e Rebecca Jane Rylett. "Attenuation of Oxidative Stress in HEK 293 Cells by the TCM Constituents Schisanhenol, Baicalein, Resveratrol or Crocetin and Two Defined Mixtures". Journal of Pharmacy & Pharmaceutical Sciences 18, n. 4 (2 novembre 2015): 661. http://dx.doi.org/10.18433/j3mw3n.

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PURPOSE: Our working hypothesis is that single bioactive phytochemicals with antioxidant properties that are important constituents of Traditional Chinese Medicine (TCM) and their defined mixtures have potential as chemoprotective agents for chronic conditions characterized by oxidative and nitrosative stress, including Alzheimer’s. Here we evaluate the ability of baicalein, crocetin, trans-resveratrol or schisanhenol and two defined mixtures of these TCM phytochemicals to attenuate the toxicity resulting from exposure to cell permeant t-butyl hydroperoxide (tBPH) in wild-type and bioengineered (to express choline acetyltransferase) HEK 293 cells. METHODS: Endpoints of tBHP-initiated oxidative and nitrosative stress in both types of HEK 293 cells and its attenuation by TCM constituents and mixtures included cytotoxicity (LDH release); depletion of intracellular glutathione (GSH); formation of S-glutathionylated proteins; oxidative changes to the disulfide proteome; and real-time changes in intracellular redox status. RESULTS: At low µM concentrations, each of the TCM constituents and mixtures effectively attenuated intracellular toxicity due to exposure of HEK 293 cells to 50 or 250 µM tBHP for 30 min to 3 h. Confocal microscopy of HEK 293 cells transfected with mutated green fluorescent protein (roGFP2) showed effective attenuation of tBHP oxidation by baicalein in real time. Three redox-regulated proteins prominent in the disulfide proteome of HEK 293 cells were identified by MALDI-TOF mass spectrometry. CONCLUSIONS: We conclude that single TCM chemicals and their simple mixtures have potential for use in adjunct chemoprotective therapy. Advantages of mixtures compared to single TCM constituents include the ability to combine compounds with varying molecular mechanisms of cytoprotection for enhanced biological activity; and to combine chemicals with complementary pharmacokinetic properties to increase half-life and prolong activity in vivo. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

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Rupel, Katia, Luisa Zupin, Andrea Colliva, Anselmo Kamada, Augusto Poropat, Giulia Ottaviani, Margherita Gobbo et al. "Photobiomodulation at Multiple Wavelengths Differentially Modulates Oxidative Stress In Vitro and In Vivo". Oxidative Medicine and Cellular Longevity 2018 (11 novembre 2018): 1–11. http://dx.doi.org/10.1155/2018/6510159.

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Photobiomodulation (PBM) is emerging as an effective strategy for the management of multiple inflammatory conditions, including oral mucositis (OM) in cancer patients who receive chemotherapy or radiotherapy. Still, the poor understanding of the mechanisms by which the light interacts with biological tissues and the heterogeneity of light sources and protocols employed worldwide significantly limits its applicability. Reactive oxygen species (ROS) are massively generated during the early phases of OM and play a major role in the pathogenesis of inflammation in general. Here, we report the results of a clinical and experimental study, aimed at evaluating the effect of laser light at different wavelengths on oxidative stress in vivo in oncologic patients suffering from OM and in vitro in two cell types abundantly present within the inflamed oral mucosa, neutrophil polymorphonuclear (PMN) granulocytes, and keratinocytes. In addition to standard ROS detection methods, we exploited a roGFP2-Orp1 genetically encoded sensor, allowing specific, quantitative, and dynamic imaging of redox events in living cells in response to oxidative stress and PBM. We found that the various wavelengths differentially modulate ROS production. In particular, the 660 nm laser light increases ROS production when applied either before or after an oxidative stimulus. In contrast, the 970 nm laser light exerted a moderate antioxidant activity both in the saliva of OM patients and in both cell types. The most marked reduction in the levels of ROS was detected in cells exposed either to the 800 nm laser light or to the combination of the three wavelengths. Overall, our study demonstrates that PBM exerts different effects on the redox state of both PMNs and keratinocytes depending on the used wavelength and prompts the validation of a multiwavelength protocol in the clinical settings.

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Takahashi, H. K., L. Prates Roma, J. Duprez e J. C. Jonas. "P2122 Effets aigus des nutriments sur l’état d’oxydation des thiols dans le cytosol et la matrice mitochondriale de cellules d’îlots pancréatiques de rat : mesures dynamiques à l’aide de la sonde GRX1-roGFP2". Diabetes & Metabolism 39 (marzo 2013): A97. http://dx.doi.org/10.1016/s1262-3636(13)72032-9.

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Roma, Leticia P., Jessica Duprez, Hilton K. Takahashi, Patrick Gilon, Andreas Wiederkehr e Jean-Christophe Jonas. "Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1". Biochemical Journal 441, n. 3 (16 gennaio 2012): 971–78. http://dx.doi.org/10.1042/bj20111770.

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Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.

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Di Marcantonio, Daniela, Jessica Vadaketh, Esteban Martinez, Prisco Mirandola, Giuliana Gobbi, Michael D. Milsom, Claudia Scholl, Stefan Fröhling, Marco Vitale e Stephen Matthew Sykes. "Pkc Epsilon Regulates Mitochondrial Redox Biology to Support the Differentiation Blockade in Acute Myeloid Leukemia". Blood 126, n. 23 (3 dicembre 2015): 444. http://dx.doi.org/10.1182/blood.v126.23.444.444.

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Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by the clonal expansion of myeloid progenitors that have impaired differentiation capacity. Determining the molecular machinery that regulate the survival and differentiation blockade of AML cells could serve as a foundation for designing novel therapies. PKCε is a serine-threonine kinase belonging to the subgroup of the Protein Kinase C family called Novel PKCs. Aberrant PKCε expression and activation is associated with the pathogenesis and chemotherapy resistance of many solid cancers. However, the contribution of PKCε in blood malignances such as AML is not well defined. To evaluate the role of PKCe in AML biology, we employed short hairpin RNA (shRNA)-mediated approaches to down-regulate PKCε expression in human and murine AML cell lines. We found that shRNA-mediated knockdown of PKCε significantly reduces the in vitro expansion of several human AML cell lines (MOLM-14, NOMO-1, OCI-AML3, THP-1 and U937). We also observed that blocking PKCε induces caspase-3 cleavage and increases the number of annexin V-positive cells (P<0.05), suggesting that PKCε antagonizes AML cell apoptosis. Additionally, we have also found that prior to cell death, AML cells expressing PKCε-targeting shRNAs display characteristics of myeloid differentiation. Specifically, down-regulation of PKCε results in altered expression of the myeloid differentiation transcription factors C/EBPa and PU.1 and increased expression of the mature myeloid marker CD11b (P<0.001). Moreover, upon PKCε inhibition, AML cells acquire morphological changes associated with differentiation, such as increased cytoplasmic volume, granule formation and nuclear segmentation. Interestingly, we observed similar phenotypic changes when we inhibited PKCε expression in murine AML cell lines driven by the leukemogenic fusion protein MLL-AF9 alone (MLL-AF9) or in combination with the internal tandem duplication mutation of murine Flt3 (MLL-AF9;Flt3-ITD). Specifically, we observed that PKCε down-modulation significantly reduces murine AML cell survival (P<0.001) and colony formation in methylcellulose (P<0.001) of both MLL-AF9 and MLL-AF9;Flt3-ITD cells compared to non-targeting shRNA-expressing cells. We are currently investigating how PKCε inhibition impacts AML progression in vivo using mouse models of AML driven by MLL-AF9 or MLL-AF9;Flt3-ITD. At the molecular level, we have found that PKCε is a key regulator of reactive oxygen species (ROS) biology in AML cells. Specifically, using a fluorogenic probe (CellRox) that indiscriminately detects most types of ROS, we have observed that PKCε knockdown in human AML cell lines results in increased steady-state levels of intracellular ROS compared to shRNA control cells (P<0.002). Total intracellular ROS levels are influenced by the production and clearance of distinct ROS types in various cellular compartments. To further characterize the localization and specific type(s) of ROS regulated by PKCε, we utilized four redox-sensitive GFP (roGFP) probes, which allow for direct measurement of cytoplasmic and mitochrondrial glutathione redox potential (Grx1-roGFP-Cyto and Grx1-roGFP-Mito, respectively) and hydrogen peroxide (H2O2) levels (Orp1-roGFP-Cyto and Orp1-roGFP-Mito, respectively) in live cells. Down-modulation of PKCε in NOMO-1 and OCI-AML-3 cells expressing each of these roGFP constructs resulted in a significant increase in the oxidation of Grx1-roGFP (P<.0007) and Orp1-roGFP-Mito (P<0.02) but not either of cytoplasmic constructs, suggesting that PKCε regulates the production of ROS in the mitochondria of AML cells. Since increased H2O2 production and glutathione oxidation results from increased superoxide (O2-) production in mitochondria, we next evaluated the impact of PKCε on O2- production. Using a fluorogenic probe (MitoSOX) that specifically detects O2- in live cells, we found that PKCε down-modulation increases the production of O2- in AML cells (P<0.05). Our future studies are focused on determining the precise molecular events that connect alterations in redox biology with the survival and differentiation of AML cells. Collectively, these results uncover the previously unrecognized role of PKCε as a critical regulator of mitochondrial redox biology and supporter of cell survival and impaired differentiation in AML. Disclosures No relevant conflicts of interest to declare.

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Eduardo, Mariana Bustamante, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B. M. M. K. Islam, Elizaveta V. Benevolenskaya et al. "Abstract 444: Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells". Cancer Research 84, n. 6_Supplement (22 marzo 2024): 444. http://dx.doi.org/10.1158/1538-7445.am2024-444.

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Abstract Introduction. A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through various metabolic reactions including those involved in serine, one-carbon, glycine (SOG) and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression. Methods. MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA. Results. Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, GSH and 2-HG after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.0001), peaking at 15 min. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01) including neural, EMT and ER- BC related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p < 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased comet tails. scRNAseq revealed OA increased the numbers of cells within all cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. Conclusions. Metabolism of OA results in a metabolic shift toward the SOG and methionine increasing the production of SAM, GSH and 2-HG which have implications for oncogenesis. The increased SAM fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress. Increased 2-HG may produce metabolic BRCAness by inhibiting 2-oxoglutarate-dependent dioxygenases. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta V. Benevolenskaya, Maxim V. Frolov, Jason Locasale, Marcelo G. Bonini, Navdeep S. Chandel, Seema Khan, Susan Clare. Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 444.

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Kim, Young-Mee, Seok-Jo Kim, Ryosuke Tatsunami, Hisao Yamamura, Tohru Fukai e Masuko Ushio-Fukai. "ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis". American Journal of Physiology-Cell Physiology 312, n. 6 (1 giugno 2017): C749—C764. http://dx.doi.org/10.1152/ajpcell.00346.2016.

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Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (H2O2) or overexpression of Nox4, which produces H2O2, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H2O2 to promote mtROS production. Mechanistically, H2O2 increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived H2O2 in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.

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Hatori, Yuta, Sachiye Inouye, Reiko Akagi e Toshio Seyama. "Local redox environment beneath biological membranes probed by palmitoylated-roGFP". Redox Biology 14 (aprile 2018): 679–85. http://dx.doi.org/10.1016/j.redox.2017.11.015.

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Hatori, Yuta, Takanori Kubo, Yuichiro Sato, Sachiye Inouye, Reiko Akagi e Toshio Seyama. "Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors". Antioxidants 9, n. 2 (3 febbraio 2020): 129. http://dx.doi.org/10.3390/antiox9020129.

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Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

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Nøstbakken, O. J., I. L. Bredal, P. A. Olsvik, T. S. Huang e B. E. Torstensen. "Effect of Marine Omega 3 Fatty Acids on Methylmercury-Induced Toxicity in Fish and Mammalian CellsIn Vitro". Journal of Biomedicine and Biotechnology 2012 (2012): 1–13. http://dx.doi.org/10.1155/2012/417652.

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Methylmercury (MeHg) is a ubiquitous environmental contaminant which bioaccumulates in marine biota. Fish constitute an important part of a balanced human diet contributing with health beneficial nutrients but may also contain contaminants such as MeHg. Interactions between the marine n-3 fatty acids eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) with MeHg-induced toxicity were investigated. Different toxic and metabolic responses were studied in Atlantic salmon kidney (ASK) cell line and the mammalian kidney-derived HEK293 cell line. Both cell lines were preincubated with DHA or EPA prior to MeHg-exposure, and cell toxicity was assessed differently in the cell lines by MeHg-uptake in cells (ASK and HEK293), proliferation (HEK293 and ASK), apoptosis (ASK), oxidation of the red-ox probe roGFP (HEK293), and regulation of selected toxicological and metabolic transcriptional markers (ASK). DHA was observed to decrease the uptake of MeHg in HEK293, but not in ASK cells. DHA also increased, while EPA decreased, MeHg-induced apoptosis in ASK. MeHg exposure induced changes in selected metabolic and known MeHg biomarkers in ASK cells. Both DHA and MeHg, but not EPA, oxidized roGFP in HEK293 cells. In conclusion, marine n-3 fatty acids may ameliorate MeHg toxicity, either by decreasing apoptosis (EPA) or by reducing MeHg uptake (DHA). However, DHA can also augment MeHg toxicity by increasing oxidative stress and apoptosis when combined with MeHg.

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Khader, Heba, Victor Solodushko, Abu Bakr Al-Mehdi, Jonathon Audia e Brian Fouty. "Overlap of Doxycycline Fluorescence with that of the Redox-Sensitive Intracellular Reporter roGFP". Journal of Fluorescence 24, n. 2 (28 novembre 2013): 305–11. http://dx.doi.org/10.1007/s10895-013-1331-6.

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Sivakumar, Krishnakumar, Manisha Mukherjee, Hsin-I. Cheng, Yingdan Zhang, Lianghui Ji e Bin Cao. "Surface display of roGFP for monitoring redox status of extracellular microenvironments inShewanella oneidensisbiofilms". Biotechnology and Bioengineering 112, n. 3 (21 ottobre 2014): 512–20. http://dx.doi.org/10.1002/bit.25471.

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Zhang, Qin, Xiaowei Li, Jiexin Lu, Liping Qiu, Shirui Pan, Xiaojun Chen e Junyang Chen. "ROG_PL: Robust Open-Set Graph Learning via Region-Based Prototype Learning". Proceedings of the AAAI Conference on Artificial Intelligence 38, n. 8 (24 marzo 2024): 9350–58. http://dx.doi.org/10.1609/aaai.v38i8.28788.

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Open-set graph learning is a practical task that aims to classify the known class nodes and to identify unknown class samples as unknowns. Conventional node classification methods usually perform unsatisfactorily in open-set scenarios due to the complex data they encounter, such as out-of-distribution (OOD) data and in-distribution (IND) noise. OOD data are samples that do not belong to any known classes. They are outliers if they occur in training (OOD noise), and open-set samples if they occur in testing. IND noise are training samples which are assigned incorrect labels. The existence of IND noise and OOD noise is prevalent, which usually cause the ambiguity problem, including the intra-class variety problem and the inter-class confusion problem. Thus, to explore robust open-set learning methods is necessary and difficult, and it becomes even more difficult for non-IID graph data. To this end, we propose a unified framework named ROG_PL to achieve robust open-set learning on complex noisy graph data, by introducing prototype learning. In specific, ROG_PL consists of two modules, i.e., denoising via label propagation and open-set prototype learning via regions. The first module corrects noisy labels through similarity-based label propagation and removes low-confidence samples, to solve the intra-class variety problem caused by noise. The second module learns open-set prototypes for each known class via non-overlapped regions and remains both interior and border prototypes to remedy the inter-class confusion problem. The two modules are iteratively updated under the constraints of classification loss and prototype diversity loss. To the best of our knowledge, the proposed ROG_PL is the first robust open-set node classification method for graph data with complex noise. Experimental evaluations of ROG_PL on several benchmark graph datasets demonstrate that it has good performance.

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