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Analysis of intracellular redox-based processes is constrained by the limited choice of appropriate biosensors. Fusion of human glutaredoxin-1 to an existing redox-sensitive GFP results in a ratiometric biosensor that allows rapid and sensitive dynamic imaging of glutathione redox potential in living cells. Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors. Conventional redox-sensitive GFPs (roGFPs) are limited by undefined specificity and slow response to changes in redox potential. In this study we demonstrate that the fusion of human glutaredoxin-1 (Grx1) to roGFP2 facilitates specific real-time equilibration between the sensor protein and the glutathione redox couple. The Grx1-roGFP2 fusion protein allowed dynamic live imaging of the glutathione redox potential ( E GSH ) in different cellular compartments with high sensitivity and temporal resolution. The biosensor detected nanomolar changes in oxidized glutathione (GSSG) against a backdrop of millimolar reduced glutathione (GSH) on a scale of seconds to minutes. It facilitated the observation of redox changes associated with growth factor availability, cell density, mitochondrial depolarization, respiratory burst activity and immune receptor stimulation.

Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that [almost equal to]65% of the total GR activity is attributed to GR1, whereas [almost equal to]35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (EGSH) in the cytosol. Using this tool, it is shown that EGSH in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.

The Arabidopsis (Arabidopsis thaliana) phytoalexin-deficient mutant pad2-1 displays enhanced susceptibility to a broad range pathogens and herbivorous insects that correlates with deficiencies in the production of camalexin, indole glucosinolates, and salicylic acid (SA). The pad2-1 mutation is localized in the GLUTAMATE-CYSTEINE LIGASE (GCL) gene encoding the enzyme of glutathione biosynthesis. While pad2-1 glutathione deficiency is not caused by a decrease in GCL transcripts, analysis of GCL protein level revealed that pad2-1 plants contained only 48% of the wild-type protein amount. In contrast to the wild type, the oxidized form of GCL was dominant in pad2-1, suggesting a distinct redox environment. This finding was corroborated by the expression of GRXl-roGFP2, showing that the cytosolic glutathione redox potential was significantly less negative in pad2-1. Analysis of oxidative stress-related gene expression showed a higher transcript accumulation in pad2-1 of GLUTATHIONE REDUCTASE, GLUTATHIONE-S-TRANSFERASE, and RESPIRATORY BURST OXIDASE HOMOLOG D in response to the oomycete Phytophthora brassicae. Interestingly, oligogalacturonide elicitation in pad2-1 revealed a lower membrane depolarization that was found to act upstream of an impaired hydrogen peroxide production. This impaired hydrogen peroxide production was also observed during pathogen infection and correlated with a reduced hypersensitive response in pad2-1. In addition, a lack of pathogen-triggered expression of the ISOCHORISMATE SYNTHASE1 gene, coding the SA-biosynthetic enzyme isochorismate synthase, was identified as the cause of the SA deficiency in pad2-1. Together, our results indicate that the padl-l mutation is related to a decrease in GCL protein and that the resulting glutathione deficiency negatively affects important processes of disease resistance.

Families of internationally adopted children may face specific problems with which general practitioners (GPs) may not be familiar. The aim of the study was to explore problems faced by families before, during and after the arrival of their internationally adopted child and to assess the usefulness of a specific medical structure for internationally adopted children, which could be a resource for the GP. We conducted a qualitative study using individual semistructured guided conversations and interviewed 21 families that had adopted a total of 26 children internationally in the Puy de Dome department, France, in 2003. Quantitative data were used to describe the pathologies diagnosed and the investigations performed.Our study showed that the history of these families, from the start of the adoption project to its achievement, is complex and warrants careful analysis. Health-care providers should not only consider the medical aspects of adoption, but should also be interested in the histories of these families, which may play a role in the forming of attachments between the adoptee and their adoptive parents and prevent further trouble during the development of the child. We also showed that adoptive parents have similar fears or transient difficulties that may be resolved quickly by listening and reassurance. Most such families would support the existence of a specific medical structure for internationally adopted children, which could be a resource for the general practitioner. However, the health-care providers interviewed were divided on the subject and expressed their fear that a special consultation could be stigmatizing to children and families. A specific consultation with well-trained and experienced practitioners acting in close collaboration with GPs and paediatricians may be of help in better understanding and supporting adopted children and their families.

• A highly negative glutathione redox potential (EGSH ) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and ironasulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear. • We dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing. • Our data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ATP-binding cassette (ABC) transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability. • We identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.

The Arabidopsis (Arabidopsis thaliana) phytoalexin-deficient mutant pad2-1 displays enhanced susceptibility to a broad range of pathogens and herbivorous insects that correlates with deficiencies in the production of camalexin, indole glucosinolates, and salicylic acid (SA). The pad2-1 mutation is localized in the GLUTAMATE-CYSTEINE LIGASE (GCL) gene encoding the first enzyme of glutathione biosynthesis. While pad2-1 glutathione deficiency is not caused by a decrease in GCL transcripts, analysis of GCL protein level revealed that pad2-1 plants contained only 48% of the wild-type protein amount. In contrast to the wild type, the oxidized form of GCL was dominant in pad2-1, suggesting a distinct redox environment. This finding was corroborated by the expression of GRX1-roGFP2, showing that the cytosolic glutathione redox potential was significantly less negative in pad2-1. Analysis of oxidative stress-related gene expression showed a higher transcript accumulation in pad2-1 of GLUTATHIONE REDUCTASE, GLUTATHIONE-S-TRANSFERASE, and RESPIRATORY BURST OXIDASE HOMOLOG D in response to the oomycete Phytophthora brassicae. Interestingly, oligogalacturonide elicitation in pad2-1 revealed a lower plasma membrane depolarization that was found to act upstream of an impaired hydrogen peroxide production. This impaired hydrogen peroxide production was also observed during pathogen infection and correlated with a reduced hypersensitive response in pad2-1. In addition, a lack of pathogen-triggered expression of the ISOCHORISMATE SYNTHASE1 gene, coding for the SA-biosynthetic enzyme isochorismate synthase, was identified as the cause of the SA deficiency in pad2-1. Together, our results indicate that the pad2-1 mutation is related to a decrease in GCL protein and that the resulting glutathione deficiency negatively affects important processes of disease resistance.

Background Cannabis is the most consumed illegal substance in France. General practitioners (GPs) are the health professionals who are most consulted by adolescents. Brief intervention (BI) is a promising care initiative for the consumption of cannabis, and could be a tool for GPs in caring for adolescents who consume cannabis. The aim of the CANABIC study is to measure the impact of a BI carried out by a GP on the consumption of cannabis by adolescents of 15 to 25 years of age. Methods A randomized clustered controlled trial, stratified over three areas (Auvergne, Languedoc-Roussillon, and Rhône - Alpes), comparing an intervention group, which carries out the BI in consultation, and a control group, which ensures routine medical care. The main assessment criterion is the consumption of cannabis by amount of joints per month, at 12 months. The amount necessary to highlight a significant difference between the two groups of 30% of consumption at 12 months is 250 patients (50 GPs, 5 patients per GP; risk α = 5%; power = 90%; intra-cluster correlation coefficient ρ = 0.2; Hawthorne effect = 15%; lost to follow-up rates for GPs = 10% and for patients = 20%). This plan is replicated for the three areas, and therefore a total of 750 patients are expected. The secondary criteria for judgment are the associated consumption of tobacco and alcohol, the perception of the consequences of consumption, and the driving of a vehicle following consumption. Discussion Research about BI for young cannabis users is underway. The aim of the CANABIC study is to validate a BI suited to adolescents who consume cannabis, which may be performed in the general practice. This would provide a tool for their treatment by a GP, which could be widely distributed during initial or further medical training. Trial registration CANABIC is a randomized clustered trial ( NCT01433692 , registered 2011 Sept 12), PHRC funded: Clinical Research Hospital Program (Governmental Fund, Health Ministry). Date first patient randomized: March 2012.

Educ Cv is a program of transfer and measurement of management skills of patients with cardiovascular risks through actions of therapeutic education. Its aim is to promote joint programs and shared therapeutic education in the different teams of the University Hospital of Clermont-Ferrand involved with this type of patient. Another goal is to promote common tools for measuring patient acquisitions, as well as for evaluating the efficacy of interventions in therapeutic education during short-term hospitalization and also during the ordinary management by general practitioners, particularly in heart failure patients. The objective of this paper is to present the process of implementation of the program and its early achievements. [PUBLICATION ABSTRACT] Reprinted by permission of Sage Publications Ltd

Educ Cv est un programme de transfert et de mesure de compétences en direction du patient à risques cardiovasculaires à travers des actions d’éducation thérapeutique. Son projet est de promouvoir des programmes communs et partagés d’éducation thérapeutique chez les différentes équipes du Centre hospitalier universitaire (CHU) de Clermont-Ferrand, intervenant auprès de ce type de patients. Il a également pour objectif de promouvoir des outils communs de mesure des acquisitions du patient mais également d’évaluation de l’efficacité des interventions en éducation thérapeutique aussi bien durant l’hospitalisation court séjour que durant la prise en charge courante du médecin généraliste, notamment chez les patients insuffisants cardiaques. L’objectif de cet article est de présenter le processus de mise en œuvre du programme et ses premières réalisations.