Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure

Physiotherapy versus placebo or no intervention in Parkinson's disease

Background: Despite medical therapies and surgical interventions for Parkinson's disease (PD), patients develop progressive disability. Physiotherapy aims to maximise functional ability and minimise secondary complications through movement rehabilitation within a context of education and support for the whole person. The overall aim is to optimise independence, safety, and well-being, thereby enhancing quality of life.  Objectives: To assess the effectiveness of physiotherapy intervention compared with no intervention in patients with PD.  Search methods: We identified relevant trials by conducting electronic searches of numerous literature databases (e.g. MEDLINE, EMBASE) and trial registers, and by handsearching major journals, abstract books, conference proceedings, and reference lists of retrieved publications. The literature search included trials published up to the end of January 2012.  Selection criteria: Randomised controlled trials of physiotherapy intervention versus no physiotherapy intervention in patients with PD.  Data collection and analysis: Two review authors independently extracted data from each article. We used standard meta-analysis methods to assess the effectiveness of physiotherapy intervention compared with no physiotherapy intervention. Trials were classified into the following intervention comparisons: general physiotherapy, exercise, treadmill training, cueing, dance, and martial arts. We used tests for heterogeneity to assess for differences in treatment effect across these different physiotherapy interventions.  Main results: We identified 39 trials with 1827 participants. We considered the trials to be at a mixed risk of bias as the result of unreported allocation concealment and probable detection bias. Compared with no intervention, physiotherapy significantly improved the gait outcomes of speed (mean difference 0.04 m/s, 95% confidence interval (CI) 0.02 to 0.06, P = 0.0002); two- or six-minute walk test (13.37 m, 95% CI 0.55 to 26.20, P = 0.04) and Freezing of Gait questionnaire (-1.41, 95% CI -2.63 to -0.19, P = 0.02); functional mobility and balance outcomes of Timed Up & Go test (-0.63 s, 95% CI -1.05 to -0.21, P = 0.003), Functional Reach Test (2.16 cm, 95% CI 0.89 to 3.43, P = 0.0008), and Berg Balance Scale (3.71 points, 95% CI 2.30 to 5.11, P < 0.00001); and clinician-rated disability using the Unified Parkinson’s Disease Rating Scale (UPDRS) (total -6.15 points, 95% CI-8.57 to -3.73, P < 0.00001; activities of daily living: -1.36, 95% CI -2.41 to -0.30, P = 0.01; and motor: -5.01, 95% CI -6.30 to -3.72, P < 0.00001). No difference between arms was noted in falls (Falls Efficacy Scale: -1.91 points, 95% CI -4.76 to 0.94, P = 0.19) or patient-rated quality of life (PDQ-39 Summary Index: -0.38 points, 95% CI -2.58 to 1.81, P = 0.73). One study reported that adverse events were rare; no other studies reported data on this outcome. Indirect comparisons of the different physiotherapy interventions revealed no evidence that the treatment effect differed across physiotherapy interventions for any of the outcomes assessed.  Authors' conclusions: Benefit for physiotherapy was found in most outcomes over the short term (i.e. < 3 months) but was significant only for speed, two- or six-minute walk test, Freezing of Gait questionnaire, Timed Up & Go, Functional Reach Test, Berg Balance Scale, and clinician-rated UPDRS. Most of the observed differences between treatments were small. However, for some outcomes (e.g. speed, Berg Balance Scale, UPDRS), the differences observed were at, or approaching, what are considered minimal clinically important changes. These benefits should be interpreted with caution because the quality of most of the included trials was not high. Variation in measurements of outcome between studies meant that our analyses include a small proportion of the participants recruited.  This review illustrates that a wide range of approaches are employed by physiotherapists to treat patients with PD. However, no evidence of differences in treatment effect was noted between the different types of physiotherapy interventions being used, although this was based on indirect comparisons. A consensus menu of 'best practice' physiotherapy is needed, as are large, well-designed randomised controlled trials undertaken to demonstrate the longer-term efficacy and cost-effectiveness of 'best practice' physiotherapy in PD

Ontwikkeling van een geïntegreerde kosten-baten analyse methode van multifunctioneel ruimtegebruik in de Noordzee en kustzone

De Noordzee is zowel een ecologisch als sociaal-economisch belangrijk gebied waarbij deze aspecten niet langer als aparte onderdelen kunnen worden beschouwd. Bij de inrichting en gebruik van de Noordzee en kustzone moet rekening worden gehouden met de verschillende belangen en factoren van de gebruikers en is een geïntegreerde aanpak nodig. Om beleidsbeslissingen met betrekking tot het ruimtelijk gebruik van de Noordzee te vereenvoudigen is het ministerie LNV in 2003 gestart met project “Noordzee en kust” (P418). In dit kader werken de leerstoelgroep Milieusysteemanalyse (MSA) van Wageningen Universiteit en Research Centre, Alterra Texel en het LEI samen. In 2003 is een geïntegreerde Kosten-Baten Analyse methode met betrekking tot multifunctioneel ruimtegebruik in het Nederlandse gedeelte van de Noordzee ontwikkeld. Deze methode zal worden verfijnd en getoetst in de resterende tijd van dit onderzoek (2004 tot en met 2006). Het raamwerk dat is ontwikkeld voor dit onderzoek bestaat uit vier onderdelen: i) de definiëring van functies, goederen, diensten en waarden, ii) belangenafweging en conflictanalyse iii) besluitvorming en iv) planning en uitvoeringsfase.In deze eerste fase is het systeem “de Noordzee” beschreven op basis van gebiedskenmerken en ecotopen. Verschillende gebieden in de Noordzee zijn onderzocht waarbij blijkt dat er nog leemtes in kennis zijn die kunnen worden aangevuld, zoals voor bijvoorbeeld de Zeeuwse banken en diepere gaten in de zuidelijke Noordzee. De systeembeschrijving dient als basis voor de eerste stap van het ontwikkelde raamwerk; de functie analyse. In deze eerste fase is ook alvast een eerste inventarisatie gemaakt van de ecologische en sociaal-economische functies van de Noordzee en kustzone. Sommige ecosysteem functies hebben vooral een economisch belang, zoals de visserij, olie-, gas-, en zandwinning, terwijl andere functies meer gerelateerd zijn aan een ecologisch belang zoals de regulatie (nutriënten huishouding) en habitat functies (kraamkamer) of het sociaal-culturele belang (kunst, esthetisch, geschiedenis etc.). Wanneer alle functies en belangen zo goed mogelijk in kaart gebracht zijn kan aan de hand daarvan een maatschappelijke discussie worden gestart om complexe problemen in de Noordzee en kustgebied aan te pakken, waarbij beleidsbeslissingen kunnen worden vereenvoudigd door een meer evenwichtige afweging van de kosten en baten die gepaard gaan met diverse scenario’s.Voor een goed beleid en beheer van de Noordzee en kustgebieden is een maatschappelijk gerichte aanpak nodig waarbij integratie op meerdere niveaus plaatsvindt, namelijk: i) integratie van ecologische, economische en sociale aspecten, ii) integratie tussen wetenschappers, beleidsmaker en burgers, iii) institutionele integratie (zowel verticale beleidsintegratie als horizontale beleidsintegratie (tussen verschillende sectoren)) en iv) een integratie over tijd en ruimte. Communicatie op alle niveaus is daarbij belangrijk om de wederzijdse afhankelijkheid tussen stakeholders zichtbaar te maken. Relevante stakeholders met een belang in de Noordzee zullen worden geïdentificeerd in de volgende fase (2004) van dit onderzoek. Deze groepen zullen worden geïnterviewd om hun ideeën, missies, doelstellingen en belangen te onderzoeken. Op die manier kunnen tegenstrijdige belangen/ conflicten en daarnaast mogelijkheden voor meervoudig ruimtegebruik worden geanalyseerd. Workshops zullen worden gebruikt om een idee te vormen hoe de “consensus building proces” zou moeten worden ontworpen om het beleid en beheer van de Noordzee te faciliteren. Dit project wordt afgerond met een case studie waarmee het ontwikkelde raamwerk zal worden getoetst

Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes.

Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon

Gentamicin Rapidly Inhibits Mitochondrial Metabolism in High-Frequency Cochlear Outer Hair Cells

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies

Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells

Confirmation of the Cardioprotective Effect of MitoGamide in the Diabetic Heart

Abstract: Purpose: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide. Methods and Results: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice. The myocardium of Akita mice had a decreased ATP/ADP ratio, elevated mitochondrial oxidative stress and increased organelle density, compared with that of wild-type mice. MitoGamide, a mitochondria-targeted 1,2-dicarbonyl scavenger, exhibited good stability in vivo, uptake into cells and mitochondria and reactivity with dicarbonyls. Treatment of Akita mice with MitoGamide for 12 weeks significantly improved the E/A ratio compared with the vehicle-treated group. Conclusion: Our work confirms that the Akita mouse model of diabetes replicates key clinical features of diabetic HFpEF, including cardiac and mitochondrial dysfunction. Furthermore, in this independent study, MitoGamide treatment improved diastolic function in Akita mice