A cell-permeable biscyclooctyne as a novel probe for the identification of protein sulfenic acids

Reactive oxygen species act as important second messengers in cell signaling and homeostasis through the oxidation of protein thiols. However, the dynamic nature of protein oxidation and the lack of sensitivity of existing molecular probes have hindered our understanding of such reactions; therefore, new tools are required to address these challenges. We designed a bifunctional variant of the strained bicyclo[6.1.0]nonyne (BCN-E-BCN) that enables the tagging of intracellular protein sulfenic acids for biorthogonal copper-free click chemistry. In validation studies, BCN-E-BCN binds the sulfenylated form of the actin-severing protein cofilin, while mutation of the cognate cysteine residues abrogates its binding. BCN-E-BCN is cell permeable and reacts rapidly with cysteine sulfenic acids in cultured cells. Using different azide-tagged conjugates, we demonstrate that BCN-E-BCN can be used in various applications for the detection of sulfenylated proteins. Remarkably, cycloaddition of an azide-tagged fluorophore to BCN-E-BCN labelled proteins produced in vivo can be visualized by fluorescence microscopy to reveal their subcellular localization. These findings demonstrate a novel and multifaceted approach to the detection and trapping of sulfenic acids

Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat

Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo

Origin of two-band chorus in the radiation belt of Earth.

Naturally occurring chorus emissions are a class of electromagnetic waves found in the space environments of the Earth and other magnetized planets. They play an essential role in accelerating high-energy electrons forming the hazardous radiation belt environment. Chorus typically occurs in two distinct frequency bands separated by a gap. The origin of this two-band structure remains a 50-year old question. Here we report, using NASA's Van Allen Probe measurements, that banded chorus waves are commonly accompanied by two separate anisotropic electron components. Using numerical simulations, we show that the initially excited single-band chorus waves alter the electron distribution immediately via Landau resonance, and suppress the electron anisotropy at medium energies. This naturally divides the electron anisotropy into a low and a high energy components which excite the upper-band and lower-band chorus waves, respectively. This mechanism may also apply to the generation of chorus waves in other magnetized planetary magnetospheres

MitoNeoD:a mitochondria-targeted superoxide probe

Mitochondrial superoxide (O2⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅−, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O2⋅− probe, MitoNeoD, which can assess O2⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅− over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O2⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅−production in health and disease

Using exomarkers to assess mitochondrial reactive species in vivo

Background: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. Scope of review: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. Major conclusions and general significance: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Abbreviations: EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium catio

A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo.

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and as a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis. To address these problems, we have developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that enables us to extract and analyse both the CoQ redox state and the magnitude of the CoQ pool with negligible changes to redox state from small amounts of tissue. This will enable the physiological and pathophysiological roles of the CoQ redox state to be investigated in vivo

A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis. To address these problems, we have developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that enables us to extract and analyse both the CoQ redox state and the magnitude of the CoQ pool with negligible changes to redox state from small amounts of tissue. This will enable the physiological and pathophysiological roles of the CoQ redox state to be investigated in vivo

Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo

There is increasing interest in the effect of energy metabolism on oxidative stress, but much ambiguity over the relationship between the rate of oxygen consumption and the generation of reactive oxygen species (ROS). Production of ROS (such as hydrogen peroxide, H2O2) in the mitochondria is primarily inferred indirectly from measurements in vitro, which may not reflect actual ROS production in living animals. Here, we measured in vivo H2O2 content using the recently developed MitoB probe that becomes concentrated in the mitochondria of living organisms, where it is converted by H2O2 into an alternative form termed MitoP; the ratio of MitoP/MitoB indicates the level of mitochondrial H2O2 in vivo. Using the brown trout Salmo trutta, we tested whether this measurement of in vivo H2O2 content over a 24 h-period was related to interindividual variation in standard metabolic rate (SMR). We showed that the H2O2 content varied up to 26-fold among fish of the same age and under identical environmental conditions and nutritional states. Interindividual variation in H2O2 content was unrelated to mitochondrial density but was significantly associated with SMR: fish with a higher mass-independent SMR had a lower level of H2O2. The mechanism underlying this observed relationship between SMR and in vivo H2O2 content requires further investigation, but may implicate mitochondrial uncoupling which can simultaneously increase SMR but reduce ROS production. To our knowledge, this is the first study in living organisms to show that individuals with higher oxygen consumption rates can actually have lower levels of H2O2

Science and Ideology in Economic, Political, and Social Thought

This paper has two sources: One is my own research in three broad areas: business cycles, economic measurement and social choice. In all of these fields I attempted to apply the basic precepts of the scientific method as it is understood in the natural sciences. I found that my effort at using natural science methods in economics was met with little understanding and often considerable hostility. I found economics to be driven less by common sense and empirical evidence, then by various ideologies that exhibited either a political or a methodological bias, or both. This brings me to the second source: Several books have appeared recently that describe in historical terms the ideological forces that have shaped either the direct areas in which I worked, or a broader background. These books taught me that the ideological forces in the social sciences are even stronger than I imagined on the basis of my own experiences. The scientific method is the antipode to ideology. I feel that the scientific work that I have done on specific, long standing and fundamental problems in economics and political science have given me additional insights into the destructive role of ideology beyond the history of thought orientation of the works I will be discussing

Measurement in Economics and Social Science

The paper discusses measurement, primarily in economics, from both analytical and historical perspectives. The historical section traces the commitment to ordinalism on the part of economic theorists from the doctrinal disputes between classical economics and marginalism, through the struggle of orthodox economics against socialism down to the cold-war alliance between mathematical social science and anti-communist ideology. In economics the commitment to ordinalism led to the separation of theory from the quantitative measures that are computed in practice: price and quantity indexes, consumer surplus and real national product. The commitment to ordinality entered political science, via Arrow’s ‘impossibility theorem’, effectively merging it with economics, and ensuring its sterility. How can a field that has as its central result the impossibility of democracy contribute to the design of democratic institutions? The analytical part of the paper deals with the quantitative measures mentioned above. I begin with the conceptual clarification that what these measures try to achieve is a restoration of the money metric that is lost when prices are variable. I conclude that there is only one measure that can be embedded in a satisfactory economic theory, free from unreasonable restrictions. It is the Törnqvist index as an approximation to its theoretical counterpart the Divisia index. The statistical agencies have at various times produced different measures for real national product and its components, as well as related concepts. I argue that all of these are flawed and that a single deflator should be used for the aggregate and the components. Ideally this should be a chained Törnqvist price index defined on aggregate consumption. The social sciences are split. The economic approach is abstract, focused on the assumption of rational and informed behavior, and tends to the political right. The sociological approach is empirical, stresses the non-rational aspects of human behavior and tends to the political left. I argue that the split is due to the fact that the empirical and theoretical traditions were never joined in the social sciences as they were in the natural sciences. I also argue that measurement can potentially help in healing this split