Oxiforms: Unique cysteine residue- and chemotype-specified chemical combinations can produce functionally-distinct proteoforms: Like how mixing primary colours creates new shades, cysteine residue- and chemotype-specified chemical combinations can produce functionally-distinct proteoforms called oxiforms: Like how mixing primary colours creates new shades, cysteine residue- and chemotype-specified chemical combinations can produce functionally-distinct proteoforms called oxiforms.

A single protein molecule with one or more cysteine residues can occupy a plurality of unique residue and oxidation-chemotype specified proteoforms that I term oxiforms. In binary reduced or oxidised terms, one molecule with three cysteines will adopt one of eight unique oxiforms. Residue-defined sulfur chemistry endows specific oxiforms with distinct functionally-relevant biophysical properties (e.g., steric effects). Their emergent complexity means a functionally-relevant effect may only manifest when multiple cysteines are oxidised. Like how mixing colours makes new shades, combining discrete redox chemistries-colours-can create a kaleidoscope of oxiform hues. The sheer diversity of oxiforms co-existing within the human body provides a biological basis for redox heterogeneity. Of evolutionary significance, oxiforms may enable individual cells to mount a broad spectrum of responses to the same stimulus. Their biological significance, however plausible, is speculative because protein-specific oxiforms remain essentially unexplored. Excitingly, pioneering new techniques can push the field into uncharted territory by quantifying oxiforms. The oxiform concept can advance our understanding of redox-regulation in health and disease.

Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime.

Duchenne muscular dystrophy (DMD) is associated with distinct mitochondrial stress responses. Here, we aimed to determine whether the prospective mitochondrial-enhancing compound Olesoxime, prevents early-stage mitochondrial stress in limb and respiratory muscle from D2.mdx mice using a proof-of-concept short-term regimen spanning 10-28 days of age. As mitochondrial-cytoplasmic energy transfer occurs via ATP- or phosphocreatine-dependent phosphate shuttling, we assessed bioenergetics with or without creatine in vitro. We observed that disruptions in Complex I-supported respiration and mH2O2 emission in D2.mdx quadriceps and diaphragm were amplified by creatine demonstrating mitochondrial creatine insensitivity manifests ubiquitously and early in this model. Olesoxime selectively rescued or maintained creatine sensitivity in both muscles, independent of the abundance of respiration-related mitochondrial proteins or mitochondrial creatine kinase cysteine oxidation in quadriceps. Mitochondrial calcium retention capacity and glutathione were altered in a muscle-specific manner in D2.mdx but were generally unchanged by Olesoxime. Treatment reduced serum creatine kinase (muscle damage) and preserved cage hang-time, microCT-based volumes of lean compartments including whole body, hindlimb and bone, recovery of diaphragm force after fatigue, and cross-sectional area of diaphragm type IIX fiber, but reduced type I fibers in quadriceps. Grip strength, voluntary wheel-running and fibrosis were unaltered by Olesoxime. In summary, locomotor and respiratory muscle mitochondrial creatine sensitivities are lost during early stages in D2.mdx mice but are preserved by short-term treatment with Olesoxime in association with specific indices of muscle quality suggesting early myopathy in this model is at least partially attributed to mitochondrial stress.

Synapse Pruning: Mitochondrial ROS with Their Hands on the Shears.

No overarching hypotheses tie the basic mechanisms of mitochondrial reactive oxygen species (ROS) production to activity dependent synapse pruning-a fundamental biological process in health and disease. Neuronal activity divergently regulates mitochondrial ROS: activity decreases whereas inactivity increases their production, respectively. Placing mitochondrial ROS as innate synaptic activity sentinels informs the novel hypothesis that: (1) at an inactive synapse, increased mitochondrial ROS production initiates intrinsic apoptosis dependent pruning; and (2) at an active synapse, decreased mitochondrial ROS production masks intrinsic apoptosis dependent pruning. Immature antioxidant defense may enable the developing brain to harness mitochondrial ROS to prune weak synapses. Beyond development, endogenous antioxidant defense constrains mitochondrial (ROS) to mask pruning. Unwanted age-related synapse loss may arise when mitochondrial ROS aberrantly recapitulate developmental pruning. Placing mitochondrial ROS with their hands on the shears is beneficial in early but deleterious in later life.

Whole-Body Vibration Training and Its Application to Age-Related Performance Decrements: An Exploratory Analysis.

Middle age is associated with a pronounced decline in power and flexibility. Whilst whole-body vibration training (WBVT) improves performance in a range of populations, whether WBVT can improve muscle power and flexibility in a middle-aged population is not known. The present study aimed to determine the influence of 5 weeks progressive WBVT in middle-aged (45-55 years) and younger (20-30 years) recreationally active females. Participants in each age group were randomly allocated to an intervention (WBVT) or control group. The WBVT groups trained for 5 weeks on a vibration platform, while the control groups performed identical exercises, with no vibration. Prior to, and after, the 5-week study vertical countermovement jump (VCMJ) and range of motion (ROM) performance were measured. WBVT significantly (p = 0.001) improved VCMJ performance when compared to the control groups. This improvement was significantly (p = 0.001) greater in the middle-aged compared with the younger WBVT group. WBVT significantly (p = 0.001) improved ROM irrespective of age. Taken together, these results suggest that WBVT can off-set age related performance decrements, which has therapeutic implications for musculoskeletal aging. Therefore, WBVT could be undertaken to minimise age-related performance deterioration in middle-aged female populations.

Exercise improves mitochondrial and redox-regulated stress responses in the elderly: better late than never!

Ageing is associated with several physiological declines to both the cardiovascular (e.g. reduced aerobic capacity) and musculoskeletal system (muscle function and mass). Ageing may also impair the adaptive response of skeletal muscle mitochondria and redox-regulated stress responses to an acute exercise bout, at least in mice and rodents. This is a functionally important phenomenon, since (1) aberrant mitochondrial and redox homeostasis are implicated in the pathophysiology of musculoskeletal ageing and (2) the response to repeated exercise bouts promotes exercise adaptations and some of these adaptations (e.g. improved aerobic capacity and exercise-induced mitochondrial remodelling) offset age-related physiological decline. Exercise-induced mitochondrial remodelling is mediated by upstream signalling events that converge on downstream transcriptional co-factors and factors that orchestrate a co-ordinated nuclear and mitochondrial transcriptional response associated with mitochondrial remodelling. Recent translational human investigations have demonstrated similar exercise-induced mitochondrial signalling responses in older compared with younger skeletal muscle, regardless of training status. This is consistent with data indicating normative mitochondrial remodelling responses to long-term exercise training in the elderly. Thus, human ageing is not accompanied by diminished mitochondrial plasticity to acute and chronic exercise stimuli, at least for the signalling pathways measured to date. Exercise-induced increases in reactive oxygen and nitrogen species promote an acute redox-regulated stress response that manifests as increased heat shock protein and antioxidant enzyme content. In accordance with previous reports in rodents and mice, it appears that sedentary ageing is associated with a severely attenuated exercise-induced redox stress response that might be related to an absent redox signal. In this regard, regular exercise training affords some protection but does not completely override age-related defects. Despite some failed redox-regulated stress responses, it seems mitochondrial responses to exercise training are intact in skeletal muscle with age and this might underpin the protective effect of exercise training on age-related musculoskeletal decline. Whilst further investigation is required, recent data suggest that it is never too late to begin exercise training and that lifelong training provides protection against several age-related declines at both the molecular (e.g. reduced mitochondrial function) and whole-body level (e.g. aerobic capacity).

50 shades of oxidative stress: A state-specific cysteine redox pattern hypothesis.

Oxidative stress is biochemically complex. Like primary colours, specific reactive oxygen species (ROS) and antioxidant inputs can be mixed to create unique "shades" of oxidative stress. Even a minimal redox module comprised of just 12 (ROS & antioxidant) inputs and 3 outputs (oxidative damage, cysteine-dependent redox-regulation, or both) yields over half a million "shades" of oxidative stress. The present paper proposes the novel hypothesis that: state-specific shades of oxidative stress, such as a discrete disease, are associated with distinct tell-tale cysteine oxidation patterns. The patterns are encoded by many parameters, from the identity of the oxidised proteins, the cysteine oxidation type, and magnitude. The hypothesis is conceptually grounded in distinct ROS and antioxidant inputs coalescing to produce unique cysteine oxidation outputs. And considers the potential biological significance of the holistic cysteine oxidation outputs. The literature supports the existence of state-specific cysteine oxidation patterns. Measuring and manipulating these patterns offer promising avenues for advancing oxidative stress research. The pattern inspired hypothesis provides a framework for understanding the complex biochemical nature of state-specific oxidative stress.

Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress.

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible "off-the-shelf" nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the "unseen" microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6-7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1 cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The "unseen" microplate data were "seen-to-be-believed" via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n = 3) and sample (n = 100) n-plex capacities in ∼4 h with 50-70 min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress.

RedoxiFluor: A microplate technique to quantify target-specific protein thiol redox state in relative percentage and molar terms.

Unravelling how reactive oxygen species regulate fundamental biological processes is hampered by the lack of an accessible microplate technique to quantify target-specific protein thiol redox state in percentages and moles. To meet this unmet need, we present RedoxiFluor. RedoxiFluor uses two spectrally distinct thiol-reactive fluorescent conjugated reporters, a capture antibody, detector antibody and a standard curve to quantify target-specific protein thiol redox state in relative percentage and molar terms. RedoxiFluor can operate in global mode to assess the redox state of the bulk thiol proteome and can simultaneously assess the redox state of multiple targets in array mode. Extensive proof-of-principle experiments robustly validate the assay principle and the value of each RedoxiFluor mode in diverse biological contexts. In particular, array mode RedoxiFluor shows that the response of redox-regulated phosphatases to lipopolysaccharide (LPS) differs in human monocytes. Specifically, LPS increased PP2A-, SHP1-, PTP1B-, and CD45-specific reversible thiol oxidation without changing the redox state of calcineurin, PTEN, and SHP2. The relative percentage and molar terms are interpretationally useful and define the most complete and extensive microplate redox analysis achieved to date. RedoxiFluor is a new antibody technology with the power to quantify relative target-specific protein thiol redox state in percentages and moles relative to the bulk thiol proteome and selected other targets in a widely accessible, simple and easily implementable microplate format.

Exercise decreases PP2A-specific reversible thiol oxidation in human erythrocytes: Implications for redox biomarkers.

New readily accessible systemic redox biomarkers are needed to understand the biological roles reactive oxygen species (ROS) play in humans because overtly flawed, technically fraught, and unspecific assays severely hamper translational progress. The antibody-linked oxi-state assay (ALISA) makes it possible to develop valid ROS-sensitive target-specific protein thiol redox state biomarkers in a readily accessible microplate format. Here, we used a maximal exercise bout to disrupt redox homeostasis in a physiologically meaningful way to determine whether the catalytic core of the serine/threonine protein phosphatase PP2A is a candidate systemic redox biomarker in human erythrocytes. We reasoned that: constitutive oxidative stress (e.g., haemoglobin autoxidation) would sensitise erythrocytes to disrupted ion homeostasis as manifested by increased oxidation of the ion regulatory phosphatase PP2A. Unexpectedly, an acute bout of maximal exercise lasting ~16 min decreased PP2A-specific reversible thiol oxidation (redox ratio, rest: 0.46; exercise: 0.33) without changing PP2A content (rest: 193 pg/ml; exercise: 191 pg/ml). The need for only 3-4 µl of sample to perform ALISA means PP2A-specific reversible thiol oxidation is a capillary-fingertip blood-compatible candidate redox biomarker. Consistent with biologically meaningful redox regulation, thiol reductant-inducible PP2A activity was significantly greater (+10%) at rest compared to exercise. We establish a route to developing new readily measurable protein thiol redox biomarkers for understanding the biological roles ROS play in humans.

N-Acetylcysteine's attenuation of fatigue after repeated bouts of intermittent exercise: practical implications for tournament situations.

Production of reactive oxygen species (ROS) during muscle contractions is associated with muscle fatigue and damage in the short term and adaptive responses in the long term. When adaptation is inconsequential acute antioxidant supplementation may be able to attenuate muscle fatigue and damage to enhance performance. This study aimed to determine the effects of acute oral N-acetylcysteine (NAC) supplementation on Yo-Yo Intermittent Recovery Test Level 1 (YIRT-L1) performance after repeated bouts of damaging intermittent exercise. In a pair-matched design, 12 recreationally trained men engaged in 6 d of either NAC (n = 6) or placebo (n = 6) supplementation. After a treatment-loading day, participants completed 3 testing sessions, on alternating days, consisting of a preexercise isokinetic dynamometry (IKD) test, a damaging intermittent-exercise protocol, YIRT-L1, and a postexercise IKD test. Another IKD test was completed on the 2 intervening d. NAC treatment resulted in a significant preservation of YIRT-L1 performance (p ≤ .0005). IKD performance significantly deteriorated over time at all contraction speeds, and this deterioration was not influenced by treatment group. Plasma creatine kinase values increased significantly over time (p = .002) and were significantly greater in the NAC group than in the placebo group (p = .029). NAC induced mild gastrointestinal side effects. NAC supplementation may be a useful strategy to enhance performance during short-term competitive situations when adaption is inconsequential. Titration studies to elucidate a treatment dose that enhances performance without inducing side effects are now required.

ALISA: A microplate assay to measure protein thiol redox state.

Measuring protein thiol redox state is central to understanding redox signalling in health and disease. The lack of a microplate assay to measure target specific protein thiol redox state rate-limits progress on accessibility grounds: redox proteomics is inaccessible to most. Developing a microplate assay is important for accelerating discovery by widening access to protein thiol redox biology. Beyond accessibility, enabling high throughput time- and cost-efficient microplate analysis is important. To meet the pressing need for a microplate assay to measure protein thiol redox state, we present the Antibody-Linked Oxi-State Assay (ALISA). ALISA uses a covalently bound capture antibody to bind a thiol-reactive fluorescent conjugated maleimide (F-MAL) decorated target. The capture antibody-target complex is labelled with an amine-reactive fluorescent N-hydroxysuccinimide ester (F-NHS) to report total protein. The covalent bonds that immobilise the capture antibody to the epoxy group functionalised microplate enable one to selectively elute the target. Target specific redox state is ratiometrically calculated as: F-MAL (i.e., reversible thiol oxidation)/F-NHS (i.e., total protein). After validating the assay principle (i.e., increased target specific reversible thiol oxidation increases the ratio), we used ALISA to determine whether fertilisation-a fundamental biological process-changes Akt, a serine/threonine protein kinase, specific reversible thiol oxidation. Fertilisation significantly decreases Akt specific reversible thiol oxidation in Xenopus laevis 2-cell zygotes compared to unfertilised eggs. ALISA is an accessible microplate assay to advance knowledge of protein thiol redox biology in health and disease.

Mechanisms of Mitochondrial ROS Production in Assisted Reproduction: The Known, the Unknown, and the Intriguing.

The consensus that assisted reproduction technologies (ART), like in vitro fertilization, to induce oxidative stress (i.e., the known) belies how oocyte/zygote mitochondria-a major presumptive oxidative stressor-produce reactive oxygen species (ROS) with ART being unknown. Unravelling how oocyte/zygote mitochondria produce ROS is important for disambiguating the molecular basis of ART-induced oxidative stress and, therefore, to rationally target it (e.g., using site-specific mitochondria-targeted antioxidants). I review the known mechanisms of ROS production in somatic mitochondria to critique how oocyte/zygote mitochondria may produce ROS (i.e., the unknown). Several plausible site- and mode-defined mitochondrial ROS production mechanisms in ART are proposed. For example, complex I catalyzed reverse electron transfer-mediated ROS production is conceivable when oocytes are initially extracted due to at least a 10% increase in molecular dioxygen exposure (i.e., the intriguing). To address the term oxidative stress being used without recourse to the underlying chemistry, I use the species-specific spectrum of biologically feasible reactions to define plausible oxidative stress mechanisms in ART. Intriguingly, mitochondrial ROS-derived redox signals could regulate embryonic development (i.e., their production could be beneficial). Their potential beneficial role raises the clinical challenge of attenuating oxidative damage while simultaneously preserving redox signaling. This discourse sets the stage to unravel how mitochondria produce ROS in ART, and their biological roles from oxidative damage to redox signaling.

The mitochondria-targeted antioxidant MitoQ, attenuates exercise-induced mitochondrial DNA damage.

High-intensity exercise damages mitochondrial DNA (mtDNA) in skeletal muscle. Whether MitoQ - a redox active mitochondrial targeted quinone - can reduce exercise-induced mtDNA damage is unknown. In a double-blind, randomized, placebo-controlled design, twenty-four healthy male participants consisting of two groups (placebo; n = 12, MitoQ; n = 12) performed an exercise trial of 4 x 4-min bouts at 90-95% of heart rate max. Participants completed an acute (20 mg MitoQ or placebo 1-h pre-exercise) and chronic (21 days of supplementation) phase. Blood and skeletal muscle were sampled immediately pre- and post-exercise and analysed for nuclear and mtDNA damage, lipid hydroperoxides, lipid soluble antioxidants, and the ascorbyl free radical. Exercise significantly increased nuclear and mtDNA damage across lymphocytes and muscle (P < 0.05), which was accompanied with changes in lipid hydroperoxides, ascorbyl free radical, and α-tocopherol (P < 0.05). Acute MitoQ treatment failed to impact any biomarker likely due to insufficient initial bioavailability. However, chronic MitoQ treatment attenuated nuclear (P < 0.05) and mtDNA damage in lymphocytes and muscle tissue (P < 0.05). Our work is the first to show a protective effect of chronic MitoQ supplementation on the mitochondrial and nuclear genomes in lymphocytes and human muscle tissue following exercise, which is important for genome stability.

Proteomic strategies to unravel age-related redox signalling defects in skeletal muscle.

Increased oxidative damage and disrupted redox signalling are consistently associated with age-related loss of skeletal muscle mass and function. Redox signalling can directly regulate biogenesis and degradation pathways and indirectly via activation of key transcription factors. Contracting skeletal muscle fibres endogenously generate free radicals (e.g. superoxide) and non-radical derivatives (e.g. hydrogen peroxide). Exercise induced redox signalling can promote beneficial adaptive responses that are disrupted by age-related redox changes. Identifying and quantifying the redox signalling pathways responsible for successful adaptation to exercise makes skeletal muscle an attractive physiological model for redox proteomic approaches. Site specific identification of the redox modification and quantification of site occupancy in the context of protein abundance remains a crucial concept for redox proteomics approaches. Notwithstanding, the technical limitations associated with skeletal muscle for proteomic analysis, we discuss current approaches for the identification and quantification of transient and stable redox modifications that have been employed to date in ageing research. We also discuss recent developments in proteomic approaches in skeletal muscle and potential implications and opportunities for investigating disrupted redox signalling in skeletal muscle ageing.

Exercise-induced muscle damage: What is it, what causes it and what are the nutritional solutions?

Exercise-induced muscle damage (EIMD) is characterized by symptoms that present both immediately and for up to 14 days after the initial exercise bout. The main consequence of EIMD for the athlete is the loss of skeletal muscle function and soreness. As such, numerous nutrients and functional foods have been examined for their potential to ameliorate the effects of EIMD and accelerate recovery, which is the purpose of many nutritional strategies for the athlete. However, the trade-off between recovery and adaptation is rarely considered. For example, many nutritional interventions described in this review target oxidative stress and inflammation, both thought to contribute to EIMD but are also crucial for the recovery and adaptation process. This calls into question whether long term administration of supplements and functional foods used to target EIMD is indeed best practice. This rapidly growing area of sports nutrition will benefit from careful consideration of the potential hormetic effect of long term use of nutritional aids that ameliorate muscle damage. This review provides a concise overview of what EIMD is, its causes and consequences and critically evaluates potential nutritional strategies to ameliorate EIMD. We present a pragmatic practical summary that can be adopted by practitioners and direct future research, with the purpose of pushing the field to better consider the fine balance between recovery and adaptation and the potential that nutritional interventions have in modulating this balance.

Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation.

Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M-1 s-1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation-an established molecular correlate of impaired enzyme activity-in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50-60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.

Mitochondrial ROS cause motor deficits induced by synaptic inactivity: Implications for synapse pruning.

Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bungarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP5+ and/or MnTnBuOE-2-PyP5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS-using mitochondria-targeted Paraquat (MitoPQ)-recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP5+. We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features.

Exercise redox biochemistry: Conceptual, methodological and technical recommendations.

Exercise redox biochemistry is of considerable interest owing to its translational value in health and disease. However, unaddressed conceptual, methodological and technical issues complicate attempts to unravel how exercise alters redox homeostasis in health and disease. Conceptual issues relate to misunderstandings that arise when the chemical heterogeneity of redox biology is disregarded: which often complicates attempts to use redox-active compounds and assess redox signalling. Further, that oxidised macromolecule adduct levels reflect formation and repair is seldom considered. Methodological and technical issues relate to the use of out-dated assays and/or inappropriate sample preparation techniques that confound biochemical redox analysis. After considering each of the aforementioned issues, we outline how each issue can be resolved and provide a unifying set of recommendations. We specifically recommend that investigators: consider chemical heterogeneity, use redox-active compounds judiciously, abandon flawed assays, carefully prepare samples and assay buffers, consider repair/metabolism, use multiple biomarkers to assess oxidative damage and redox signalling.

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.