ABSTRACT
Redox modifications to the plasma protein albumin have the potential to be used as biomarkers of disease progression and treatment efficacy in pathologies associated with inflammation and oxidative stress. One such pathology is Duchenne muscular dystrophy (DMD), a fatal childhood disease characterised by severe muscle wasting. We have previously shown in the mdx mouse model of DMD that plasma albumin thiol oxidation is increased; therefore, the first aim of this paper was to establish that albumin thiol oxidation in plasma reflects levels within mdx muscle tissue. We therefore developed a method to measure tissue albumin thiol oxidation. We show that albumin thiol oxidation was increased in both mdx muscle and plasma, with levels correlated with measures of dystropathology. In dystrophic muscle, albumin content was associated with areas of myonecrosis. The second aim was to test the ability of plasma thiol oxidation to track acute changes in dystropathology: we therefore subjected mdx mice to a single treadmill exercise session (known to increase myonecrosis) and took serial blood samples. This acute exercise caused a transient increase in total plasma albumin oxidation and measures of dystropathology. Together, these data support the use of plasma albumin thiol oxidation as a biomarker to track active myonecrosis in DMD.
ABSTRACT
Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. The objective of the study was to evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero proinflammatory stimulus. Fetal lambs (n = 51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mg·kg-1·h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline 7 days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a two-way ANOVA. Fetal creatine supplementation increased lung creatine content by 149% (PCr < 0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS < 0.0001) and increased levels of CD45+ leukocytes (PLPS < 0.0001) and MPO+ (PLPS < 0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr = 0.045) and MPO+ cells (PCr = 0.012) in the lungs and reduced thiol oxidation in plasma (PCr < 0.01) and lung tissue (PCr = 0.02). In conclusion, fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.NEW & NOTEWORTHY We evaluated the effect of antenatal creatine supplementation to reduce pulmonary inflammation and oxidative stress in the fetal lamb lungs arising from lipopolysaccharide (LPS)-induced chorioamnionitis. Fetal creatine supplementation increased lung creatine content and had no adverse effects on systemic fetal physiology and overall lung architecture. Importantly, fetuses that received creatine had significantly lower levels of inflammation and oxidative stress in the lungs, suggesting an anti-inflammatory and antioxidant benefit of creatine.
Subject(s)
Chorioamnionitis , Creatine , Dietary Supplements , Lipopolysaccharides , Lung , Oxidative Stress , Animals , Chorioamnionitis/drug therapy , Chorioamnionitis/metabolism , Chorioamnionitis/pathology , Creatine/pharmacology , Female , Oxidative Stress/drug effects , Pregnancy , Sheep , Lung/drug effects , Lung/metabolism , Lung/pathology , Pneumonia/metabolism , Pneumonia/prevention & control , Pneumonia/drug therapy , Pneumonia/pathology , Disease Models, Animal , Fetus/metabolism , Fetus/drug effectsABSTRACT
PURPOSE: Exercise-induced muscle damage (EIMD) results in the generation of reactive oxygen species (ROS), but little is known about the temporal profile of change in ROS post-EIMD and how ROS levels relate to the onset of and recovery from EIMD. Our primary aim was to examine the effect of EIMD on the pattern of change in the blood level of thiol-oxidised albumin, a marker of oxidative stress. METHODS: Seven male participants were subjected on separate days to eccentric muscle contraction to cause EIMD or a no-exercise condition. After each session, the participants collected daily dried blood spots to measure thiol-oxidised albumin and returned to the laboratory every 2 days for the assessment of indirect markers of EIMD, namely maximal voluntary contraction (MVC), delayed onset muscle soreness (DOMS), creatine kinase (CK), and myoglobin. RESULTS: Eccentric exercise resulted in a significant decrease in MVC and increase in DOMS, CK, myoglobin, and thiol-oxidised albumin with the latter reaching above baseline level within 24-48 h post-exercise. All the markers of EIMD returned to baseline level within 6 days post-exercise, but not the level of thiol-oxidised albumin which remained elevated for 10 days after exercise. There was a moderate correlation between changes in thiol-oxidised albumin and DOMS, but no significant relationship between any other markers of muscle damage. CONCLUSION: The levels of thiol-oxidised albumin increase in response to EIMD and remain elevated for several days post-exercise. The temporal pattern of change in the level of thiol-oxidised albumin suggests that this may be a useful biomarker of muscle repair post-EIMD.
ABSTRACT
Duchenne muscular dystrophy (DMD) is a fatal X-linked disease characterised by severe muscle wasting. The mechanisms underlying the DMD pathology likely involve the interaction between inflammation, oxidative stress and impaired Ca2+ signalling. Hypochlorous acid (HOCl) is a highly reactive oxidant produced endogenously via myeloperoxidase; an enzyme secreted by neutrophils that is significantly elevated in dystrophic muscle. Oxidation of Ca2+ -handling proteins by HOCl may impair Ca2+ signalling. This study aimed to determine the effects of HOCl on skeletal muscle function and its potential contribution to the dystrophic pathology. Extensor digitorum longus (EDL), soleus and interosseous muscles were surgically isolated from anaesthetised C57 (wild-type) and mdx (dystrophic) mice for measurement of ex vivo force production and intracellular Ca2+ concentration. In whole EDL muscle, HOCl (200 µM) significantly decreased maximal force and increased resting muscle tension which was only partially reversible by dithiothreitol. The effects of HOCl (200 µM) on maximal force in slow-twitch soleus were lower than found in the fast-twitch EDL muscle. In single interosseous myofibres, HOCl (10 µM) significantly increased resting intracellular Ca2+ concentration and decreased Ca2+ transient amplitude. These effects of HOCl were reduced by the application of tetracaine, Gd3+ or streptomycin, implicating involvement of ryanodine receptors and transient receptor potential channels. These results demonstrate the potent effects of HOCl on skeletal muscle function potentially mediated by HOCl-induced oxidation to Ca2+ signalling proteins. Hence, HOCl may provide a link between chronic inflammation, oxidative stress and impaired Ca2+ handling that is characteristic of DMD and presents a potential therapeutic target for DMD. KEY POINTS: Duchenne muscular dystrophy is a fatal genetic disease with pathological mechanisms which involve the complex interaction of chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations. Hypochlorous acid can be endogenously produced by neutrophils via the enzyme myeloperoxidase. Both neutrophil and myeloperoxidase activity are increased in dystrophic mice. This study found that hypochlorous acid decreased muscle force production and increased cytosolic Ca2+ concentrations in isolated muscles from wild-type and dystrophic mice at relatively low concentrations of hypochlorous acid. These results indicate that hypochlorous acid may be key in the Duchenne muscular dystrophy disease pathology and may provide a unifying link between the chronic inflammation, increased reactive oxygen species production and increased cytosolic Ca2+ concentrations observed in Duchenne muscular dystrophy. Hypochlorous acid production may be a potential target for therapeutic treatments of Duchenne muscular dystrophy.
Subject(s)
Muscular Dystrophy, Duchenne , Animals , Mice , Hypochlorous Acid/pharmacology , Hypochlorous Acid/metabolism , Hypochlorous Acid/therapeutic use , Peroxidase/metabolism , Mice, Inbred mdx , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Inflammation/metabolism , Disease Models, AnimalABSTRACT
New medicines are urgently required to treat the fatal neuromuscular disease Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested 2 weeks of daily 100 mg/kg DMF versus 5 mg/kg standard-care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology, and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED, warranting follow-up longitudinal studies to evaluate disease-modifying impact.
Subject(s)
Dimethyl Fumarate , Muscular Dystrophy, Duchenne , Animals , Mice , Mice, Inbred mdx , Dimethyl Fumarate/pharmacology , Dimethyl Fumarate/therapeutic use , Muscular Dystrophy, Duchenne/drug therapy , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Prednisone , Muscles/pathologyABSTRACT
Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma samples and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.
ABSTRACT
Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.
Subject(s)
Muscular Dystrophy, Duchenne , Mice , Animals , Mice, Inbred mdx , Muscular Dystrophy, Duchenne/metabolism , Muscle, Skeletal/metabolism , Disease Models, Animal , Reactive Oxygen Species/metabolism , Proteins/metabolism , Biomarkers/metabolism , Necrosis/metabolism , Necrosis/pathologyABSTRACT
Inflammation and neonatal hypoxia-ischemia (HI) are important etiological factors of perinatal brain injury. However, underlying mechanisms remain unclear. Sirtuins are a family of nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylases. Sirtuin-6 is thought to regulate inflammatory and oxidative pathways, such as the extracellular release of the alarmin high mobility group box-1 (HMGB1). The expression and role of sirtuin-6 in neonatal brain injury are unknown. In a well-established model of neonatal brain injury, which encompasses inflammation (lipopolysaccharide, LPS) and hypoxia-ischemia (LPS+HI), we investigated the protein expression of sirtuin-6 and HMGB1, as well as thiol oxidation. Furthermore, we assessed the effect of the antioxidant N-acetyl cysteine (NAC) on sirtuin-6 expression, nuclear to cytoplasmic translocation, and release of HMGB1 in the brain and blood thiol oxidation after LPS+HI. We demonstrate reduced expression of sirtuin-6 and increased release of HMGB1 in injured hippocampus after LPS+HI. NAC treatment restored sirtuin-6 protein levels, which was associated with reduced extracellular HMGB1 release and reduced thiol oxidation in the blood. The study suggests that early reduction in sirtuin-6 is associated with HMGB1 release, which may contribute to neonatal brain injury, and that antioxidant treatment is beneficial for the alleviation of these injurious mechanisms.
ABSTRACT
Duchenne muscular dystrophy (DMD) is a severe X-linked muscle wasting disease with no cure. While the precise mechanisms of progressive dystropathology remain unclear, oxidative stress caused by excessive generation of oxidants is strongly implicated. Blood biomarkers that could track oxidant levels in tissues would be valuable to measure the effectiveness of clinical treatments for DMD; our research has focused on developing such biomarkers. One target of oxidants that has the potential to be harnessed as a clinical biomarker is the thiol side chain of cysteine 34 (Cys34) of the blood protein albumin. This study using the mdx mouse model of DMD shows that in plasma, albumin Cys34 undergoes thiol oxidation and these changes correlate with levels of protein thiol oxidation and damage of the dystrophic muscles. A comparison with the commonly used biomarker protein carbonylation, confirmed that albumin thiol oxidation is the more sensitive plasma biomarker of oxidative stress occurring in muscle tissue. We show that plasma albumin oxidation reflects muscle dystropathology, as increased after exercise and decreased after taurine treatment of mdx mice. These data support the use of albumin thiol oxidation as a blood biomarker of dystropathology to assist with advancing clinical development of therapies for DMD.
ABSTRACT
Isotope-coded affinity tags (ICATs) are valuable tools for mass spectrometry-based quantitative proteomics, in particular, for comparison of protein (cysteine-residue) thiol oxidation state in normal, stressed, and diseased tissue. However, the iodoacetamido electrophile used in most commercial ICATs suffers from poor thiol-selectivity and modest rates of adduct formation, which can lead to spurious results. Hence, we designed and synthesized three ICATs containing thiol-selective N-alkylmaleimide electrophiles (isotope-coded maleimide affinity tags = ICMATs) and assessed these as mass spectrometry probes for ratiometric analysis of lysozyme and muscle proteomes. Two ICMAT pairs containing butylene/D8-butylene linkers were effective MS probes, but not ideal for typical proteomics workflows, because peptides bearing these tags frequently did not coelute with HPLC. A switch to a phenylene/13C6-phenylene linker solved this issue without compromising the efficiency of adduct formation.
Subject(s)
Carbon Isotopes/chemistry , Isotope Labeling/methods , Maleimides/chemistry , Muscle Proteins/metabolism , Proteomics/methods , Animals , Chromatography, Liquid , Dogs , Gene Expression Regulation , Male , Mice , Mice, Inbred mdx , Models, Molecular , Muscle Proteins/chemistry , Muscle Proteins/genetics , Muscle, Skeletal , Protein Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500-6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies.
Subject(s)
Biomarkers/urine , Muscular Dystrophy, Duchenne/pathology , Oxidative Stress , Animals , Antibodies/immunology , Biomarkers/metabolism , Creatine Kinase/blood , Disease Models, Animal , Dogs , Female , Hydrazines/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/metabolism , Neutrophils/metabolism , Peroxidase/metabolism , Protein CarbonylationABSTRACT
Duchenne muscular dystrophy (DMD) is a lethal genetic muscle wasting disorder, which currently has no cure. Supplementation with the drug taurine has been shown to offer therapeutic benefit in the mdx model for DMD, however the mechanism by which taurine protects dystrophic muscle is not fully understood. Mdx muscle is deficient in taurine, however it is not known if this deficiency occurs in the extracellular space, in other cells present in the tissue (such as immune cells) or in the myofibre itself. Likewise, the tissue location of taurine enrichment in taurine treated mdx muscle is not known. In this study we applied X-ray absorption near edge spectroscopy (XANES) at the sulfur K-edge in an imaging format to determine taurine distribution in muscle tissue. XANES is the only technique currently capable of imaging taurine directly in muscle tissue, at a spatial resolution approaching myocyte cell size (20-50 µm). Using a multi-modal approach of XANES imaging and histology on the same tissue sections, we show that in mdx muscle, it is the myofibres that are deficient in taurine, and taurine supplementation ameliorates this deficiency. Increasing the taurine content of mdx myofibres was associated with a decrease in myofibre damage (as shown by the percentage of intact myofibres) and inflammation. These data will help drive future studies to better elucidate the molecular mechanisms through which taurine protects dystrophic muscle; they also support the continued investigation of taurine as a therapeutic intervention for DMD.
Subject(s)
Muscular Dystrophy, Duchenne , Animals , Dietary Supplements , Disease Models, Animal , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle, Skeletal , Synchrotrons , Taurine/pharmacologyABSTRACT
Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease that causes severe loss of muscle mass and function in young children. Promising therapies for DMD are being developed, but the long lead times required when using clinical outcome measures are hindering progress. This progress would be facilitated by robust molecular biomarkers in biofluids, such as blood and urine, which could be used to monitor disease progression and severity, as well as to determine optimal drug dosing before a full clinical trial. Many candidate DMD biomarkers have been identified, but there have been few follow-up studies to validate them. This Review describes the promising biomarkers for dystrophic muscle that have been identified in muscle, mainly using animal models. We strongly focus on myonecrosis and the associated inflammation and oxidative stress in DMD muscle, as the lack of dystrophin causes repeated bouts of myonecrosis, which are the key events that initiate the resultant severe dystropathology. We discuss the early events of intrinsic myonecrosis, along with early regeneration in the context of histological and other measures that are used to quantify its incidence. Molecular biomarkers linked to the closely associated events of inflammation and oxidative damage are discussed, with a focus on research related to protein thiol oxidation and to neutrophils. We summarise data linked to myonecrosis in muscle, blood and urine of dystrophic animal species, and discuss the challenge of translating such biomarkers to the clinic for DMD patients, especially to enhance the success of clinical trials.
Subject(s)
Biomarkers/metabolism , Inflammation/pathology , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/pathology , Oxidative Stress , Animals , Humans , Muscular Dystrophy, Duchenne/physiopathology , Necrosis , RegenerationABSTRACT
Introduction: In order to better understand the physiological and pathophysiological roles of reactive oxygen species (ROS), multiple blood and urine biomarkers of oxidative stress have been developed. The single free thiol (Cys34) in plasma albumin is a useful biomarker of oxidative stress because thiol groups are particularly sensitive to oxidation by ROS. The primary aim of this study was to develop a gel electrophoresis-based method (mPEG assay) that would be more widely accessible than existing chromatography techniques to assay the oxidation state of albumin Cys34.Method: Blood samples were collected into a solution containing polyethylene glycol maleimide (malpeg). Plasma samples were divided into two aliquots, with a reducing agent added to one aliquot. Albumin bound to malpeg was separated from albumin by gel electrophoresis. The proportion of albumin in reduced form (-SH), disulphide form (-SSX) and irreversibly oxidised form (-SO2, -SO3) could then be calculated.Results: Data for the mPEG assay was comparable to data from chromatographic and mass spectrometric assays. The mPEG assay was more sensitive than the albumin carbonyl assay for the detection of changes in albumin oxidation level in response to exposure to hydrogen peroxide or hypochlorous acid. This assay could also be performed on small blood samples (less than 10 µL) from fingerprick, thus facilitating longitudinal tracking of changes in albumin Cys34 oxidation level.Conclusion: The mPEG assay is a user-friendly, highly sensitive, specific, cost-effective gel electrophoresis-based method for the assay of the oxidations state of albumin Cys34 as a biomarker of oxidative stress.HighlightsProtein thiol groups are sensitive to oxidation by reactive oxygen species.Plasma albumin contains a reduced cysteine residue (Cys34) sensitive to oxidation.A novel gel electrophoresis-based method (mPEG) has been developed to measure the oxidation state of Cys34.The mPEG assay can be run on a drop of blood collected by fingerprick.
Subject(s)
Biomarkers/blood , Cysteine/metabolism , Oxidative Stress/physiology , Serum Albumin/metabolism , Humans , Oxidation-ReductionABSTRACT
PURPOSE: This study aimed to examine the effect of taurine ingestion on maximal voluntary muscle torque and power in trained male athletes with different caffeine habits. METHODS: Fourteen male athletes 21.8 ± 2.5 yr old were separated into caffeine and noncaffeine consumers to control for the effect of caffeine withdrawal on muscle function. On separate occasions, participants performed four isokinetic or three maximal isometric knee extensions with and without taurine (40 mg·kg body mass) after a double-blind, counterbalanced design. Muscle contractile performances were compared between the first sets as well as between the sets where these variables scored best. RESULTS: In response to isokinetic contraction, taurine treatment in the noncaffeine consumers resulted in a significant fall in first (-16.1%; P = 0.013) and best peak torque (-5.0%; P = 0.016) as well as in first (-17.7%; P = 0.015) and best power output (-8.0%; P = 0.008). In the caffeine consumers deprived of caffeine, taurine intake improved best power (5.2%; P = 0.045). With respect to the isometric variables, there was a significant decrease in the first (-5.1%; P = 0.002) and best peak torque (-4.3%; P = 0.032) in the noncaffeine group, but no effect in the group of caffeine consumers deprived of caffeine. Taurine ingestion increased blood taurine levels but had no effect on plasma amino acid levels. CONCLUSIONS: Taurine ingestion is detrimental to maximal voluntary muscle power and both maximal isokinetic and isometric peak torque in noncaffeine consumers, whereas taurine ingestion in caffeine-deprived caffeine consumers improves maximal voluntary muscle power but has no effect on other aspects of contractile performance.
Subject(s)
Muscle Contraction/drug effects , Muscle Strength/drug effects , Muscle, Skeletal/drug effects , Taurine/administration & dosage , Adult , Caffeine/administration & dosage , Double-Blind Method , Humans , Male , Muscle, Skeletal/physiology , Taurine/pharmacology , Torque , Young AdultABSTRACT
Assessment is a central component of course curriculums and is used to certify student learning, but it can also be used as a tool to improve teaching and learning. Many laboratory courses are structured such that there is only a grade for a particular laboratory, which limits the insights that can be gained in student learning. We developed a laboratory program that incorporates assessments designed to probe student understanding of different components of the individual modules making up the program. The challenge was to analyze and present grades from these assessment tasks in a format that was readily interpretable by academics. We show that a simplified synthesis of grade distributions (grade distribution digests) provides sufficient information to make decisions about changes in course components. The main feature of the digests is its data visualization approach, where student grades for individual laboratory practicals, individual assessment tasks or individual assessment items are graphically presented as an overall average grade, an average top quartile grade and an average bottom quartile grade, and relative averages across all assessments. This ability to visualize student grades in variety of contexts enables academics with many other demands on their time (e.g. research and administration) to more efficiently identify ways to improve teaching delivery and learning outcomes. Examples are presented of the use of such data to identify and improve deficiencies in both student skills and teaching practice, resulting in improved learning outcomes. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):130-140, 2018.
Subject(s)
Educational Measurement , Laboratories , Learning , Teaching/education , Humans , Students , UniversitiesABSTRACT
Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disease manifested in young boys, for which there is no current cure. We have shown that the amino acid taurine is safe and effective at preventing dystropathology in the mdx mouse model for DMD. This study aimed to establish if treating growing mdx mice with a higher dose of taurine was more effective at improving strength and reducing inflammation and oxidative stress. Mice were treated with a dose of taurine estimated to be 16 g/kg/day, in drinking water from 1-6 weeks of age, after which in vivo and ex vivo muscle strength was assessed, as were measures of inflammation, oxidative stress and taurine metabolism. While the dose did decrease inflammation and protein oxidation in dystrophic muscles, there was no improvement in muscle strength (in contrast with benefits observed with the lower dose) and growth of the young mice was significantly restricted. We present novel data that a high taurine dose increases the cysteine content of both mdx liver and plasma, a possible result of down regulation of the taurine synthesis pathway in the liver (which functions to dispose of excess cysteine, which is toxic). These data caution that a high dose of taurine can have adverse effects and may be less efficacious than lower taurine doses. Therefore, monitoring of taurine dosage needs to be considered in future pre-clinical trials, in anticipation of using taurine as a clinical therapy for growing DMD boys (and other conditions).
Subject(s)
Growth , Muscular Dystrophy, Animal/drug therapy , Taurine/therapeutic use , Animals , Mice , Mice, Inbred mdx , Muscular Dystrophy, Animal/pathology , Oxidative Stress/drug effectsABSTRACT
KEY POINTS: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a therapeutic intervention for DMD boys, but potential adverse effects of NAC have not been widely investigated. We used young (6 weeks old) growing mdx mice to investigate the capacity of NAC supplementation (2% in drinking water for 6 weeks) to improve dystrophic muscle function and to explore broader systemic effects of NAC treatment. NAC treatment improved normalised measures of muscle function, and decreased inflammation and oxidative stress, but significantly reduced body weight gain, muscle weight and liver weight. Unexpected significant adverse effects of NAC on body and muscle weights indicate that interpretation of muscle function based on normalised force measures should be made with caution and careful consideration is needed when proposing the use of NAC as a therapeutic treatment for young DMD boys. ABSTRACT: Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 weeks old) mdx and non-dystrophic C57 mice receiving 2% NAC in drinking water for 6 weeks were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 week old mice were anaesthetised and extensor digitorum longus (EDL) muscles were excised for functional analysis and tissues were sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC-treated mdx [3.13 (0.58) vs 4.87 (0.78) g body weight (bw)-1 ; P < 0.001] and C57 mice [3.90 (0.32) vs 5.32 (0.60) g bw-1 ; P < 0.001]. Maximum specific force was significantly greater in NAC-treated mdx muscles [9.80 (2.27) vs 13.07 (3.37) N cm-2 ; P = 0.038]. Increased force in mdx mice was associated with reduced thiol oxidation and inflammation in fast muscles, and increased citrate synthase activity in slow muscle. Importantly, NAC significantly impaired body weight gain in both strains of young growing mice, and reduced liver weight in C57 mice and muscle weight in mdx mice. These potentially adverse effects of NAC emphasise the need for caution when interpreting improvements in muscle function based on normalised force measures, and that careful consideration be given to these effects when proposing NAC as a potential treatment for young DMD boys.
Subject(s)
Acetylcysteine/adverse effects , Free Radical Scavengers/adverse effects , Muscular Dystrophy, Duchenne/drug therapy , Acetylcysteine/administration & dosage , Acetylcysteine/therapeutic use , Animals , Body Weight/drug effects , Drug Evaluation, Preclinical , Free Radical Scavengers/administration & dosage , Free Radical Scavengers/therapeutic use , Liver/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Strength , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , Muscular Dystrophy, Duchenne/genetics , Oxidative StressABSTRACT
Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. Because thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolyzed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized or hydrolyzed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (â¼14%) of gels and proteolytic digestion buffers (â¼20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol-UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that it is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidized peptides.