Inhibition of collagen I accumulation reduces glomerulosclerosis by a Hic-5-dependent mechanism in experimental diabetic nephropathy

Glomerulosclerosis of any cause is characterized by loss of functional glomerular cells and deposition of excessive amounts of interstitial collagens including collagen I. We have previously reported that mesangial cell attachment to collagen I leads to upregulation of Hic-5 in vitro, which mediates mesangial cell apoptosis. Furthermore, glomerular Hic-5 expression was increased during the progression of experimental glomerulosclerosis. We hypothesized that reducing collagen I accumulation in glomerulosclerosis would in turn lower Hic-5 expression, reducing mesangial cell apoptosis, and thus maintaining glomerular integrity. We examined archive renal tissue from rats undergoing experimental diabetic glomerulosclerosis, treated with the transglutaminase-2 inhibitor NTU281. Untreated animals exhibited increased glomerular collagen I accumulation, associated with increased glomerular Hic-5 expression, apoptosis, and mesangial myofibroblast transdifferentiation characterized by a-smooth muscle actin (a-SMA) expression. NTU281 treatment reduced glomerular collagen I accumulation, Hic-5 and a-SMA expression, and apoptosis. Proteinurea and serum creatinine levels were significantly reduced in animals with reduced Hic-5 expression. In vitro studies of Hic-5 knockdown or overexpression show that mesangial cell apoptosis and expression of both a-SMA and collagen I are Hic-5 dependent. Together, these data suggest that there exists, in vitro and in vivo, a positive feedback loop whereby increased levels of collagen I lead to increased mesangial Hic-5 expression favoring not only increased apoptosis, but also mesangial myofibroblast transdifferentiation and increased collagen I expression. Prevention of collagen I accumulation interrupts this Hic-5-dependent positive feedback loop, preserving glomerular architecture, cellular phenotype, and function. © 2013 USCAP, Inc All rights reserved.

Sympathetic innervation of ciliary muscle and oculomotor function in emmetropic and myopic young adults

Purpose: Evidence exists for an additional inhibitory accommodative control system mediated by the sympathetic branch of the autonomic nervous system (ANS). This work aims to show the relative prevalence of sympathetic inhibition in young emmetropic and myopic adults, and to evaluate the effect of sympathetic facility on accommodative and oculomotor function. Methods: Profiling of ciliary muscle innervation was carried out in 58 young adult subjects (30 emmetropes, 14 early onset myopes, 14 late onset myopes) by examining post-task open-loop accommodation responses, recorded continuously by a modified open-view infrared optometer. Measurements of amplitude of accommodation, tonic accommodation, accommodative lag at near, AC/A ratio, and heterophoria at distance and near were made to establish a profile of oculomotor function. Results: Evidence of sympathetic inhibitory facility in ciliary smooth muscle was observed in 27% of emmetropes, 21% of early-onset myopes and 29% of late-onset myopes. Twenty-six percent of all subjects demonstrated access to sympathetic facility. Closed-loop oculomotor function did not differ significantly between subjects with sympathetic facility, and those with sympathetic deficit. Conclusions: Emmetropic and myopic groups cannot be distinguished in terms of the relative proportions having access to sympathetic inhibition. Presence of sympathetic innervation does not have a significant effect on accommodative function under closed-loop viewing conditions. © 2005 Elsevier Ltd. All rights reserved.

Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies

The purpose of this study was to produce a well-characterised electrospun polystyrene scaffold which could be used routinely for three-dimensional (3D) cell culture experimentation. A linear relationship (p<0.01p<0.01) between three principal process variables (applied voltage, working distance and polymer concentration) and fibre diameter was reliably established enabling a mathematical model to be developed to standardise the electrospinning process. Surface chemistry and bulk architecture were manipulated to increase wetting and handling characteristics, respectively. X-ray photoelectron spectroscopy (XPS) confirmed the presence of oxygen-containing groups after argon plasma treatment, resulting in a similar surface chemistry to treated tissue culture plastic. The bulk architecture of the scaffolds was characterised by scanning electron microscopy (SEM) to assess the alignment of both random and aligned electrospun fibres, which were calculated to be 0.15 and 0.66, respectively. This compared to 0.51 for collagen fibres associated with native tissue. Tensile strength and strain of approximately of 0.15 MPa and 2.5%, respectively, allowed the scaffolds to be routinely handled for tissue culture purposes. The efficiency of attachment of smooth muscle cells to electrospun scaffolds was assessed using a modified 3-[4,5-dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide assay and cell morphology was assessed by phalloidin-FITC staining of F-actin. Argon plasma treatment of electrospun polystyrene scaffold resulted in significantly increased cell attachment (p<0.05p<0.05). The alignment factors of the actin filaments were 0.19 and 0.74 for the random and aligned scaffold respectively, compared to 0.51 for the native tissue. The data suggests that electrospinning of polystyrene generates 3D scaffolds which complement polystyrene used in 2D cell culture systems.

An experimental and theoretical analysis of nitric oxide availability in the microcirculation

Nitric Oxide (NO) is produced in the vascular endothelium where it then diffuses to the adjacent smooth muscle cells (SMC) activating agents known to regulate vascular tone. The close proximity of the site of NO production to the red blood cells (RBC) and its known fast consumption by hemoglobin, suggests that the blood will scavenge most of the NO produced. Therefore, it is unclear how NO is able to play its role in accomplishing vasodilation. Investigation of NO production and consumption rates will allow insight into this paradox. DAF-FM is a sensitive NO fluorescence probe widely used for qualitative assessment of cellular NO production. With the aid of a mathematical model of NO/DAF-FM reaction kinetics, experimental studies were conducted to calibrate the fluorescence signal showing that the slope of fluorescent intensity is proportional to [NO]2 and exhibits a saturation dependence on [DAF-FM]. In addition, experimental data exhibited a Km dependence on [NO]. This finding was incorporated into the model elucidating NO 2 as the possible activating agent of DAF-FM. A calibration procedure was formed and applied to agonist stimulated cells, providing an estimated NO release rate of 0.418 ± 0.18 pmol/cm2s. To assess NO consumption by RBCs, measurements of the rate of NO consumption in a gas stream flowing on top of an RBC solution of specified Hematocrit (Hct) was performed. The consumption rate constant (kbl)in porcine RBCs at 25°C and 45% Hct was estimated to be 3500 + 700 s-1. kbl is highly dependent on Hct and can reach up to 9900 + 4000 s-1 for 60% Hct. The nonlinear dependence of kbl on Hct suggests a predominant role for extracellular diffusion in limiting NO uptake. Further simulations showed a linear relationship between varying NO production rates and NO availability in the SMCs utilizing the estimated NO consumption rate. The corresponding SMC [NO] level for the average NO production rate estimated was approximately 15.1 nM. With the aid of experimental and theoretical methods we were able to examine the NO paradox and exhibit that endothelial derived NO is able to escape scavenging by RBCs to diffuse to the SMCs.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Regulatory factors controlling muscle mass : competition between innate immune function and anabolic signals in regulation of atrogin-1 in Atlantic salmon

The research was supported by an industrial PhD studentship between University of Aberdeen and by BioMar Ltd., for Z. Heidari.

Epigenetic Therapy for the Treatment of Hypertension-Induced Cardiac Hypertrophy and Fibrosis

BACKGROUND: The development of heart failure is associated with changes in the size, shape, and structure of the heart that has a negative impact on cardiac function. These pathological changes involve excessive extracellular matrix deposition within the myocardial interstitium and myocyte hypertrophy. Alterations in fibroblast phenotype and myocyte activity are associated with reprogramming of gene transcriptional profiles that likely requires epigenetic alterations in chromatin structure. The aim of our work was to investigate the potential of a currently licensed anticancer epigenetic modifier as a treatment option for cardiac diseases associated with hypertension-induced cardiac hypertrophy and fibrosis.

METHODS AND RESULTS: The effects of DNA methylation inhibition with 5-azacytidine (5-aza) were examined in a human primary fibroblast cell line and in a spontaneously hypertensive rat (SHR) model. The results from this work allude to novel in vivo antifibrotic and antihypertrophic actions of 5-aza. Administration of the DNA methylation inhibitor significantly improved several echocardiographic parameters associated with hypertrophy and diastolic dysfunction. Myocardial collagen levels and myocyte size were reduced in 5-aza-treated SHRs. These findings are supported by beneficial in vitro effects in cardiac fibroblasts. Collagen I, collagen III, and α-smooth muscle actin were reduced in a human ventricular cardiac fibroblast cell line treated with 5-aza.

CONCLUSION: These findings suggest a role for epigenetic modifications in contributing to the profibrotic and hypertrophic changes evident during disease progression. Therapeutic intervention with 5-aza demonstrated favorable effects highlighting the potential use of this epigenetic modifier as a treatment option for cardiac pathologies associated with hypertrophy and fibrosis.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

CaV channels and cancer: canonical functions indicate benefits of repurposed drugs as cancer therapeutics

The importance of ion channels in the hallmarks of many cancers is increasingly recognised. This article reviews current knowledge of the expression of members of the voltage-gated calcium channel family (CaV) in cancer at the gene and protein level and discusses their potential functional roles. The ten members of the CaV channel family are classified according to expression of their pore-forming α-subunit; moreover, co-expression of accessory α2δ, β and γ confers a spectrum of biophysical characteristics including voltage dependence of activation and inactivation, current amplitude and activation/inactivation kinetics. CaV channels have traditionally been studied in excitable cells including neurones, smooth muscle, skeletal muscle and cardiac cells, and drugs targeting the channels are used in the treatment of hypertension and epilepsy. There is emerging evidence that several CaV channels are differentially expressed in cancer cells compared to their normal counterparts. Interestingly, a number of CaV channels also have non-canonical functions and are involved in transcriptional regulation of the expression of other proteins including potassium channels. Pharmacological studies show that CaV canonical function contributes to the fundamental biology of proliferation, cell-cycle progression and apoptosis. This raises the intriguing possibility that calcium channel blockers, approved for the treatment of other conditions, could be repurposed to treat particular cancers. Further research will reveal the full extent of both the canonical and non-canonical functions of CaV channels in cancer and whether calcium channel blockers are beneficial in cancer treatment.

Le cordon ombilical humain, source de cellules pour le génie tissulaire : Isolement, caractérisation et production de substituts humains

Le cordon ombilical humain suscite beaucoup d’intérêt comme source de cellules à des fins de recherche et de thérapie. Quatre types cellulaires majeurs - les cellules épithéliales, stromales, musculaires lisses et endothéliales - composent les tissus solides du cordon ombilical. Quelques-uns de ces types cellulaires ont été utilisés en recherche scientifique depuis longtemps, alors que d’autres commencent à peine à dévoiler leur potentiel. Nous avons développé un protocole unique pour l’extraction séquentielle de tous ces types cellulaires d’un seul cordon ombilical, permettant ainsi la reconstruction à partir d’une même source. La combinaison des techniques de perfusion, immersion et explants a mené à la mise en culture et à l’expansion de ces cellules, dont les cellules épithéliales et les cellules stromales de la gelée de Wharton qui ont été caractérisées plus en détail par l’immunomarquage de protéines spécifiques. Leur potentiel pour la médecine régénératrice a été démontré par la production de tissus par génie tissulaire. Un vaisseau sanguin composé de cellules stromales et de cellules musculaires lisses du cordon ombilical démontra une résistance substantielle à l’éclatement. Les capacités de différenciation des cellules épithéliales ont été étudiées dans le contexte d’une peau bilamellaire reconstruite en combinaison avec des kératinocytes, des fibroblastes dermiques, et des cellules stromales de la gelée de Wharton. Les cellules épithéliales ont montré une différenciation similaire à celle des kératinocytes lorsque cultivées sur des fibroblastes dermiques et exposées à l’air, tandis que sur des cellules stromales du cordon, elles ont subi une désorganisation. Finalement, la différenciation des cellules stromales a été induite en culture vers plusieurs types cellulaires afin de compléter cette étude. L’ensemble des résultats fait ressortir l’importance non seulement de l’influence du milieu physique sur la croissance et la différenciation des cellules, mais également de l’impact de la provenance des cellules sur la qualité des tissus reconstruits.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Theoretical investigation of intra- and inter-cellular spatiotemporal calcium patterns in microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined.^ Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. ^ From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.^

Interactive processes link the multiple symptoms of fatigue in sport competition

Muscle physiologists often describe fatigue simply as a decline of muscle force and infer this causes an athlete to slow down. In contrast, exercise scientists describe fatigue during sport competition more holistically as an exercise-induced impairment of performance. The aim of this review is to reconcile the different views by evaluating the many performance symptoms/measures and mechanisms of fatigue. We describe how fatigue is assessed with muscle, exercise or competition performance measures. Muscle performance (single muscle test measures) declines due to peripheral fatigue (reduced muscle cell force) and/or central fatigue (reduced motor drive from the CNS). Peak muscle force seldom falls by >30% during sport but is often exacerbated during electrical stimulation and laboratory exercise tasks. Exercise performance (whole-body exercise test measures) reveals impaired physical/technical abilities and subjective fatigue sensations. Exercise intensity is initially sustained by recruitment of new motor units and help from synergistic muscles before it declines. Technique/motor skill execution deviates as exercise proceeds to maintain outcomes before they deteriorate, e.g. reduced accuracy or velocity. The sensation of fatigue incorporates an elevated rating of perceived exertion (RPE) during submaximal tasks, due to a combination of peripheral and higher CNS inputs. Competition performance (sport symptoms) is affected more by decision-making and psychological aspects, since there are opponents and a greater importance on the result. Laboratory based decision making is generally faster or unimpaired. Motivation, self-efficacy and anxiety can change during exercise to modify RPE and, hence, alter physical performance. Symptoms of fatigue during racing, team-game or racquet sports are largely anecdotal, but sometimes assessed with time-motion analysis. Fatigue during brief all-out racing is described biomechanically as a decline of peak velocity, along with altered kinematic components. Longer sport events involve pacing strategies, central and peripheral fatigue contributions and elevated RPE. During match play, the work rate can decline late in a match (or tournament) and/or transiently after intense exercise bursts. Repeated sprint ability, agility and leg strength become slightly impaired. Technique outcomes, such as velocity and accuracy for throwing, passing, hitting and kicking, can deteriorate. Physical and subjective changes are both less severe in real rather than simulated sport activities. Little objective evidence exists to support exercise-induced mental lapses during sport. A model depicting mind-body interactions during sport competition shows that the RPE centre-motor cortex-working muscle sequence drives overall performance levels and, hence, fatigue symptoms. The sporting outputs from this sequence can be modulated by interactions with muscle afferent and circulatory feedback, psychological and decision-making inputs. Importantly, compensatory processes exist at many levels to protect against performance decrements. Small changes of putative fatigue factors can also be protective. We show that individual fatigue factors including diminished carbohydrate availability, elevated serotonin, hypoxia, acidosis, hyperkalaemia, hyperthermia, dehydration and reactive oxygen species, each contribute to several fatigue symptoms. Thus, multiple symptoms of fatigue can occur simultaneously and the underlying mechanisms overlap and interact. Based on this understanding, we reinforce the proposal that fatigue is best described globally as an exercise-induced decline of performance as this is inclusive of all viewpoints.