Potential mechanisms are explored including diffuse ventricular fibrosis, focal myocardial scarring, and impaired myocardial perfusion and perfusion reserve. The complex relationship between AF, systemic inflammation, as well as endothelial/microvascular dysfunction and the effects of AF on ventricular calcium handling and oxidative stress are also addressed. Finally, consideration is given to the clinical implications of these observations and concepts, with particular reference to rate vs. rhythm control.Atrial fibrillation (AF) is the most common sustained clinical arrhythmia and is associated with significant morbidity, mostly secondary to heart failure and stroke, and an estimated two-fold increase in premature death. Efforts to increase our understanding of AF and its complications have focused on unravelling the mechanisms of electrical and structural remodelling of the atrial myocardium. Yet, it is increasingly recognized that AF is more than an atrial disease, being associated with systemic inflammation, endothelial dysfunction, and adverse effects on the structure and function of the left ventricular myocardium that may be prognostically important. Here, we review the molecular and  Efforts to increase our pathophysiological understanding of AF have focused on unravelling the mechanisms responsible for these progressive atrial abnormalities.6 However, it is now increasingly recognized that AF can also adversely affect ventricular function.7 While some patients develop overt left ventricular (LV) dysfunction and heart failure, others present with subclinical alterations in ventricular structure and function, which may only be revealed by detailed investigation.9The discovery that AF triggers are located in the pulmonary veins in the majority of cases led to the development of pulmonary vein isolation via catheter ablation,11 One such potential mechanism is impaired myocardial perfusion and perfusion reserve due to microvascular coronary dysfunction in the presence of AF.12 This could underlie the clinical observation of ischaemic chest pain, ST depression, and troponin release in patients with AF and no obstructive epicardial coronary artery disease.14Ventricular remodelling may be contributed to by the adverse haemodynamic effect of loss of coordinated atrial contraction and/or tachycardia-mediated cardiomyopathy from a persistently elevated ventricular rate. The possibility of additional mechanisms is suggested by the observation that some patients with AF and a controlled ventricular rate develop abnormalities in ventricular function, which are reversible following restoration of sinus rhythm (SR).17 A deeper understanding of the relationships between inflammation, endothelial function, and ventricular function in AF and the reversibility or otherwise of these alterations following restoration of SR may lead to more rational therapeutic strategies and ultimately to improved outcomes for patients.In addition to the LV abnormalities described, AF is also associated with systemic inflammation and impaired endothelial function.Figure 1) with a focus on AF-associated changes in ventricular structure, perfusion, and overall function. The relationship between AF and systemic inflammation and endothelial dysfunction is also briefly addressed. Finally, we discuss the implications of these findings for preclinical and basic research and provide a current clinical perspective.Here, we review the systemic correlates of AF  in heart failure trials,26 although it remains controversial whether this effect is truly independent.22AF is associated with impaired ventricular function, with a spectrum of severity ranging from congestive heart failure to subtle subclinical alterations in LV performance. Causation is much more challenging to ascertain, particularly when the index presentation is with both AF and LV dysfunction. The possibilities are that AF may promote ventricular impairment by raising the ventricular rate , or by other mechanisms such as endothelial dysfunction and impaired myocardial perfusion. Alternatively, a pre-existing ventricular cardiomyopathy may lead to AF, via mechanical stretch and structural remodelling of the atria and altered myocardial intracellular calcium handling.27 Instead, a substantial increase in the heterogeneity of conduction during atrial pacing is noted due to extensive atrial interstitial fibrosis, a finding not seen following short-term rapid atrial pacing.27 In clinical practice, a significant improvement in ventricular function following reduction in AF burden by cardioversion or ablation is perhaps the best identifier of a causal role of AF in the associated LV dysfunction, demonstrating that ventricular impairment is a reversible secondary effect. This notwithstanding, there are often multiple interacting causative factors in human AF and current classification schemes are unable to capture the inherent complexity and heterogeneity. This complicates the study of human AF, and underlines the importance of translating findings from animal models, each of which partially reproduces the pathophysiological spectrum of clinical AF.28Studies in a canine heart failure model have demonstrated that AF induced by a primary impairment in LV function is not associated with characteristic atrial electrical abnormalities such as the reduced atrial refractory period seen in AF induced by rapid atrial pacing with a controlled ventricular response.3.in vitro experiments on such preparations may not be fully reflective of in vivo conditions, these investigations have provided relevant insights into the changes in ion channel expression and function that accompany human AF.29The systematic evaluation of ventricular molecular and cellular abnormalities in AF is limited by poor access to human tissue samples. Atrial cardiomyocytes or trabeculae can be isolated from samples of the right atrial appendage excised at the time of cardiac surgery and cardiopulmonary bypass. Although the results of 30 Even when undertaken, biopsy carries the risk of sampling error, and patchy abnormalities may remain undetected.31 The limited human data in the literature largely represent small case series of ventricular biopsy32 or studies of tissue from explanted hearts following cardiac transplant.20 Interpretation of these data is made more difficult by the lack of matched tissue from healthy control subjects.The situation is even more challenging for investigations of the ventricular myocardium, as ventricular biopsy is rarely clinically indicated and is difficult to justify purely for research purposes due to the risk of complications.4.4.132 Importantly, all patients had structurally normal hearts without atrial or ventricular dilatation and a normal LV ejection fraction. Three of 14 had evidence of focal myocarditis (eosinophilic or lymphocytic) and initiation of corticosteroid therapy in addition to antiarrhythmic medications induced recovery of SR in all of this group. A further three patients had evidence of a diffuse cardiomyopathic process, whereas the remaining eight patients had a non-specific diffuse fibrotic change with or without evidence of cardiomyocyte necrosis. However, such changes may not be specific to AF as a separate study documented similar histological findings in the majority of patients with other forms of supraventricular tachycardia.33Although human data reporting ventricular changes in the presence of AF are sparse, one series that investigated 14 young patients with apparent ‘lone’ AF refractory to initial medical therapy demonstrated abnormal histology in all cases.T1 mapping,34 usually requiring administration of a gadolinium-based contrast agent. Several methods have been described: some rely on T1 measurement following contrast administration alone, while others combine these data with native (i.e. pre-contrast) T1 measurement and haematocrit to define myocardial volume of distribution or myocardial extracellular volume fraction (ECV).36 Contrast diffuses freely in the extracellular space and a low post-contrast T1 value reflects an elevated ECV, which is usually indicative of diffuse fibrosis. This method has been validated against histological assessment of fibrosis in right ventricular endomyocardial biopsy specimens taken from subjects who had undergone orthotopic heart transplantation.19 A study on post-contrast T1 mapping in subjects with paroxysmal (n = 40) and persistent AF (n = 27) as well as healthy controls (n = 23) demonstrated significantly lower post-contrast T1 values in both sets of AF patients compared with age- and sex-matched controls.37 Additionally, patients with persistent AF had lower post-contrast T1 values than the paroxysmal AF group. In keeping with established data showing that diffuse myocardial fibrosis increases with age, there was a negative correlation between increasing age and post-contrast T1 value in the healthy controls. In a multivariate model, only age, AF class, and LV ejection fraction were independent correlates of post-contrast ventricular T1 values. These data suggest that increasing AF burden is associated with progressive diffuse LV fibrosis, and may play a role in adverse cardiac remodelling.More data are available from non-invasive studies using MRI techniques, which have aimed to identify ventricular fibrosis in AF. Diffuse fibrosis cannot be visualized by the widely used method of late gadolinium enhancement (LGE) cardiac MRI, which relies on the presence of areas of normal myocardium to identify discrete areas of focal fibrosis, scarring, or infarction. However, diffuse fibrosis can be detected by the recently developed methods of 38 Heart failure and ventricular fibrosis have been reported in a similar dog model, despite the use of oral digoxin and metoprolol with the aim of achieving ventricular rate control.39 However, the degree of ventricular fibrosis was less marked than the observed atrial fibrosis , and the ventricular rates of up to 180 bpm reported in this study may have contributed to a tachycardia-mediated cardiomyopathy; no corresponding increase in ventricular fibrosis was detected by the same investigators in a pig model.39Data from animal studies suggest that the development of LV fibrosis is related to an elevated ventricular rate in the presence of AF. Induction of AF by rapid atrial pacing in dogs in the presence of atrioventricular (AV) node ablation and a paced ventricular rate of 80 bpm was sufficient to induce atrial but not ventricular fibrosis, whereas dogs without AV node ablation and a more rapid ventricular rate did develop significant ventricular fibrosis.40 Focal atrial tachycardia provides a good model for the study of human atrial arrhythmia as long-term ‘cure’, and maintenance of SR is feasible following ablation. The subgroup with LV cardiomyopathy at baseline had lower post-contrast T1 values on CMR imaging at a mean of 5 years following ablation, indicating a greater burden of diffuse ventricular fibrosis that was irreversible despite restoration of normal SR and improvement in LV ejection fraction. Interestingly, there was no significant difference in mean heart rate on 24-h ECG monitoring prior to ablation between the patients with and without cardiomyopathy. While this may reflect the lack of statistical power to detect a true difference, it is also possible that the maximum ventricular rate tolerated before the development of ventricular fibrosis and impaired LV function varies between individuals.Findings consistent with the paradigm of tachycardia-induced ventricular fibrosis have also been described in patients who had undergone previous ablation for incessant focal atrial tachycardia.41 Thus, ventricular tissue appears inherently less susceptible to the development of fibrosis than atrial tissue, though prolonged exposure to an elevated ventricular rate can nevertheless still result in ventricular fibrosis. In summary, although it is currently not possible to definitively determine causality, there is strong circumstantial evidence that AF can induce diffuse ventricular fibrosis.42Atrial and ventricular fibrosis in response to AF may be regulated by different mechanisms. The study of cultured rat atrial and ventricular fibroblasts shows that NADPH oxidase 4 (NOX-4) is more highly expressed in the atria than the ventricles, and this mediates the enhanced collagen and fibronectin production of atrial fibroblasts compared with ventricular fibroblasts in response to TGF-β.43 Intriguingly, the presence of LGE was a powerful predictor of mortality with each 1% increase in LGE associated with a 15% increased risk of death. The patterns of LGE seen in this study included an ischaemic pattern, which probably reflects silent myocardial infarction, and a non-ischaemic pattern, which may relate to inclusion of patients with heart failure and/or reduced ejection fraction or may reflect the presence of an AF-related ventricular cardiomyopathy in some of the studied cohort. The presence of focal ventricular fibrosis in patients with AF is not well described, and further studies are needed to identify whether these findings are more prevalent in this population than in a matched cohort of patients in SR.Furthermore, a recent study emphasized the prevalence and prognostic significance of focal myocardial scar, as detected by LGE. LV LGE was present in 13% of patients with AF and without known prior myocardial infarction undergoing a routine cardiac MRI study prior to pulmonary vein isolation.44 and also strongly predicts recurrence of AF following catheter ablation in hypertensive patients.45 If AF caused diffuse LV fibrosis and adverse remodelling, targeting an aggressive rhythm control strategy to the subgroup of patients in whom AF is the primary pathology (rather than a consequence of another disease state) could clearly be important. The hypothesis that this strategy would improve clinical outcomes would need to be examined in an appropriately powered clinical trial.There are also data defining the prognostic value of diffuse LV fibrosis in AF. ECV expansion (mainly due to diffuse fibrosis) is associated with increased mortality and heart failure in broadly unselected patients undergoing clinical cardiac MRI independently of age, ejection fraction, and previous infarct size,4.214 implying the presence of either microvascular coronary dysfunction or arrhythmia-induced coronary artery spasm. In a pig pacing model of AF, 6 h of rapid atrial pacing (600 bpm) are sufficient to elicit a substantial reduction in coronary flow reserve (CFR) in the absence of any significant change in fractional flow reserve (FFR).46 CFR is influenced by abnormal flow in the epicardial coronary arteries as well as the microcirculation, whereas FFR is specific to epicardial disease; hence, these data support the hypothesis that AF can rapidly adversely affect microvascular coronary function. The investigators report that this reduction in CFR was abolished by pretreatment with dronedarone (but not amiodarone), and suggest that this may reflect an attenuating effect of dronedarone on oxidative stress and consequent microvascular flow abnormalities.46 However, as ventricular rates were not reported, it is not possible to identify the extent to which tachycardia (and reduced diastole duration) may have contributed to the reduction in CFR.Patients with AF and without obstructive epicardial coronary artery disease may still demonstrate clinical evidence of ischaemia, such as chest pain, ST depression, or troponin release, often at the onset of arrhythmia,12 Myocardial blood flow at rest was markedly reduced in the AF group compared with age- and risk-matched controls as was adenosine-induced hyperaemic flow . Minimal coronary vascular resistance at rest and under hyperaemia was also significantly increased in AF patients compared with controls. Finally, in the subgroup of patients that maintained SR following cardioversion, myocardial blood flow at rest normalized and hyperaemic flow and coronary vascular resistance were significantly improved from baseline. These human findings are in accordance with the animal data, and suggest that the presence of AF induces abnormalities in microvascular coronary function that are at least partially reversible with restoration of SR. However, the subgroup that maintained SR following cardioversion was small (10 patients), and confirmation of these findings in a larger cohort is awaited.Myocardial perfusion and perfusion reserve have been evaluated using PET and radioactively labelled water in patients with persistent AF after exclusion of epicardial coronary artery disease.47 A coronary Doppler catheter was placed and measurements taken in SR, during AF induced by bursts of atrial pacing, and during regular atrial tachycardia with a rate of pacing equal to the overall ventricular rate observed during AF. The relative contribution of the irregularity of ventricular contraction in AF was also assessed by the inclusion of a second group of patients with complete AV block and dual-chamber pacemakers in situ; in this group, the ventricle was paced regularly at 115 bpm before and after induction of AF. Acute induction of AF led to an increase in coronary flow, which was lower than that seen in the presence of rapid atrial pacing and was associated with a reduction in CFR and vascular resistance. In contrast, only a small effect on coronary flow and an increase in CFR were seen in the group with pacemakers in situ. The increase in coronary flow following induction of AF is likely to be explained by the elevated ventricular rate following acute induction of AF, as patients in this study were not receiving rate control medications. However, this absolute rise in coronary flow appears insufficient relative to the increase in ventricular rate and a consequent increase in myocardial metabolic demand. A study in pigs demonstrated a similar finding of ‘supply-demand ischaemia’ in the atria following acute induction of AF,48 evidenced by an increase in atrial lactate production despite compensatory rises in both atrial coronary conductance and oxygen extraction.In a separate study, the effect of AF on coronary flow, flow reserve, and vascular resistance was assessed invasively in patients in SR with no ventricular or valvular dysfunction, and no significant epicardial coronary artery disease.48 Indeed, chronic ventricular ischaemia could underlie the impaired ventricular function commonly seen in AF.Atrial supply-demand ischaemia has also been proposed as a potential trigger for atrial structural remodelling and has a molecular phenotype that resembles changes seen in hibernating ventricular myocardium.4.347 leaving the possibility that irregular ventricular contraction might cause coronary vasoconstriction, possibly by increasing the release of vasoactive peptides such as angiotensin II or endothelin. Studies in a pig model of rapid atrial pacing have suggested that the angiotensin receptor blocker, irbesartan, abolishes the impairment of microvascular flow otherwise seen with rapid atrial pacing , as well as preventing ventricular oxidative stress.49 The link between AF and impaired vascular function is not limited to the coronary circulation, and there are numerous reports confirming an association between AF and systemic endothelial dysfunction. Non-invasive assessment of endothelial function is most readily undertaken using flow-mediated dilatation (FMD), which assesses nitric oxide production in response to shear stress and by measurement of plasma levels of markers of endothelial damage or dysfunction, such as the von Willebrand factor and circulating endothelial cells. Endothelium-dependent FMD is abnormal in patients with AF,15 and a causative role for AF in endothelial dysfunction is suggested by the finding that cardioversion52 and successful catheter ablation of AF16 are associated with significant improvements in endothelial function.The irregular pulse interval that is typical of AF seems to play a role in the impaired coronary flow observed in these patients, even when the overall ventricular rate is well controlled. Irregular pulse intervals can adversely affect blood pressure, but there was no correlation between blood pressure and coronary flow in the invasive study discussed,53 This group was compared with patients who underwent regular atrial pacing at 150 bpm, a control group of patients with paroxysmal AF who were not paced and remained in SR, and a further control group of patients with no history of AF undergoing ablation of left-sided accessory pathways who were also not paced and in SR. The study showed that even at the very short time point of 15 min, rapid atrial rates  were associated with increased local platelet activation and thrombin generation. Furthermore, AF  additionally led to local and systemic endothelial dysfunction (as assessed by levels of asymmetric dimethylarginine) and inflammation (as assessed by levels of soluble CD40L). These data suggest that even a short duration of AF may cause inflammation and endothelial dysfunction. Further studies are needed to investigate whether this may be an important mechanism underlying impaired myocardial perfusion and perfusion reserve and the thromboembolic risk of asymptomatic short-lasting episodes of AF.54Inflammation represents a potential link between AF, impaired endothelial function, and a reduction in myocardial perfusion and perfusion reserve. A recent study investigated the acute effects of AF induced by burst atrial pacing immediately prior to catheter ablation in patients with a history of paroxysmal AF but in SR at the time of the procedure.4.456 More recently, data from animal and human studies have suggested that, even in the absence of persistently elevated ventricular rates, AF can be associated with adverse atrial and ventricular remodelling and impairment of LV systolic and diastolic function.39 The relationship between AF and LV dysfunction is complex and potentially bi-directional, leading to a vicious electromechanical cycle.7 Causality in individual cases cannot be determined at a single time point, but a number of studies have undertaken serial evaluation around an intervention aimed at maintaining SR, and these have provided an important insight into causal relationships in the studied cohorts.It is well recognized that AF and other supraventricular tachycardias with an uncontrolled ventricular rate can cause a transient and reversible ventricular dysfunction that is termed ‘tachycardia-induced cardiomyopathy’.n = 26) compared with those randomized to medical rate control (n = 24).10 A similar improvement was seen in peak oxygen consumption and was mirrored by reductions in plasma B-type natriuretic peptide concentrations and NYHA class. Importantly, all patients were assessed for adequate rate control at baseline by ambulatory monitoring and exercise testing, thereby demonstrating that even rate-controlled AF has deleterious effects on LV function. The improvement in LV ejection fraction shown in this study is in keeping with the results of other  studies of patients with AF undergoing catheter ablation .A recent randomized study of patients with persistent AF, symptomatic heart failure, and LV ejection fraction <50% demonstrated a significant improvement in LV ejection fraction by 1 month (and persisting at 6 months) in those randomized to catheter ablation .58 The findings in the latter group may reflect the relatively low AF burden and the poor sensitivity of ejection fraction for detecting subtle changes in LV systolic function. Support for the latter possibility comes from a study by Tops et al.59 who utilized speckle-tracking to investigate LV strain in selected patients undergoing ablation for AF. Longitudinal and circumferential LV strain and strain rate were impaired at baseline compared with healthy controls. Intriguingly, maintenance of SR at follow-up was associated with improvement in these parameters, whereas recurrence of AF was associated with further deterioration. These data support the presence of subtle LV dysfunction in patients with AF and maintained LV ejection fraction; its reversibility following AF ablation strongly implies that AF is playing a causal role.A subsequent study which used cardiac MRI to evaluate LV function before and after ablation for short-lasting paroxysmal AF confirmed impaired LV systolic function at baseline and improvement in LV ejection fraction following ablation in all but patients with a normal ejection fraction at baseline  loss of atrial systole and AV synchrony, (ii) elevated overall ventricular rate, (iii) irregularity in the pulse interval, (iv) valvular regurgitation, and (v) neurohormonal effects. The role of some of these mechanisms has been directly assessed in human studies. In particular, in an invasive study involving ventricular pacing in patients with AF undergoing AV node ablation, AF and all forms of pacing were associated with reduced cardiac output compared with SR, emphasizing the importance of AV synchrony.61 While heart rate is slowed immediately and atrial contractility recovers by 1 week and remains unchanged thereafter, recovery of LV systolic function takes 1 month. Improvements in exercise tolerance and peak oxygen consumption mirrored the time course of recovery in LV function. These data suggest that the impairment in LV function seen in persistent AF may be due to a transient ventricular cardiomyopathy or stunning, due in part to an elevated ventricular rate but perhaps also to the irregular pulse interval.A separate study showed that, in patients with persistent AF and impaired LV function, improvement of atrial and ventricular function after successful cardioversion each follows a different time course.5.The molecular and cellular effects of AF on the ventricular myocardium are challenging to the study in humans due to limited clinical indications for ventricular biopsy and few other means of accessing ventricular tissue. In comparison, it is relatively straightforward to assess atrial tissue that is frequently excised in common cardiothoracic operations such as on-pump coronary artery bypass grafting. As a result, there are few studies which have explored this area in humans, although several findings have been obtained in animal models of AF.5.165 Less is known regarding the potential importance of ventricular oxidative stress in AF. Limited data are available from a study by Cai et al.,66 conducted in a pig model of rapid atrial pacing. The protein level of endothelial nitric oxide synthase was significantly decreased in LV tissue from animals with pacing-induced AF compared with controls, although this difference was less pronounced than in the left atrium. Importantly, this finding could not be attributed to an elevated ventricular rate as both groups of animals underwent AV node ablation and the ventricles were paced at 100 bpm in both the AF and control groups. A separate study, also conducted in a pig model of rapid atrial pacing, further demonstrated a significant increase in ventricular NADPH oxidase 2 (NOX2) expression, a finding that was prevented by treatment with irbesartan.49 Ventricular concentrations of F2-isoprostanes and LOX-1 protein, both markers of oxidative stress, were also elevated by rapid atrial pacing. These observations suggest that rapid atrial pacing induces oxidative stress in LV tissue by an angiotensin-mediated mechanism. Previous work demonstrated that plasma levels of angiotensin II are elevated within 6 h of rapid atrial pacing. Angiotensin II appears to be an important mediator of oxidative stress in AF67 by acting via the AT1 receptor to activate mitogen-activated protein kinases and stimulate proliferation of fibroblasts, cellular hypertrophy, and apoptosis as well as inducing calcium release and PKC activation, which in turn promotes activation of NOX2 and NOX1 NADPH-oxidases.68 A further study in the same model also reported a reduction in LV oxidative stress in the presence of dronedarone.46 In contrast to Cai et al., these studies cannot discriminate between the effect of AF and that of rapid ventricular rates induced by rapid atrial pacing with a competent AV node.Oxidative stress refers to the chronic disturbance of redox circuits and redox-responsive signal transduction pathways, and atrial oxidative stress is known to play an important pathophysiological role in the induction and maintenance of AF.5.269 Briefly, calcium handling abnormalities may be induced by oxidative stress70 and can promote AF via (i) induction of delayed afterdepolarizations and triggered events, (ii) enhanced Ca2+/calmodulin kinase type 2 activity and associated hyperphosphorylation and increased open probability of the type 2 ryanodine receptor, and (iii) up-regulation of the Na+/Ca2+ exchanger, increasing the size of the inward current for a given amount of aberrant Ca2+ release.71Calcium handling abnormalities in the atrial myocardium have been implicated in the initiation and maintenance of AF, and have been recently reviewed.n = 6 in SR and n = 6 in AF) who underwent cardiac transplantation as well as neonatal rat ventricular cardiomyocytes subjected to regular or irregular electrical stimulation ex vivo, to simulate SR and AF, respectively .20 To exclude effects of rate, all cardiomyocytes paced regularly or irregularly were stimulated the same number of times in total over a 24- h period. The mRNA and protein levels of sarcoplasmic reticulum ATPase 2a pump (SERCA), an important determinant of intracellular calcium levels, were significantly lower in the ventricular myocardium of the heart failure patients with AF compared with those in SR. This finding was recapitulated in paced rat ventricular cardiomyocytes, suggesting that irregularity of ventricular contraction influences SERCA expression beyond any effect of heart failure or ventricular rate. Additionally, in both models, AF was associated with a significant decrease in the phosphorylation of phospholamban (PLB), a key regulator of SERCA. Confirmation that these changes affect intracellular calcium handling was obtained from calcium imaging of rat cardiomyocytes, which demonstrated significantly reduced calcium transient amplitude and increased time-to-peak calcium transient amplitude in cardiomyocytes exposed to irregular pacing, compared with unpaced and regularly paced groups. Surprisingly, the rate of decay of the intracellular calcium transient was not significantly different between the groups.In contrast, very sparse data are available on calcium handling in ventricular myocardium in AF. Some insights are provided by a small study which examined human LV myocardial tissue obtained from end-stage heart failure patients with or without AF  failed to demonstrate any significant improvement in cardiovascular outcomes from a rhythm control strategy over a rate-control strategy. So, is there no rationale for trying to achieve SR in any patient with AF? There are several reasons that this may not be an appropriate conclusion to draw from these data.Human studies indicate that control of rhythm with cardioversion or ablation can, at least in selected groups of patients, restore LV function. Of particular importance for the clinician is determining whether this translates into long-term prognostic benefit. Experience over the last decade has not been encouraging: several large randomized clinical trials . The corollary of this is that a rhythm control strategy is less likely to successfully maintain SR the longer AF has been established or in disease states where primary atrial structural remodelling is the main substrate for AF (i.e. in the presence of heart failure).First, younger and more symptomatic patients are underrepresented in these trials. This population is more likely to have lone AF and to be at an earlier stage in the natural history of AF, and thus more likely to respond successfully to a rhythm control strategy. It is well established from animal models that the presence of AF induces progressive electrical and structural remodelling that augments AF stability , a large randomized trial of 3000 patients that is currently recruiting, will inform whether catheter ablation offers prognostic benefit beyond conventional medical strategy.8.AF is a common disease with significant clinical heterogeneity. Mechanistic and translational studies are providing important insights into its aetiology, and suggest that AF has important adverse effects on LV structure and function, in addition to a complex relationship with systemic inflammation and endothelial function. It is hoped that an improved understanding of the mechanisms underlying these observations will provide a platform for the development of much-needed advances in the therapeutic armamentarium against AF.Conflict of interest: none declared.The authors acknowledge support from the BHF Centre of Research Excellence, Oxford (RE/08/004).


Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy. Our best effort for containment of the global HIV epidemic is the development of a broadly protective vaccine. Current research has focused on vaccines that can generate a protective immune response against numerous strains of the virus. For this reason, vaccines with centralized HIV genes as immunogens, which merge HIV genetic information and potentially protect against multiple viral strains in a single inoculation, are an increasing area of interest to the field. Existing published studies have not evaluated centralized immunogens in the context of attenuated vaccines, which to date, have demonstrated the highest level of vaccine protection in lentiviral studies. Furthermore, centralized immunogen studies have also not included protective efficacy findings accomplished through challenge with highly pathogenic virus strains. In this study we not only examine the immunogenicity of these immunogens in an animal model, but we also for the first time evaluate the ability of centralized immunogens to induce protection against virulent virus challenge. The scientific community has aggressively sought after the development of a universal HIV vaccine that can prevail over the extraordinary levels of antigenic diversity in the fight against HIV and AIDS. The considerable extent of genomic variation found between isolates and within clades, and to a larger extent within the circulating recombinant forms, make for an effectual blockade to vaccine protection. Different strategies of vaccine composition and delivery have been proposed that are actively and widely being examined. A majority of these vaccines target the Env protein, as lentiviral antigenic variation is most pronounced in the viral Env proteins that serve as initial primary targets for host immune responses Centralized Env immunogens are one of the more promising contemporary approaches to overcoming HIV antigenic diversity Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes a persistent infection and chronic disease in equids While the disease processes for EIAV and HIV have distinguishing dynamics, key similarities between the two virus systems make EIAV an extremely valuable tool and model for AIDS vaccine development S2 accessory gene (EIAVD9) D9 viral vaccine were 100% protected from disease by virulent, albeit homologous, EIAV challenge. Thus, the EIAV system mirrors other animal lentivirus vaccine models, which consistently identify attenuated vaccines as producing the highest level of vaccine protection. D9 data demonstrated the effects of challenge virus Env sequence variation on vaccine protection We previously reported serial studies evaluating the efficacy of an attenuated EIAV proviral vaccine containing a mutation in the viral D9 vaccine studies. Our attenuated vaccine model, coupled with the well-characterized genomic and phylogenetic ancestry of the Env gene of our EIAV strain, enabled a thorough, unparalleled evaluation of centralized sequence vaccine efficacy not as readily modelled in other lentiviral systems. The presented studies evaluated multiple derivatives of centralized Env immunogens, both consensus and ancestral, in our proviral attenuated vaccine backbone. The studies were designed to first, develop and test a consensus immunogen for functionality through examination of replication and pathogenic potential in proviral backbones; second, compare the protective efficacy of the consensus immunogen in an attenuated backbone to attenuated strains containing our ancestral Env Despite numerous studies on the immunogenicity of centralized Env proteins, use of these noteworthy immunogens in an attenuated vaccine model, accompanied by virulent virus challenge, has yet to be reported. In the current study, we sought to directly build upon our current model and the series of described EIAVConsensus gene development of the EIAV Env protein focused on the gp90 region of the gene as genomic evolution and antigenic variation in the transmembrane (gp45) protein has been shown to be minimal among characterized longitudinal EIAV isolates D9 backbone UK3 backbone in vitro replication kinetics in vitro kinetics, emulating their parental and variant strain counterparts, and peaked in virus production at approximately ten DPI.To fully assess the competency of the ConEnv protein to function indistinguishably from a naturally occurring Env protein, ConEnv was evaluated in the context of both attenuated and pathogenic EIAV proviruses. Commercially synthesized ConEnv was cloned into the attenuated EIAVIn vivo analysis of the proviral pathogenic and attenuated ConEnv strains, by experimental infections of equids, confirmed characteristic EIAV clinical and virological profiles of both pathogenic and attenuated infections (2–103 copies RNA/ml) which failed to progress to clinical disease over a 100 day observation period were observed. Conversely, pathogenesis and virulence, characterized by standard viremic replication kinetics (averaging between103–104 copies RNA/ml and peaking at 106 copies RNA/ml), including the induction of acute, and progression to chronic disease were observed with the EVCon strain in vitro and in vivo, but also capable of inducing traditional and virulent lentiviral disease.fections . TypicalThe final pre-vaccine trial evaluation was an assessment of ConEnv's immunogenicity. Assays of antibody responses elicited by EVCon, EV0, EV6, and EV13 experimental infections, with variant and consensus viruses, indicated primarily distinct neutralization phenotypes for the individual variant Envs; each variant virus was neutralized by immune serum from homologous virus infections, but not from heterologous virus infections, except for marginal neutralization of the EVCon strain by the EV0 heterologous serum . HoweverD9 or D9). Proficiency of the centralized immunogens was further scrutinized by inclusion of a third attenuated vaccine regimen. The third arm of the study, a polyvalent attenuated strain mixture, was chosen as the most rigorous match to the centralized immunogens. The polyvalent attenuated quasispecies was constructed utilizing the D9 backbone. A trivalent attenuated mixture was assembled with the D9 as one of three strains. The EV6 and EV13 Envs were cloned into the D9 backbone to create 6D9 and 13D9, respectively, the final two strains of the polyvalent mix. The polyvalent attenuated mixture, a 1∶1∶1 mix of D9, 6D9, and 13D9, otherwise termed TriD9, was tested in vivo for TCID50 dosage verification in a group of eight ponies.To directly evaluate the consensus Env as well as the general premise of centralized immunogens, we compared the consensus Env attenuated strain (ConD9) with an ancestral Env attenuated vaccine strain , and not from the consensus ConD9 regimen . Polyvalent TriD9 vaccinates demonstrated the highest levels of protection that was significantly different from the unvaccinated controls and the consensus ConD9 curves . While the consensus ConD9 group had the lowest level of protection, it was significant as compared to unvaccinated subjects (P = 0.001). Analysis of the trend revealed a significant relationship between the complexity of immunogen and protective efficacy (P = 0.02). Ultimately, the consensus ConD9 strain, while pre-trial appearing to be more broadly immunogenic, demonstrated the lowest level of protection. The polyvalent TriD9 regimen demonstrated the highest level of protection against a quasispecies challenge. The current study not only highlighted important information towards HIV vaccine development and highlighted the importance of rigorous challenge strain engineering.The percentage of animals within each trial group protected from clinical EIA was plotted as a function of days post-challenge for survival analysis . Both cehallenge . OverallThe collective success of attenuated vaccine regimens makes it the ideal modality to rigorously test novel vaccine concepts. Previous work by our group and others have demonstrated attenuated vaccines to be an extremely useful tool for vaccine development, regardless of the potential for marketable advancement of the attenuated platform due to the safety concerns associated with HIV vaccines. Consequently, we resolved to examine the efficacy of centralized Env immunogens in our well-established EIAV attenuated vaccine model. Results presented here reveal, for the first time, a consensus Env in a fully replication-competent attenuated virus backbone that can confer protective efficacy against virulent virus challenge; however, it does not induce the highest level of protection as compared to ancestral or polyvalent vaccines.MX quasispecies challenge strain, EV0, EV6, and EV13 In vitro analysis of the virus constructed from the second-generation consensus gp90, assembled from the 90-isolate alignment and cloned into a proviral backbone, was found to infect equine cells, but was not fully replication competent. Therefore, if we were to analyze this Env in a single-round infection assay it would falsely appear to be fully functional. Hence, the 90-isolate sequence alignment was re-evaluated and a higher level of hand-editing performed, especially in the highly variable V3 region, prior to consensus generation. This thorough computational analysis and consideration of the consensus Env limited the potential sampling bias that obfuscates computational engineering of protein immunogens A concept not articulated in most reports on centralized Env sequences are the specifics involved in the determination of the consensus sequence. Creation of an ideal consensus sequence requires careful consideration of various parameters. Our original attempts to construct a consensus gp90 gene focused on a more basic approach than what we utilized for the current study. Initial alignments were performed utilizing the three sequences that comprise the EVMX challenge quasispecies virus strain composition, the polyvalent TriD9 regimen displaying the highest levels of protective efficacy might be anticipated; however, the ancestral D9 vaccinates also protected to a higher degree than the consensus ConD9 vaccinates. Pre-challenge analysis of clinical and virological factors would not have predicted these results. All three attenuated vaccine regimens displayed similar levels of pre-challenge viral strain replication. Pre-trial evaluation of the capability to induce neutralizing antibodies  and amino acid sequences from naturally arisen EIAV isolates originated from an experimental infection (pony #567) 3 TCID50 of either chimeric strain ConD9 or EVCon. Rectal temperatures and clinical status were recorded daily. Platelet numbers were determined using the IDEXX VetAutoread Hematology Analyzer . Clinical EIA (fever) episodes were determined on the basis of rectal temperature and platelet count  in combination with the viremic presence of infectious plasma virus (≥105) All equine procedures were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health at the Gluck Equine Research Center of the University of Kentucky according to protocols approved by the University of Kentucky IACUC (#01058A2006). The animals were monitored daily and maintained as described previously MX quasispecies was produced by combining equivalent infectious titers (TCID50) of the variant challenge strains EV0, EV6, and EV13 The challenge strain EVD9 stock was produced and vaccinations performed as described 3 TCID50 of EVMX. The ponies were monitored daily for clinical symptoms of EIA, and blood was drawn at regular intervals  for assays of platelets, viral replication, and virus-specific immune responses. During the course of these experiments ponies that demonstrated severe disease-associated symptoms resulting in distress as outlined by the University of Kentucky IACUC were euthanized.Equine procedures were conducted at the Gluck Equine Research Center of the University of Kentucky according to protocols approved by the University of Kentucky IACUC. Thirty-six mixed age and gender outbred ponies, serognegative for EIAV, were utilized. Daily rectal temperatures and clinical status were recorded. CBC analysis of whole blood was performed using an IDEXX QBC Vet Autoreader. Hematocrit and platelet numbers were monitored weekly. The EIAVgag-specific amplification primers 1 molecules as a lower limit and 108 molecules as an upper limit.Plasma samples from all subjects were analyzed for the levels of viral RNA per milliliter of plasma using a previously described quantitative real-time multiplex RT-PCR assay based on in vivo method for assessing immune reactivity to specific peptides, described previously D9 gp90 were generated . An additional 15 peptides, specific for the variable regions ConD9 gp90, were included in the pool for analysis of those vaccinates. Vaccinates were screened for gp90 specific cellular immune responses one week prior to day of challenge. A 2 mm skin biopsy was collected and homogenized and RNA extracted. IFNγ gene expression was determined by real-time PCR, as previously described −ΔΔCT.Our PV, and vaccine-specific virus stocks EVCon and EVMX, mediated by immune sera, was assessed in an indirect cell-ELISA based infectious center assay using a constant amount of infectious EIAV and sequential 2-fold dilutions of serum Serum IgG antibody reactivity to EIAV envelope glycoproteins was assayed quantitatively (end point titer) and qualitatively  using our standard concanavalin A (ConA) ELISA procedures as described previously Significance of protection from disease was performed by survival curve analysis as implemented in GraphPad Prism version 6.0d . Significance of survival curves were determined utilizing One-way ANOVA with Bonferroni's multiple comparison's test as well as survival analysis of Kaplan Meier plots with Logrank test for trend.All equine procedures were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health at the Gluck Equine Research Center of the University of Kentucky according to protocols approved by the University of Kentucky IACUC (#01058A2006).S1 FigDay of challenge humoral immune responses in protected versus unprotected vaccinates. Characterization of the quantitative and qualitative properties of induced EIAV envelope-specific humoral responses on the day of challenge were conducted in ConA serological ELISA assays of serum antibody (A) endpoint titer, (B) avidity, and (C) conformational dependence; (D) and 50% serum neutralization titer determinations, all as described in A) Mean serum antibody titers are presented as the log10 of the highest reciprocal dilution yielding reactivity two standard deviations above background. (B) Mean avidity index measurements are presented as percentages of the antibody-antigen complexes resistant to disruption with 8M urea. (C) Mean conformation dependence values are calculated as the ratio of serum antibody reactivity with native envelope compared to denatured envelope antigen. Conformation ratios greater than 1.0 indicate predominant antibody specificity for conformational determinants, while ratios less than 1.0 indicate predominant antibody specificity for linear envelope determinants. (D) The mean reciprocal dilutions of serum from vaccinated horses, which neutralized 50% of input EIAVPV, as measured in an infectious center assay. The grey dashed line denotes the cut off (≥25) value for valid 50% neutralization titers.(TIF)Click here for additional data file.


Perinatal exposure to endocrine disrupting chemicals (EDCs) can induce promiscuous neurobehavioral disturbances. Bisphenol A and phthalates are two widely prevalent and persistent EDCs reported to lead to such effects. Parental and social behaviors are especially vulnerable to endocrine disruption, as these traits are programmed by the organizational-activational effects of testosterone and estrogen. Exposure to BPA and other EDCs disrupts normal maternal care provided by rodents and non-human primates, such as nursing, time she spends hunched over and in the nest, and grooming her pups. Paternal care may also be affected by BPA. No long-term study has linked perinatal exposure to BPA or other EDC and later parental behavioral deficits in humans. The fact that the same brain regions and neural hormone substrates govern parental behaviors in animal models and humans suggests that this suite of behaviors may also be vulnerable in the latter. Social behaviors, such as communication, mate choice, pair bonding, social inquisitiveness and recognition, play behavior, social grooming, copulation, and aggression, are compromised in animal models exposed to BPA, phthalates, and other EDCs. Early contact to these chemicals is also correlated with maladaptive social behaviors in children. These behavioral disturbances may originate by altering the fetal or adult gonadal production of testosterone or estrogen, expression of ESR1, ESR2, and AR in the brain regions governing these behaviors, neuropeptide/protein hormone  and their cognate neural receptors, and/or through epimutations. Robust evidence exists for all of these EDC-induced changes. Concern also exists for transgenerational persistence of such neurobehavioral disruptions. In sum, evidence for social and parental deficits induced by BPA, phthalates, and related chemicals is strongly mounting, and such effects may ultimately compromise the overall social fitness of populations to come. The perinatal environment can dramatically shape later adult behaviors, and these disruptions can be propagated through transgenerational transmission to future generations . Strong conservation exists in brain development and function across species spanning from rodents to humans  and phthalates. Therefore, these two will be the primary focus, although, where appropriate, effects of other EDCs on these traits will be discussed. BPA is one of the most widely prevalent EDCs with production currently reported to be in excess of 15 billion pounds per year  phthalate (DEHP) is another ubiquitous EDC present in plastic products personal care products, paints, pharmaceuticals, food products, and textiles /day for gestational days 14–18) spend less time nursing their pups and huddling over them while in the nest  may also be potential factors. Finally, differences in animal husbandry  might account for the disparate rodent results.These conflicting rodent model findings may be attributed to various factors. First, all four of these above studies used rats or mice of different strains, and there is evidence of strain-specificity and phenotypic differences in response to BPA and other estrogenic EDC  male infants prenatally  exposed to BPA (10 μg/kg bw/day via osmotic pump) were more reminiscent of females, and these mothers nursed their “feminized” sons less than those rearing control males  rat pups prenatally exposed through their biological dam to the environmental chemical 3, 4, 3′, 4′-tetrachlorobiphenyl  resulted in their foster dams spending an increased frequency nursing them and allogrooming , where the male is responsible for tending the eggs, endocrine disruption of paternal care behaviors has been reported , males use territorial marking to communicate and protect their home range and mate from intruders. Males pericoceptionally through perinatally exposed (2 weeks prior to breeding of the dam through weaning at PND 30) to BPA  show reduction of this behavior when a control male is present in the testing arena  and introduced red shiner fish (C. venusta) disrupts normal visual communication signals and abolishes the species-dependent sexually selected behavioral traits with the net potential for hybridization to occur between these two otherwise isolated species  selectively reject males developmentally exposed to BPA    females treated for a 3 week duration (from pairing to day 21 of cohabituation) with daily oral administration of either BPA (2 or 20 μg/kg bw/day) or EE2 (0.04 μg/kg bw/day) exhibit heightened investigation of their partner and less exploration than untreated controls  prolonged the duration of time juvenile F1 and F3 C57BL/6 mice, respectively, spend investigating a novel animal , which indicates that this chemical can defeminize select female social behaviors , were sexually incompetent as adults, but no deficits in female sexual behavior were noted in this study  copulatory behaviors, in ovo treatment with other estrogenic chemicals, including estradiol benzoate (10 or 25 μg per egg), DES (700 ng per egg), and the soy phytoestrogen, genistein (1000 μg per egg), abolished this behavior in pubertal males , who were treated each day for 26 days, and mummichog, a small killifish, (Fundulus heteroclitus), who were treated daily for 4 weeks  exposure through the water  altered the shoaling behaviors  in threespine stickleback  of Caiman latirostris eggs elevated E2 and lowered T concentrations and concomitantly reversed the normal temperature sex determination mechanisms with a predominance of females occurring in these groups  and estrogen (E2) by male and female gonads   led to sex dependent effects on circulating corticosterone concentrations  and neural  of Crh) mRNA expression, but these changes were not evident in exposed females. Following subsequent exposure to a mild stressor, corticosterone and ACTH concentrations increased further in BPA males; whereas, these hormones decreased in BPA females. In non-stressed animals, Gr mRNA was increased in the hippocampus and hypothalamic paraventricular nucleus of BPA females. In contrast, these transcripts decreased in BPA males. The collective results suggest that BPA exposure might affect the HPA axis in males and females but the specific effects are sex-dependent.Another study in rats where dams were subcutaneously injected with 2 μg BPA/kg bw/day from gestational day 10 to lactational day 7 showed sex-dependent alterations in the adreno-cortical axis  increased concentrations of ACTH and corticosterone in treated male rats at 20 and 40 days of age; whereas no effects were observed in adult (60 days of age) males , and humans  are obligatory for the expression of maternal behavior, and variation in OXTR levels in the medial preoptic area of the hypothalamus (MPOA) are functionally linked to differences in degree of maternal care  mice exposed to BPA express less oxytocin, OXTR, and vasopressin than control males  through both DNA methylation via MECP2 and histone protein (H3K9) binding to this gene  RNAs  in the brain, there is evidence that this chemical can affect expression of these biomolecules in other cells, including MCF7 , reproduction, and likelihood of successfully rearing young. Thus, early exposure of animal and humans to these widely prevalent chemicals may lead to insidious behavioral effects. Moreover, there is ample evidence in animal models that EDCs (and humans in the case of DES) can induce transgenerational effects (Newbold et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The initiation and progression of human prostate cancer are highly associated with aberrant dysregulations of tumor suppressors and proto-oncogenes. Despite that deletions and mutations of tumor suppressors and aberrant elevations of oncogenes at the genetic level are reported to cause cancers, emerging evidence has revealed that cancer progression is largely regulated by posttranslational modifications (PTMs) and epigenetic alterations. PTMs play critical roles in gene regulation, cellular functions, tissue development, diseases, malignant progression and drug resistance. Recent discoveries demonstrate that ubiquitination and SUMOylation are complicated but highly-regulated PTMs, and make essential contributions to diseases and cancers by regulation of key factors and signaling pathways. Ubiquitination and SUMOylation pathways can be differentially modulated under various stimuli or stresses in order to produce the sustained oncogenic potentials. In this review, we discuss some new insights about molecular mechanisms on ubiquitination and SUMOylation, their associations with diseases, oncogenic impact on prostate cancer (PCa) and clinical implications for PCa treatment. Posttranslational modifications (PTMs) of proteins include enzymatic changes such as the addition of chemical group adducts such as ubiquitin (Ub), small ubiquitin-like modifiers (SUMO), phosphor, acetyl and methyl moieties ,2. PTMs C-terminal glycine of Ub and the amino group of lysine [C terminus of Ub, and Ub is transferred to the cysteine residue of E1; (b) Ub-conjugation: Ub is conjugated with E2, the Ub-conjugating enzymes . In this reaction, E2 physically interacts with both Ub and E1 to transfer Ub from E1 to the active site cysteine of E2; (c) Ub-ligation: Ub is transferred to the substrate by an E3 Ub ligase , through an isopeptide linkage between substrate proteins and Ub moieties. In this reaction, the E3 enzyme interacts with both the substrate protein and the E2~Ub thioester to catalyze Ub transfer in the ubiquitination process. An E3 enzyme recognizes its substrate protein to mark it for protein trafficking among subcellular compartments in cellular functions (K63) or to designate it as a target of degradation through the proteasome (K48), depending on the lysine linkage of ubiquitination. Several hundreds of E3 Ub ligases have been reported in eukaryotes, and the linkages of ubiquitination are determined by the functional specificities of the E3 Ub enzymes. There are three subfamilies of E3 Ub enzymes: the homology to E6-AP carboxyl terminus (HECT), the really interesting new gene (RING) and U-box. The HECT domain of E3 enzymes first binds Ub at the active site of E3, and then the RING domain catalyzes the Ub transfer from the E2 enzyme to the substrate protein. RING domain-containing E3s  consist of more than 600 members, while HECT-type E3s  only have 28 members. Cullin-RING ligases (CRLs) are the major group of RING-type E3 ligases to mediate proteasomal degradation. Recently, U-box type Ub ligases (such as UFD2a) are termed as E4 Ub ligases, a new class of ubiquitination enzymes [Ubiquitination is the biochemical pathway in which a small protein called ubiquitin (Ub) is enzymatically linked to a substrate protein . Ub is af lysine . For low enzymes . Given t enzymes .C terminal carboxyl group on the SUMO moiety and the amino group on the lysine residue of the substrate protein. Although SUMOylation has been reported in many proteins, SUMO modification is much less commonly detected for most proteins in cells as compared to Ub modification. The major reason for this difference is that SUMO modification does not directly target substrates for degradation [SUMOylation refers to the biochemical procedure in which a small ubiquitin-like modifier (SUMO) protein moiety is enzymatically conjugated to a substrate protein ,16. As aradation . The SUMradation . The SUMradation .Ubiquitination and SUMOylation pathways, together with deubiquitination and deSUMOylation reversing steps, are tightly regulated to maintain a balanced homeostasis on proteins in normal cells. In addition, recent studies have revealed that dysregulation of ubiquitination and SUMOylation contributes to the initiation and progression of various human cancers including prostate cancer (PCa) by directly attenuating protein functions of tumor suppressors and/or potentiating the hyperactive roles of oncogenes. Several essential signaling pathways are aberrantly regulated by ubiquitination and SUMOylation modifications in cancer cells through affecting the binding abilities and levels of some key co-factors. Altered forms of ubiquitination and/or SUMOylation cause an abnormal accumulation of oncoproteins and transcriptional factors in subcellular compartments, which drives cancer progression through regulation of gene expression and cell reprogramming. Recent studies report that the ubiquitination pathway can work in concert with the SUMOylation to modulate cellular functions when these two modifications target the same substrate protein, and the modification preference may generate influential oncogenic impacts in cells. Both ubiquitination and SUMOylation make important contributions to the regulation of key tumor suppressors, oncogenes and the associated signaling pathways in PCa.Emerging evidence revealed that aberrant alterations of enzymes in the ubiquitination pathway directly promote the initiation and progression of various human cancers. Therefore, targeting ubiquitin enzyme activities at all three steps may be a potential and novel chemotherapeutic approach for cancer control . KnockdoThe ubiquitination pathway is essential to maintain protein function, protein trafficking and protein homeostasis of tumor suppressors and oncogenes in various human cancers including PCa. Inactivation and mutations of p53 and PTEN tumor suppressors, and aberrant elevations of AKT and androgen receptor (AR) oncogenes lead to development and progression of PCa ,28,29,30in vitro and a suppression of prostate tumorigenesis in vivo. However it has been observed that SKP2 deficiency also contributes to dysregulation of other signaling pathways in PCa cells that in turn may antagonize the suppressive effects. Concomitant inactivation of Pten and p53 genes leads to a highly aggressive phenotype of PCa in mice, and Skp2 deficiency partially reduces, but does not completely block prostate tumorigenesis in Pten/p53 mutant mice [AR is a steroid hormone receptor and its aberrant elevation drives the development of PCa by upregulating downstream targets and co-factors in cell proliferation. The levels and activities of AR protein are controlled by several E3 Ub ligases through ubiquitination for protein degradation  or the augmentation of transcriptional activity  ,45,46. Rant mice ,50. Furtant mice , implicaant mice . The autCompared to studies on E3 Ub ligases in ubiquitination, advances in deubiquitination and the Ub proteasome system (UPS) are less documented, particularly for their functional roles in cancers. Several studies demonstrated that dysregulation of deubiquitination processes cause aberrant alterations of signaling cascades and cellular functions, which may accelerate the development of diseases and cancers. For example, overexpression of USP7 deubiquitinase promotes hepatocellular carcinoma (HCC) progression , and USPPten/p53 mutant mice by decreasing the stability and SUMOylation of SLUG protein [Aberrant regulation of multiple signaling pathways including PTMs governs the initiation and progression of PCa ,58,59,60 protein .It is well known that aberrant alterations of the PTEN/PI3K/AKT pathway plays a pivotal role on PCa, and that loss of the PTEN tumor suppressor drives the hyperactivation of PI3K/AKT/mTOR signaling to cause PCa . Recent The AR signaling pathway makes essential contributions to the development of normal prostate and PCa progression through regulation of PTMs including SUMOylation ,82,83. TSENP1 and SENP2 are reported to be the deSUMOylation enzymes that cleave the SUMO-1 group from SUMOylated AR protein , and theIn addition, SUMOylation is involved in the regulation of many other transcription factors and non-transcription factors for cellular functions in PCa malignancy . One stuIn addition to its potent roles in cancers, the ubiquitination pathway also plays important roles in human diseases including immune disorders, aging, diabetes and cardiomyopathies as it is involved in the regulation of some key signaling pathways  3,113,1,1113,114Dysregulation of ubiquitination is associated with the development and progression of neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s diseases . The abeThe ubiquitination pathway contributes to senescence and aging by regulation of oxidative stress and proteolysis processes. A reduction of the Ub proteasome system (UPS) and autophagy is recognized as the biochemical feature of aging, which results in a decline of cellular function and an accumulation of damaged and aggregated proteins. Knockdown of UBA1, an Ub E1 enzyme, results in typical features of aging: impaired motion and decreased lifespan in adult Drosophila . HoweverThe SUMOylation pathway plays essential roles in human diseases through regulation of genome stability, gene expression, protein-protein interactions and nuclear functions ,21,119. in vitro and in vivo deserves further investigation.Advances in mechanistic insights into both ubiquitination/deubiquitination and SUMOylation/deSUMOylation pathways have provided valuable information for the development of novel drugs to effectively combat cancers and other diseases. Tremendous progress has been achieved to successfully target these pathways for therapeutic purposes ,10,126. etc. In addition, the imbalanced procedures of ubiquitination/deubiquitination and SUMOylation/deSUMOylation can generate additional influence on dysregulation of multiple signaling pathways. Importantly, the ubiquitination and SUMOylation modifications of one target protein can be modulated differentially depending on stimuli or environments: (1) in a synchronized manner in which SUMOylation assists/determines the ubiquitin-mediated degradation [Overall, ubiquitination and SUMOylation as unique machineries of PTMs make important contributions to the initiation, development and progression of many human diseases and malignancy including PCa. Although present in only a small percentage of cells (less than 10%), target proteins modified with SUMO or Ub moieties in various linkages display dramatic impacts in the regulation of gene expression, genome instability, cellular functions, cellular senescence and stem cell reprogramming radation ; (2) in radation ,112; (3)radation ,80. Howe


It can be argued that adaptive designs are underused in clinical research. We have explored concerns related to inadequate reporting of such trials, which may influence their uptake. Through a careful examination of the literature, we evaluated the standards of reporting of group sequential (GS) randomised controlled trials, one form of a confirmatory adaptive design.st January 2001 to 23rd September 2014, supplemented with trials from an audit study. We included parallel group, confirmatory, GS trials that were prospectively designed using a Frequentist approach. Eligible trials were examined for compliance in their reporting against the CONSORT 2010 checklist. In addition, as part of our evaluation, we developed a supplementary checklist to explicitly capture group sequential specific reporting aspects, and investigated how these are currently being reported.We undertook a systematic review, by searching Ovid MEDLINE from the 1high impact” peer-reviewed journals. Examination of trials established that 46(68%) were stopped early, predominantly either for futility or efficacy. Suboptimal reporting compliance was found in general items relating to: access to full trials protocols; methods to generate randomisation list(s); details of randomisation concealment, and its implementation. Benchmarking against the supplementary checklist, GS aspects were largely inadequately reported. Only 3(7%) trials which stopped early reported use of statistical bias correction. Moreover, 52(76%) trials failed to disclose methods used to minimise the risk of operational bias, due to the knowledge or leakage of interim results. Occurrence of changes to trial methods and outcomes could not be determined in most trials, due to inaccessible protocols and amendments.Of the 284 screened trials, 68(24%) were eligible. Most trials were published in “There are issues with the reporting of GS trials, particularly those specific to the conduct of interim analyses. Suboptimal reporting of bias correction methods could potentially imply most GS trials stopping early are giving biased results of treatment effects. As a result, research consumers may question credibility of findings to change practice when trials are stopped early. These issues could be alleviated through a CONSORT extension. Assurance of scientific rigour through transparent adequate reporting is paramount to the credibility of findings from adaptive trials. Our systematic literature search was restricted to one database due to resource constraints. Appropriate use of adaptive designs has the potential to improve efficiency in the conduct of randomised clinical trials (RCTs). However, this can involve considerable extra work and effort during their planning, implementation, and reporting . One forVarious initiatives have been undertaken to facilitate discussions, and to address some of the barriers to the use of adaptive designs: predominately from a pharmaceutical drug development perspective ,4,8–14. rd September 2014, at least 30 reporting related guidance documents were being developed, to enhance transparency in the reporting and conduct of studies  that were completely reported was 26(70%) [24(65%) to 28(76%)], and a minimum of 15(41%). However, this distribution was 30(81%) [29(78%) to 32(87%)], and a minimum of 24(65%) for checklist items that met partial compliance criterion. The median number (proportion) [95% CI] of items that met complete compliance increased by 2 (5%)  post-publication of the CONSORT 2010 statement. The median difference (proportion) [95% CI] in items that met complete compliance in favour of journals that endorse the CONSORT statement as part of their publication policy was 1.5(4.1%) .group sequential” in the Title or Abstract. An additional 39(57%) were identifiable by the terms “interim analyses” or “interim analysis”. The rationale for choosing a group sequential design  was only explained in 11(16%) RCTs.Most items relating to group sequential aspects were poorly reported see . AdditioJust 11(16%) RCTs adequately reported the mechanism used to minimise operational bias due to the knowledge or leakage of the interim results; 7 of these cited relevant prior publications. Of the 33 RCTs that were reporting interim results after the first interim, 9(27%) reported or disclosed prior interim results. Only 3 RCTs reported unplanned deviations from planned group sequential design and its potential implications on the findings. However, unplanned deviations could not be assessed in 41(60%) RCTs due to inaccessibility of protocols and associated amendments. Only 2 RCTs described the lessons learned from using the group sequential design, and its value in helping the planning of future group sequential RCTs.The following aspects of a group sequential design were deemed to be adequately reported: description of the total (and per group) sample size, the planned number of interims and associated interim sample sizes (or number of events), and clarification on whether the RCT was stopped early with reasons where applicable. In addition, planned stopping criteria and rules or boundaries were deemed fairly reported. These particular interim analyses related items are well covered in the CONSORT 2010 checklist.Of the 68 RCTs, it was found that 46(68%) were stopped early; predominantly for futility, 61% (28/46), and efficacy, 22% (10/46) see . The proStopping criteria and rules or boundaries planned at the design stage, were unreported in 22(32%) and 24(35%) of RCTs, respectively. Of the RCTs that reported planned stopping criteria and/or stopping boundaries, 11(16%) cited additional relevant information accessible in the form of prior publications or protocols. Thirty-three (49%) RCTs were planned with at least some form of futility early stopping criteria; 9(13%) for futility only, 20(29%) for either futility or efficacy, and 4(6%) for futility, efficacy or safety.Twenty-four 35%) RCTs did not disclose the stopping rules or boundaries used. Of the 44(65%) RCTs that reported stopping rules or boundaries, the most frequently used stopping boundaries were: 1534%) Lan-DeMets (LD) [% Lan-DeM% RCTs diThe majority (75%) of RCTs were planned with either one or two interim analyses. There were very few RCTs planned with large numbers of interim analyses see . Only 5 reported the use of appropriate statistical methods for bias correction of point estimates of the intervention effect, and associated CIs and P-values; 2 of these were stopped early for futility and/or safety. Only 1 of the 10 RCTs that were stopped early for efficacy reported the use of bias corrected statistical methods to conduct inference.Although there were no publications that met complete compliance on all checklist items, we found exemplars of group sequential RCTs that reported most items adequately –47. The Roger et al  gave a cThe study design is based on a group-sequential test procedure with pre-planned analyses after 220, 320 and 428 patients meeting one of the off-study criteria. An alpha-spending approach as suggested by Lan and DeMets [with an O’Brien/Fleming-like alpha spending function was used to define the test boundaries of the group-sequential procedure. The primary analysis regarding OS uses a Cox Proportional Hazard Model with treatment and prognosis groups as predictor variables to calculate the Z score needed for the group- sequential procedure. Stagewise ordering was used to compute the unbiased median estimate and confidence limits for the prognosis-group-adjusted hazard rates [“d DeMets  with an rd rates .”One exemplar explained deviations from the planned interim analyses and the implications on their findings . Finallyhigh impact” peer-reviewed medical journals, and were often stopped early, predominantly for futility or efficacy.We found a significant number of confirmatory RCTs that employ interim analyses using group sequential methods, particularly in oncology, although the therapeutic areas of application appear to be diverse. Moreover, the majority of the group sequential RCTs were published in “We found inadequate reporting of CONSORT checklist items relating to the disclosure and access to trial protocols, methods used to generate the randomisation list(s), details of randomisation concealment and its implementation, and trial design aspects. Most concerning, is the lack of access to full trial protocols, with related amendments, in the public domain, for most of the examined group sequential RCTs. Hence, we could not ascertain the completeness in reporting of other key checklist items, such as changes to methods and outcomes. Despite this, compliance in reporting of most CONSORT 2010 checklist items appeared to be very high.In general, additional important features of group sequential RCTs are poorly reported. These encompass; rationale for choosing a group sequential design with any other add-on planned adaptations, mechanisms put in place to minimise operational bias due to the knowledge of interim results, lessons learned from using a group sequential design and its value, and clarification on whether sample size was adjusted for interim analyses. Most importantly, we found suboptimal reporting of the use of appropriate statistical methods for early stopping bias correction of point estimates, with associated CIs and P-values.Reporting of planned stopping criteria, and stopping rules or boundaries employed, is still unsatisfactory, despite the fact that aspects are covered in the CONSORT 2010 statement. Nonetheless, some aspects of interim analyses, such as clarification of early stopping with reasons where applicable, description of; interim sample sizes (or number of events), number of planned interim analyses and timing, were adequately reported.high impact” medical journals, particularly in oncology. Hence, the general quality of compliance in reporting, may exaggerate what might be observed based on reports in other therapeutic areas, or lower impact journals for some checklist items [Our review findings are predominantly based upon group sequential RCTs, published in “st items –59. For st items ,59. Regast items –61. SimiMost importantly, our findings of very poor reporting and use, of statistical methods for bias correction for early stopping, are consistent with previous findings of a systematic review, focusing on trials which stopped early for benefit . This diWe believe that our findings on areas requiring improvement provide a conservative picture, of the scale of the problem regarding compliance in reporting of group sequential specific aspects, which are vital for research consumers to make informed judgements about the quality of findings.high impact” medical journals. To some extent, this may provide assurance to sceptical researchers, who may have concerns pertaining to poor receptiveness by journal editors and reviewers towards adaptive designs, when RCTs are stopped early [Our review uncovered group sequential RCTs published predominantly in “ed early . In conted early ,62.The phenomenon of exaggeration of the intervention effects in group sequential RCTs, following early stopping when a naïve statistical approach is used, has been widely debated and highlighted –69. AlthAlthough there does not exist a unique solution to early stopping correction of statistical bias, various statistical procedures have been proposed ,70–76, aOne purpose of this article is to right a wrong I committed about a decade ago as part of the group reporting the results of The Randomized Aldactone Evaluation Study (RALES). … The trial stopped early after crossing an O’Brien–Fleming-like boundary , and we reported the results as we saw them; we corrected neither the p-value nor the effect size for having stopped early. I have consoled myself for this lapse because I knew that other trials that had stopped early had also failed to correct the observed results for having stopped early.”“The increasing use of add-on trial adaptations (such as sample size re-estimation and treatment selection in multiple arm studies), within the group sequential framework, adds more complexities requiring more transparency in the design, conduct and reporting of these trials, beyond the checklist items covered by the CONSORT 2010 statements .Our findings support initiatives for mandatory publication of, not only full trial protocols, but also all related protocol amendments. Assessing the quality of reporting of some key aspects of RCTs, proved challenging without access to these important trial documents, most imperative for complex adaptive designs. We absolutely concur with previous findings, that assessment of methodological quality should be based on evaluation of both protocols and publications .Interim analyses heighten anxiety among some research consumers, due to the potential for introducing operational bias to the conduct of RCTs, thereby undermining the scientific integrity, credibility and validity of the findings. The potential introduction of operational bias, due to the leaking or knowledge of interim results in adaptive RCTs, has been well described ,77. HoweOur findings of poor reporting or inaccessibility of, prior interim results at the interim reporting, or point of early stopping, could hinder the ability of consumers of research findings, to assess trends of the direction of the treatment effects and the potential effect of population drift. Although it is challenging to distinguish between natural and population drift induced by operation bias, access to prior interim results may help research consumers to make their own informed judgements, and alleviate some of the cited concerns.Based upon our findings and desire to improve: the reproducibility and planning of future trials, the acceptability of findings from group sequential RCTs and to reduce waste in trials research ; we reco▪group sequential” or at least “interim analys(i/e)s” in the Title or Abstract for easy identification of group sequential RCTs for future systematic reviews and meta-analyses;The use of the term “▪Provision for the rationale of choosing a group sequential design and any add-on adaptations used;▪Detailed description of the mechanisms put in place to minimise (and control for) operational bias due to the leaking or knowledge of the interim results;▪Clarification on the use of, and description of, appropriate statistical methods to obtain unbiased results  when trials are stopped early. This should also encompass description of appropriate statistical inferential methods used to account for add-on trial adaptations where appropriate;▪Disclosure of prior interim results on the primary endpoint(s) and related decisions made. Summary tables or figures showing trends in the results or citation of previous related publications may suffice;▪Discussion of the lessons learned by using a group sequential design, and any other add-on adaptations where appropriate, and its value, with implications to aid the planning of future trials;▪Discussion of the deviations from the planned group sequential design and any other add-on adaptations, and their implications on generalisability of the findings.We also encourage better reporting of group sequential aspects, which are already covered briefly in the CONSORT 2010 statement, such as planned stopping criteria and rules or boundaries used. Finally, more attention by journal editors and reviewers, and researchers, should be given to complete reporting of trial design, including any add-on adaptations; methods to generate randomisation list(s); randomisation concealment, and its implementation; and disclosure of full trial protocols (with related amendments).The need for this review has been based upon concerns raised by researchers, decision makers and policymakers regarding robustness and credibility of adaptive designs in decision making, to change practice when trials are stopped early . We provThere are issues with suboptimal reporting of key group sequential specific characteristics, such as disclosure of the use of appropriate adjusted inferential methods following early stopping. Suboptimal reporting of bias correction methods could potentially imply most group sequential trials stopping early are giving biased results of treatment effects. These issues may partly explain the cited concerns about robustness and acceptability of the methodology to change practice, when trials are stopped early. These concerns could be alleviated by modifications to the CONSORT 2010 statement. Assurance of scientific rigour through transparent and adequate reporting of RCTs is paramount to the acceptability of findings from adaptive designs in general. There is an urgent need for CONSORT statement(s) tailored for adaptive design(s). Improvements in the reporting of general CONSORT checklist items relating to access to trial protocols, trial design aspects, methods to generate randomisation list(s), concealment of the randomisation and its implementation is required.We hope our findings will enlighten researchers on potential ways to address some of the concerns we highlighted. We hope our results will also inform the CONSORT working group regarding the need for modifications to enhance the conduct and reporting of group sequential RCTs and adaptive trials in general.S1 Appendix(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS) development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent With the advent of induced pluripotent stem cells (iPSCs) and direct conversion techniques to create induced neurons (iNs), researchers now have the ability to examine cellular and molecular pathways in a completely human context with extraordinary genetic, pharmacological, and physiological control. For example, novel genetic engineering approaches such as zinc finger and TALE nucleases, as well as CRISPR/cas9, allow researchers to “correct” mutations in cell lines from diseased patients  or creat functional properties of neurons. As one of two electrically excitable cells in mammals, many disease-related phenotypes are thought to manifest as deficits in membrane excitability or synaptic communication between various neuronal populations. Many disorders such as autism, schizophrenia, and epilepsy are increasingly known as channelopathies or synaptopathies i. As Ca2+ acts as a second messenger to trigger long-term changes in cellular function, Timothy syndrome neurons displayed significant alterations in depolarization-induced gene expression compared with controls. These changes were correlated with differences in neuronal differentiation both in vitro and compared to transgenic mice carrying Cav1.2 mutation.One of the earliest examinations using hPSNs to model functional deficits was performed by Paşca et al. (2011) to study the effect of a missense mutation of the voltage-gated Casyndrome . While tsyndrome . NeuronsA4V mutation compared with unrelated wild-type cells. ALS hPSNs showed significantly greater spiking with no changes observed for passive membrane properties, which could be corrected via genetic correction of SOD1A4V mutation. Interestingly, delayed rectifier potassium currents driven by the KCNQ family of Kv7 channels were markedly reduced in ALS hPSNs, and administration of the Kv7 agonist retigabine reduced hyperexcitability and caused marked hyperpolarization (~6 mV) with EC50 of 1.5 μM , excitatory neurotransmission was impaired as indicated by reduced amplitude and frequency of sEPSCs , 13. In 2+ (LR) and those that were nonresponsive (NR). Interestingly, both populations of hPSNs showed hyperexcitable properties, with increased spontaneous and evoked AP frequency and amplitude, lower threshold of activation, and increased INa compared to control hPSNs. Interestingly, treatment of cultures with 1 mM LiCl eliminated these differences selectively in hPSNs derived from LR patients, but not from NR patients. However, cells from NR patients did show responsiveness to the antiepileptic drug lamotrigine. The authors went on to characterize the gene expression changes induced by Li2+ treatment and found several potential pathways altered, including genes involved with energy homeostasis and mitochondrial function, PKA/PKC signaling, and K+ channels.More recently, Mertens and colleagues examined the phenotypes of hPSNs differentiated from cells taken from patients with type I bipolar disorder . In an  in vitro-generated cells can recapitulate many aspects of disease with high degrees of specificity ( in vitro supports the furthered use of hPSNs and iNs to uncover novel and potentially human-specific molecular pathways governing functional maturation, dysfunction, and degeneration.Since the creation of iPSCs in 2007 and iNs in 2010, they have been used to examine disease phenotypes in hundreds of publications. Thus, in a short time these platforms have had a significant impact on our understanding of disease pathology and treatment and will likely change the direction of translational research going forward. With respect to functional phenotypes, it is remarkable that fullycificity . hPSNs aHowever, with any new tool we must caution against overinterpretation of the significance of any individual finding, particularly in light of the immature nature of the neurons derived and the variability of timing and cell type developed. As others and I have indicated , hPSNs aWith respect to variability in hPSN, efforts are currently underway to generate single cell transcriptomic and morphological signatures to correlate with functional properties of hPSNs in an effort to help identify subclasses of neurons. Through combined use of directed differentiation, cell sorting, and genetically encoded reporter lines, derivation of pure populations of transmitter- and functional phenotype-specific neurons is an achievable goal for some laboratories. These techniques will be particularly useful for cell-based therapies. Together with continued improvements in reducing tumorgenicity and aberrant differentiation through the use of insertion-free approaches and screening, iPSCs appear poised to revolutionize replacement therapies via functional integration with appropriate neural circuits. And, despite the cautionary notes, it is exciting to see that many stem cell researchers recognize the importance of functional assays as a complement to biochemical studies. With the improvements noted here and elsewhere , 80, dev


Oxidative stress as a result of elevated levels of reactive oxygen species (ROS) has been observed in almost all cancers, including leukaemia, where they contribute to disease development and progression. However, cancer cells also express increased levels of antioxidant proteins which detoxify ROS. This includes glutathione, the major antioxidant in human cells, which has recently been identified to have dysregulated metabolism in human leukaemia. This suggests that critical balance of intracellular ROS levels is required for cancer cell function, growth, and survival. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor plays a dual role in cancer. Primarily, NRF2 is a transcription factor functioning to protect nonmalignant cells from malignant transformation and oxidative stress through transcriptional activation of detoxifying and antioxidant enzymes. However, once malignant transformation has occurred within a cell, NRF2 functions to protect the tumour from oxidative stress and chemotherapy-induced cytotoxicity. Moreover, inhibition of the NRF2 oxidative stress pathway in leukaemia cells renders them more sensitive to cytotoxic chemotherapy. Our improved understanding of NRF2 biology in human leukaemia may permit mechanisms by which we could potentially improve future cancer therapies. This review highlights the mechanisms by which leukaemic cells exploit the NRF2/ROS response to promote their growth and survival. Acute myeloid leukaemia (AML) is primarily a disease of the elderly with 75% of cases being diagnosed in patients over 60 years of age . AML comOxidative stress is described as a change in the balance between reactive oxygen species and antioxidant defence mechanisms, where the balance is disturbed for the support of the oxidants . TogetheReactive oxygen species (ROS) are oxygen-containing chemical species with reactive properties, including free radicals such as superoxide and nonradical molecules such as hydrogen peroxide . These rThere is a complex interaction between ROS generation, signaling, and toxicity that results in the initiation, growth, and survival of cancer. Cancer may be induced through oxidative damage to cellular macromolecules as a result of overproduction of ROS, which subsequently affects antioxidant and/or DNA repair mechanisms . In addiAML develops from a common myeloid progenitor, a cell which would physiologically differentiate to form monocytes, granulocytes, platelets, and erythrocytes in the bone marrow , 18. AMLThe discovery of specific mutant genes in AML has provided increased biologic understanding, new potential targets for drug development , and newκB) are able to alter the redox balance of human cancer cells including leukaemic cells [A number of oncogenes such as KRAS, cMYC, BCR/ABL, NRF2, and NF-kappaB  induces an increase in ROS in AML cells as part of their mechanism of cytotoxic action . FurtherNRF2 regulates the expression of over 200 genes including many antioxidant genes and phase II enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) , 44 and GSH has emerged as an important regulator of chemotherapy resistance in human cancer. GSH is present in all mammalian tissues at 1–10 millimolar concentrations and protects against oxidative stress . In the It is becoming apparent that NRF2 is the main transcription factor that controls the regulation of many aspects of GSH synthesis and regeneration , 61. ImpHydrogen peroxide is one of the main activators of the NRF2-KEAP1 pathway. It is metabolised by GPX in the cytosol resulting in GSH being oxidized to GSSG in the mitochondria. GSSG can be reduced back to GSH by GR at the expense of NADPH, thereby forming a redox cycle, where organic peroxides can be reduced by either GPX or GSH S-transferase (GST) . GSTs arAlthough NRF2 is protective against tumorigenesis by reducing the amount of ROS and DNA damage in cells, tumour cells were found to be capable of harnessing the protective function of NRF2 for their own protection and survival , 70. IndPrimitive hematopoietic stem and progenitor cells reside within the bone marrow and express the CD34 surface antigen , 74. MorThe prognostic value of GST deletions in adult AML, including individuals with GSTM1 or GSTT1 deletions (or deletions of both), is found to have enhanced resistance to chemotherapy, lower complete remission, and a shorter survival ; this fuROS play an important functional role in human leukaemia. NRF2 and its control of GSH regulate ROS. Recent data suggests that GSH is fundamental to NRF2 function in AML suggesting that this pathway may yield future therapeutic targets for leukaemia cells in which GSH is dysregulated.