The potential role of inflammation and increased blood–brain barrier permeability in the pathophysiology of PTE is also discussed. A better understanding of mechanisms that promote the generation of epileptic activity versus those that promote compensatory brain repair and functional recovery should aid development of successful new therapies for PTE.Traumatic brain injury (TBI) greatly increases the risk for a number of mental health problems and is one of the most common causes of medically intractable epilepsy in humans. Several models of TBI have been developed to investigate the relationship between trauma, seizures, and epilepsy-related changes in neural circuit function. These studies have shown that the brain initiates immediate neuronal and glial responses following an injury, usually leading to significant cell loss in areas of the injured brain. Over time, long-term changes in the organization of neural circuits, particularly in neocortex and hippocampus, lead to an imbalance between excitatory and inhibitory neurotransmission and increased risk for spontaneous seizures. These include alterations to inhibitory interneurons and formation of new, excessive recurrent excitatory synaptic connectivity. Here, we review  Post-traumatic epilepsy (PTE) is a common long-term consequence of traumatic brain injury (TBI), for which there are few effective therapies. More than one million people are presented for medical care each year in the United States after sustaining a head injury . While tHere, we review experimental animal models used in PTE research, including the neurophysiological and structural abnormalities believed to underlie the increased propensity of the injured brain to generate spontaneous seizures. Emphasis is placed on modifications of synaptic networks in the injured dentate gyrus that are associated with post-traumatic epileptogenesis. The hippocampus has long been recognized as an important structure in epilepsy . The denin vivo, that develop following a sudden, mechanical injury  seems to be necessary minimal criteria for a “post-traumatic epilepsy” model. The presence of relatively high numbers of animals in which the behavioral, anatomical, and physiological characteristics of epileptic pathology in humans are reproduced, including short but distinct latencies between initial injury and spontaneous seizure onset, also has obvious advantages. Ideally, a model will effectively reproduce the range of tissue deformation and damage observed in mild, moderate, and severe TBI in a controllable way so that the dual pathology of cellular and molecular factors associated with injury dynamics and epileptogenesis might be dissociated. Using these selection criteria, many traditional experimental epilepsy models, such as pharmacologically induced status epilepticus, do not qualify as models of PTE, because they do not induce spontaneous seizures by a mechanical insult. This is not to negate these models, because they have provided valuable information about cellular mechanisms of epileptogenesis. Rather, these criteria provide a more restrictive and specific basis for differentiating between epileptogenesis mediated by a mechanically induced lesion versus brain insults by other causes . It is important to make this distinction, because there are currently few effective treatments for PTE  model is one of the best characterized in terms of epilepsy development after closed-head TBI . In thissevere FPI , but do not generally develop tonic–clonic convulsive seizures , 2009. SThe weight drop model, also referred to as an impact-acceleration injury, has been examined as a model of closed-head post-traumatic hyper-excitability . Trauma Controlled cortical impact (CCI) injury is a widely used experimental model of closed-head injury that was recently identified as a model of injury-induced epilepsy . First dSeveral laboratories have independently demonstrated the presence of spontaneous seizures in mice or rats after CCI. Early seizures, within 24 h of injury, have been reported in rats  and miceIn vivo electrographic epileptiform activity develops in intact regions of the neocortex within hours after injury in cats . An important goal of studies which investigate post-traumatic epileptogenesis is to dissociate injury-induced cellular alterations that promote seizure generation from compensatory and “self-repair” responses.The term “epileptogenesis” refers to a transformation process by which the normal brain develops an increased propensity for generating spontaneous seizures . This prPrimary injury refers to the immediate tissue deformation and compression that occurs within seconds to minutes after mechanical brain insult . There iSecondary injury involves a myriad of cellular and physiological factors associated with progressive tissue damage . Over tiGenetics, gender, age, acute medical treatments, and the agent of injury all likely influence the epileptogenic processes, but the contribution of these personal traits and injury dynamics in epilepsy have not been well established . A betteThe central dogma in epilepsy research has long been that seizures occur due to some type of imbalance between excitatory and inhibitory neurotransmission . In humaFigure 2). Dentate granule cells, which are not normally connected to each other, sprout axon collaterals into the inner molecular layer to form functional recurrent excitatory connections with nearby granule cells during epileptogenesis  and tonic (extrasynaptic) inhibition . Phasic inhibition refers to the transient activation of postsynaptic GABAA receptors at the synaptic junction following exposure to high concentrations of GABA released from presynaptic vesicles. This form of synaptic communication allows for rapid transmission of information from the presynaptic terminal to the postsynaptic membrane in a spatially and temporally restricted manner. In whole-cell voltage-clamp recordings, synaptic events can be easily identified as inhibitory postsynaptic currents (IPSCs). Small amounts of GABA escape the synaptic cleft and activate high-affinity extrasynaptic GABAA receptors on the same or adjacent neurons, an event termed “spillover.” Tonic inhibition refers to the persistent activation of these extrasynaptic GABAA receptors by low concentrations of ambient GABA. Phasic and tonic currents can be isolated by high concentrations of GABAA receptor antagonists. In whole-cell voltage-clamp recordings the tonic current is reflected by a shift in “holding” current.GABAergic interneurons form robust local synaptic connections with principal cells and play an important role in controlling cortical activity. Two primary modes of GABAA receptor subunit levels after CCI injury -6 results in elevated serum levels of tumor necrosis factor alpha (TNF-α) whereas treatment with antibodies against TNF-α results in decreased serum levels of IL-6 . IndividThe PTE field can be guided by reports that characterize or manipulate immune processes in non-PTE models and models of trauma. To date, many links between innate immunity and seizure expression in non-PTE models have been made studying cytokine signaling . CytokinIncreases in BBB permeability and tissue adhesion molecules may also reduce seizure threshold or have pro-epileptogenic effects . The BBB+ current in astrocytes that reduces their ability to buffer extracellular K+  may also contribute to the development of epilepsy. Chemokines are chemotactic proteins that are characterized by the relative location of their cysteine (Cys) residues within the NH residue . There i residue . The cen residue . Importa residue . Many ch residue . Unfortu residue . CCL3 an residue . Adminis residue . These r residue . TargeteCollectively, a number of studies suggest that inflammation may contribute to the development of PTE. Inflammation or other immune changes are shared features of many seizure disorders and these changes often precede the manifestation of behavioral seizures (i.e., during the latent period; The development of medically intractable epilepsy is one of the most common long-term health problems associated with head injury in humans. Recent work using animal models of TBI has shown substantial evidence for the formation of new, excessive recurrent excitatory synaptic connectivity and alterations to inhibitory interneurons in this disorder, although these studies have primarily focused on the dentate gyrus and other regions of the injured brain should be investigated in more detail. However, there are a number of important aspects of post-traumatic epileptogenesis that are not fully understood. For example, there is a need for a more complete profile of how individual components of inflammation and BBB permeability contribute to post-traumatic epileptogenesis. Thus, much progress has been made in modeling epilepsy in rodents after mechanical injury and basic mechanisms of neural circuit reorganization associated with epilepsy have been identified in these models, but new developments in this field should continue to identify precise cellular or molecular mechanisms that control the development of epilepsy after head injury.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the growing body of literature and putative links between the use of ergogenic nutritional supplements, doping and illicit drugs, it remains unclear whether, in athletes' minds, doping aligns with illicit behaviour or with functional use of chemical or natural preparations. To date, no attempt has been made to quantitatively explore athletes' mental representation of doping in relation to illegality and functionality.A convenience sample of student athletes from a large South-Eastern Australian university responded to an on-line survey. Competitive athletes (n = 46) were grouped based on self-reported use as follows: i) none used (30%), ii) supplement only (22%), iii) illicit only (26%) and iv) both supplements and illicit drug use (22%). Whereas no athlete reported doping, data provided on projected supplement-, doping- and drug use by the four user groups allowed evaluation of doping-related cognition in the context of self-reported supplement- and illicit drug taking behaviour; and comparison between these substances.A significantly higher prevalence estimation was found for illicit drug use and a trend towards a biased social projection emerged for supplement use. Doping estimates by user groups showed mixed results, suggesting that doping had more in common with the ergogenic nutritional supplement domain than the illicit drug domain.Assessing the behavioural domain to which doping belongs to in athletes' mind would greatly advance doping behaviour research toward prevention and intervention. Further investigation refining the peculiarity of the mental representation of doping with a larger study sample, controlling for knowledge of doping and other factors, is warranted. One of the key constraints in designing effective anti-doping programs is the lack of conceptual clarity of the psychological mechanisms that influence doping behaviour. For example, preventing doping use in sport on the basis of fair play vs. cheating naturally lends to prevention and intervention programs that focus on the ethics of anti-doping and values, coupled with the consequences of being caught - not necessarily limited to sanctions but including dishonour, shame and guilt. Other programs may emphasise the potential hazards and detrimental health effects as consequences of doping use, which are omnipresent, independently from doping testing and sanctioning. What is largely unknown is what approach (if any) works as an effective deterrent . StudiesIn the recent years, a number of athletes have talked publicly about their reasons and motives for doping use, contrasting perceived obligations and duty to perform well with guilt and the shame of lying. Studies conducted among professional athletes, particularly cyclists, offer valuable insight into how athletes perceive doping; and how this perception varies in different contexts -19. In aThe question of whether doping behaviour has the character of illicit substance use, ergogenic substance use, neither or both has been recently raised in connection with anabolic steroids . The ongThis pilot study is part of a larger research programme investigating social projection in various samples and context -37 and aPrevious results investigating social projection in performance enhancing and illicit drug use suggest that projected prevalence of doping and drug use was higher among self-admitted users respectively but absent for nutritional supplement use , and thaThe importance of the social project lies with the question of whether an elevated and potentially distorted social projection leads to a congruent behavioural choice or resulted from it . The facWith the view of gaining some insight into athletes' implicit mental representation, we focused on social projection as an indirect indicator of mental representation of doping. Whilst recent evidence suggests that social projection cannot be safely used as a proxy for behavioural measures ,37, declvice versa (the positive case). Conversely if doping belongs to another domain, then the estimates of doping would occur independently of nutritional supplement use (the negative case). This suggests the relatedness of doping with either illicit or ergogenic substance use behaviours can be determined by emergent patterns of the FCE across behaviours.The FCE is a perceptual bias where people who engage in particular behaviours tend to overestimate the proportion of the population who also engage in that behaviour. People who abstain either underestimate or correctly estimate prevalence. For example, marijuana users tend to overestimate the proportion of the population who use marijuana, and non-users are more accurate or underestimate ,41. A fuIn order to elucidate the peculiarities observed in the supplement-doping-drug triangle, this paper aims to test the domain specificity of doping relative to illicit substance and ergogenic nutritional supplement use via the FCE to explore whether it is an illicit or functional phenomenon. A proposed schema of mental representation of doping is depicted in Figure Following ethics approval, an on-line survey collected data from students attending a South-Eastern Australian university. Every student of the student population of > 40,000 who logged into the website during the 4-week period in 2009 received the link to the survey, with no tracking option as imposed on the project by the institutional ethics committee. This convenience sampling method yielded 186 responses, of which n = 127 (86 usable) were athletes and n = 59 were from the general student population (comparison group for selected questions). A number of respondents provided variably incomplete data across the question set.rd grade sport or above (n = 46) imposed for investigating the mental representation. This selection criterion was based on the level of sport involvement. Australian 3rd grade sport represents a point at which club sport begins to feed into professional or elite sport, or serve as a transition after injury or is used to regain form for professional or elite athletes. Students who reported participating at 3rd grade or above captures student athletes who may experience the competitive pressures of sport differently to those who participate in recreational or social sport. Recruitment was via a link on the top page of the administrative web site used by students for enrolment, examination timetables and so on. The on-line survey was part of a larger study on responses to the use of different kinds of substances in sporting contexts. Only the measures of relevance are reported, namely: i) age, gender, sport and level of participation; ii) questions on illicit drug use in the general population and personal use; iii) questions on ergogenic supplement use in the general population and personal use; and iv) questions on doping use in the athlete population and personal use.The athlete group (86 usable) was further restricted to those in 3Nutritional supplements' are vitamins , minerals  and non-vitamin non-mineral substances including herbals and botanicals . 'Doping' or 'banned substances' are those substances that are prohibited by the World Anti-Doping Agency or other governing body in training and/or competition. 'Social drugs' are psychoactive drugs  used for recreational purposes rather than for work, medical or spiritual reasons. Although caffeine, alcohol and tobacco are also social drugs, are excluded from the definition in this survey. These definitions were provided at the start of the questionnaire and used in this manuscript. The terms 'social drugs' and 'illicit drugs'; 'doping' and 'prohibited performance enhancing substances' are used interchangeably throughout the paper.The dependent variables were estimated population prevalence of nutritional supplement, illicit drug use and doping. These substances were defined as follows: 'What % of others in your sport has used a banned substance?'. The responses were recorded on a percentage scale where 0% means that nobody and 100% means that everybody uses a substance.The FCE was gauged by asking respondents to make an estimation of the level of fellow athletes using prohibited performance enhancing substances, illicit drugs and nutritional supplements independently asking 'Do you believe that most people need supplementation to balance their diet?' (Yes/No/I don't know) and ii) 'Do you believe that it is possible to win in high level sport competitions without doping?' (Yes/No/I don't know).In addition, athletes were asked about their beliefs regarding the effectiveness and necessity of various performance enhancing substances using the following questions: i) 'The FCE tests compared estimated lifetime prevalence rates by personal use (independent variables with levels "yes" and "no"). Domain specificity was assessed by examining whether the FCE emerged by personal use within and across substances. In operational terms, domain specificity emerges from the absence of the FCE when users of one substance (e.g. illicit drug users) estimate the prevalence of another unaccepted drug (e.g. doping). Estimations for substances with similar ergogenic effects to doping but with generally accepted use  were expected to show no difference between users and non-users.L), where L is the likelihood for an estimated model with k parameters. Thus the AIC is not testing the model with a traditional null hypothesis but rather, it affords ranking and comparing competing statistical models taking complexity into account. Statistical analyses were performed using PASW Statistics 17 and R Statistical Computing.One-way analysis of variance assessed differences between groups with Tukey's HSD post hoc test when required. To test domain specificity, logistic regression was used for the prediction of the probability of an event  by fitting a logistic curve to a predictor variable . Model fThe mean age of the athletes in the sample (n = 86) was 23.07 . The prevalence rates for self-reported nutritional supplement and social drug use are presented in Table rd grade or above . Self-reported supplement use (43.5%) was below that reported by the only epidemiological study on Australian supplement use (52.2%) [Among athletes who responded to the survey, 46 met the criteria of sport participation 3 (52.2%) . Self-rement use .5% was b (52.2%) . AggregaEvidence supporting domain specificity was found among self-admitted illicit drug users. The simple models of fitted logistic regression curves predicting the probability of social drug use based on social drug use estimate and doping use estimate are depicted in Figure The model based on self-reported illicit drug use and illicit drug use estimate showed a good fit ; whereas the model for predicting doping use based on self-admitted social drug use was unsatisfactory . In practical terms, the model fit (or the lack of fit in the case of cross drug type prediction) provides some evidence for domain specificity. The social drug use estimations given by those who admitted its use were congruent with the previously observed FCE and resulted in a well fitting prediction model. On the contrary, social drug users' estimation of doping use failed to show an acceptable relationship between behaviour and estimation. Should there be no distinction made in athletes' minds about these two classes of drugs , we would expect to see good fitting models across all four possible combinations of doping use, drug use, doping estimates and social drug use estimates.As expected, differences in social projection by the user groups, including mixed groups, reflected the group characteristics. All groups overestimated population and sample prevalence of illicit drug use. No statistically distinct FCE emerged for supplement use. Means, standard deviations and ANOVA test statistics with corresponding p-values are presented in Table In terms of assessing the behavioural domain for doping, the results are ambiguous. Overestimation of doping by non-users suggests something other than substance use drove the estimation. The relative underestimation  by supplement users supports this notion. The pattern of doping estimation approximates that of illicit drugs with all groups overestimating prevalence. This pattern fails to provide firm evidence whether doping is related or unrelated to other substance use behaviours.The FCE was found for illicit substance use and was evident as a trend for ergogenic supplement use. It is unclear whether the results point to a relationship between doping and either or neither of the other substances. The results associated with respondents who used supplements suggested that doping estimates may be influenced by ergogenic supplement use. Individuals who used supplements tend to inflate the percentage of individuals who dope but to a much smaller degree than those who use other substances. In addition to these main results, illicit drug use and doping were overestimated. This indicates that, self-report notwithstanding; Australian university athletes may have unrealistic perceptions of illicit drug use and doping . While tvice versa.The presence of the FCE within rather than between substances provides an indication that nutritional supplement use and illicit drug use come from different behavioural domains. Individuals who admitted using one particular type of drug tend to inflate the percentage of individuals who uses the same drug to a much larger degree than those who use other substances. This suggests the FCE could provide an expedient way of identifying when interventions designed to influence one behaviour could influence another. The results indicate that interventions aimed at illicit drug use are unlikely to have much effect on supplement use, and Estimates of those who used both supplements and illicit drugs were more akin to illicit drug users, suggesting illicit drug use may be a dominant behavioural domain. As Table 3 shows, users of 'both' illicit drugs and NS gave similar social projections (67%) for illicit drug use to the projected figure of those who use illicit drugs only (74%), compared to a definitely lower estimate (55%) given by NS-only users, suggesting that users of 'both' may have behaved like illicit drug users due to that domain requiring a different psychological mechanism. For example, users of both may do so as a function of substance use, whereas supplement only users may do so for ergogenic reasons. The dominance of illicit drug use may have implications for the domain specificity of doping behaviour. A reversed pattern was observed for NS use projection, where NS-only users gave higher estimation (46%) compared to those who use both (34%), thus providing further evidence that in mixed behavioural categories, and the self-anchored behavioural domain is context specific.et al [The effort to determine whether doping was more akin to illicit drug or ergogenic supplement use led to mixed results. Out of these mixed results, the marked lower estimate by supplement users may be a clue. Mazanov et al  showed tThe observation that the sample overestimated illicit drug and doping use is concerning. Such overestimation has been observed elsewhere  and reprThe sample sizes themselves are a clear indication of the low statistical power. In this instance, the statistical tool is used to establish direction for future research rather than establish definitive results that require a higher level of statistical integrity. The intention of this short report was to draw researchers' and policy makers' attention to this potentially important aspect of doping behaviour to facilitate further targeted research. Future research building on this pilot study should involve collecting data from a representative larger sample that delivers statistical certainty. Assessing knowledge of doping and anti-doping to determine if it impacts doping estimates would strengthen the findings.This pilot study provided an indication that harnessing the FCE might be a fruitful avenue to further examine whether doping behaviour has more in common with illicit drug use or ergogenic supplement use. The pilot nature of the study suggests that doping may be an ergogenic phenomenon, however further testing with an improved research design and sample is needed to establish any such claim. The importance of having a precise picture of the mental representation of doping is underscored by the increasing need for effective anti-doping prevention and intervention. Further research is required to establish if some athletes project their own behavioural tendencies or actual behavior onto other athletes and assume that many others feel or do the same and indeed are using prohibited performance enhancing substances. As a consequence, their own doping tendency or behaviour appears normal and normative, so that they can follow it without compromising their own self-esteem and social acceptance. In this vein, FCE has importance beyond being a useful vector to understanding the position of doping in athletes' minds. These considerations, coupled with the mental representation of doping in athletes' minds, suggest possible intervention strategies to increase compliance with anti-doping initiatives.The authors declare that they have no competing interests.AP drafted and finalised the paper, and with DPN designed the study and the questionnaire. JM collected and analysed the data, and developed early drafts. All authors approved the final version the paper.


The present study reports the case of a 33-year-old male who presented with Terson syndrome with no cerebral hemorrhage secondary to traumatic brain injury (TBI). A computed tomography scan of the patient, who had sustained an impact injury to the right occipital region, showed no cerebral lesion. Ophthalmoscopy clearly demonstrated vitreous hemorrhage in both eye globes. Vitreous hemorrhage, which results from an abrupt increase in intracranial pressure (ICP), is associated with TBI. In this case, the visual disturbance was attributed to Terson syndrome secondary to TBI. Therefore, close ophthalmological and radiological evaluation is required in patients with TBI, in order to enable the diagnosis of Terson syndrome and an early vitrectomy. Terson syndrome was originally defined as a vitreous hemorrhage arising from an aneurysmal subarachnoid hemorrhage (SAH) ; howeverThe presence of vitreous and preretinal hemorrhage without cerebral hemorrhage occurring secondary to TBI, creates challenges for oculists and neurosurgeons and reduces the likelihood of Terson syndrome being considered. This study presents a rare case of Terson syndrome with no cerebral hemorrhage secondary to TBI.The patient, a 33-year-old male miner, was hit in the right occipital region by a boulder while mining, three hours prior to admission to a local clinic. Computed tomography showed no cerebral lesion. One week later, the patient presented with sudden visual disturbance. Ophthalmoscopic examination detected vitreous hemorrhage in both eyes and the vitreous hemorrhage was subsequently managed with conservative treatment. The patient was referred to Tangshan Ophthalmology Hospital  due to visual deterioration six months subsequent to the onset of the symptoms. Visual acuity was 20/400 in each eye. The patient’s ophthalmic signs were as follows: Peripheral vitreous hemorrhage, peripheral preretinal hemorrhage, macula lutea and optic papilla traction and tractional retinal detachment . As a reTerson syndrome, a known complication of cerebral hemorrhage, may lead to loss of vision. Vision loss develops subsequent to cerebral hemorrhage, as a result of vitreous hemorrhage caused by retinal capillary disruption. The pathogenesis of Terson syndrome has been suggested to be associated with an impairment of the retinal microcirculation as a result of increased ICP following cerebral hemorrhage, which ultimately results in venous hypertension and hemorrhage . Therefoet al(The present case showed that the absence of cerebral hemorrhage secondary to TBI may induce an abrupt increase in ICP, resulting in Terson syndrome. Hoving et al describeet al. In the et al.The immediate examination of patients with TBI for intraocular hemorrhage is important due to the fact that severe, non-clearing vitreous hemorrhage may result in visual loss. A prompt diagnosis of Terson syndrome for patients with vitreous hemorrhage suffering from TBI with no cerebral hemorrhage is required to enable vitrectomy to be performed earlier.


The present investigation was undertaken to test whether exercise training (ET) associated with AMPK/PPAR agonists (EM) would improve skeletal muscle function in mdx mice. These drugs have the potential to improve oxidative metabolism. This is of particular interest because oxidative muscle fibers are less affected in the course of the disease than glycolitic counterparts. Therefore, a cohort of 34 male congenic C57Bl/10J mdx mice included in this study was randomly assigned into four groups: vehicle solution (V), EM [AICAR  and GW 1516 ], ET (voluntary running on activity wheel) and EM+ET. Functional performance (grip meter and rotarod), aerobic capacity (running test), muscle histopathology, serum creatine kinase (CK), levels of ubiquitined proteins, oxidative metabolism protein expression  and intracellular calcium handling  protein expression were analyzed. Treatments started when the animals were two months old and were maintained for one month. A significant functional improvement (p<0.05) was observed in animals submitted to the combination of ET and EM. CK levels were decreased and the expression of proteins related to oxidative metabolism was increased in this group. There were no differences among the groups in the intracellular calcium handling protein expression. To our knowledge, this is the first study that tested the association of ET with EM in an experimental model of muscular dystrophy. Our results suggest that the association of ET and EM should be further tested as a potential therapeutic approach in muscular dystrophies. Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disease caused by the absence of the protein dystrophin Many investigators suggest that exercise training is beneficial in muscular dystrophies, since it results in better intracellular calcium handling, activation of compensatory or antagonistic signaling pathways, increased antioxidant capacity and angiogenesis, improved ability to blunt alpha-adrenergic vasoconstriction, increased iNOS activity and membrane protective effects AMPK (AMP-activated protein kinase) and PPAR (peroxisome proliferator-activated receptor) agonists are exercise mimetics. In 2009, Miura et al. demonstrated that a PPAR agonist reduced the number of skeletal muscle fiber membrane lesions and decreased force drop due to eccentric contractions in mdx mice As exercise training and its mimetics present both coincident and different beneficial effects www.cobea.org.br) and was approved by the University of São Paulo, Institute of Biosciences Ethical Committee (087/2009).This study was conducted in accordance with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation  and GW 1516  every other day and the ET animals stayed in individual cages with activity wheel and a cycle computer to register time and intensity of exercise. The mice were maintained in a 12:12 h dark-light cycle and temperature-controlled environment (22°C) with free access to tap water and standard laboratory chow . Before being killed, the animals stayed for 24 hours in metabolic cages for measurements of food intake, feces excretion, water intake and urine excretion. Furthermore, after a night fasting (one week before to be killed), their blood was harvested for glucose, triglycerides and cholesterol analysis .A cohort of 34 two-months old male congenic C57Bl/10J mdx mice were randomly assigned into four groups: vehicle solution (V), exercise mimetics (EM), exercise training (ET) and EM+ET. Analysis started when the animals were three months-old. During all the month before the EM mice received AICAR  with the forepaws as the experimenter gently pulled on their tails - the result is the maximal force before the animal releases the forepaws of the bar (mean of three measurements of maximum pull) After the tests, the mice were killed and triceps braquialis muscle was harvested, immediately frozen in melting isopentane and stored in liquid nitrogen. The frozen muscles were cut into 10-µm cross sections from the proximal to distal region using a cryostat . Sections of muscle were then stained with hematoxylin and eosin In order to evaluate aspects related to muscular structure, oxidative metabolism and calcium handling, immunoblots of triceps braquialis muscle homogenates were performed according to Bacurau et al. In order to evaluate mice renal function, we evaluated creatinine clearance All values are presented as means ± SE. Two-way analysis of variance  and Duncan post-hoc test  were used to compare the groups. Statistical significance was considered as p≤0.05.As presented in the In animals that had a better functional capacity (exercise mimetics + training), skeletal muscle showed statistically increased cross sectional area , 0.015, Since an improvement in oxidative metabolism is a proposed mechanism to explain the beneficial effects of both exercise mimetics and exercise training, we evaluated the expression of proteins related to oxidative metabolism. As observed in Since impaired transsarcolemmal calcium flux and sarcoplasmic reticulum calcium release/reuptake has been identified as main contributors to skeletal muscle functional and structural abnormalities As a potential adverse effect of different drugs can be an impaired renal function, we analyzed creatinine clearance. Interestingly, the exercise mimetics group displayed better renal function than the other groups .Finally, as both aerobic exercise training and AMPK/PPAR agonists accelerate the oxidative metabolism, an expected consequence would be a reduction in the amount of fat in the body. In order to test this hypothesis, we analyzed the carcass fat of the animals. The results are presented in the The main result of the present study was that voluntary exercise training combined with AMPK/PPAR agonists improves the functional performance and the aerobic capacity of mdx mice. Of particular interest is the fact that these improvements might be associated to reduced muscle degeneration  and improved efficiency of the aerobic metabolism. To our knowledge, this is the first study that tested the association of exercise training with AMPK/PPAR agonists in a disease murine model.Here we show that the association of exercise training and AMPK/PPAR agonists resulted in functional improvements related to grip meter, rotarod and aerobic capacity (running test). The animals submitted only to the exercise training showed improved aerobic capacity, supporting the efficiency of the training, and the animals that received only the drugs had an improvement in the grip meter test. Our data in the running test corroborated Narkar et al. Interestingly, the exercise training group presented increased values of training intensity in wheel activity when compared to the animals that also received exercise mimetics - the time of activity per day was similar in both groups. This can help explain, at least in part, the results where the combined therapy did not result in a better outcome as compared to the isolated approaches. Although different intensities between the groups can be a limitation of the present study, we did the running test before the treatment to certify that both groups presented similar aerobic capacity (data not shown). Furthermore, the absence of exercise intensity control is inherent to the activity wheel method and this kind of voluntary training was chosen because it has been demonstrated that it avoid excessive overload to the dystrophic muscle. Exercise intensity is a prominent factor to induce muscle adaptations, but dystrophic patients are not able to perform intense exercises because their muscles are more susceptible to mechanical stress Although strength training is more efficient to induce hypertrophic response, aerobic exercise training also can increase muscle mass, particularly in animals with skeletal muscle abnormalities As stated before, glycolitic muscle fibers are more affected by the dystrophy when compared to the oxidative counterparts and only the exercise mimetics group presented increased percentage of type I fibers than the control group. This effect of drugs is in accordance with Narkar et al. We have no explanation for the apparently decreased activity in serum CK in the mimetics group. It could be due to the small sample size or to the natural fluctuation that is seen in this enzyme which might not be directly related to the dystrophic process We also observed that in the group in which exercise training was associated with AMPK/PPAR agonists, there was an increased expression of AMPK, phosphorylated AMPK and PPARδ. The exercise training group also presented increased AMPK activity evaluated by phosphorylation and these results corroborate the running test data since both groups with highest running performance presented increased AMPK activity. Additionally, it is known that mice with dysfunction in the skeletal muscle AMPK signaling present reduced running capacity However, the high levels of triglycerides in this group do not represent a metabolic disorder - these animals do not present increased cholesterol and glucose values, for example. The result can be related to decreased lipogenesis (discussed before) and the time that blood was harvested for analysis. In addition, Narkar et al. demonstrated that triglycerides levels may be increased by exercise Although they were not analyzed in previous studies with AMPK/PPAR agonists We also demonstrated that the ratio between protein expressions of phosphorylated and total acetyl-CoA carboxylase (Ser79) was increased in the three groups submitted to the treatments, which indicate inhibition of carboxylation of acetyl-CoA to malonyl-CoA in the biosynthesis of fatty acids. These results might explain the results from the carcass fat. In addition, these data reinforce the effectiveness of the exercise mimetics treatment because an AMPK downstream activity is the phosphorylation of acetyl-CoA carboxylase In short, a beneficial effect of combination of exercise training and AMPK/PPAR agonists was observed in mdx mice. Although exercise training alone or alternatively its mimetics showed some improvement in skeletal muscle histology and aerobic capacity of mdx mice, the combination of both strategies seems more effective. In addition, our results suggest that a favorable protein turnover  as well an improved efficiency of the oxidative metabolism might explain, at least in part, the observed functional improvements. It will be interesting to repeat these studies in other experimental models of muscular dystrophy. Although most dystrophic patients are not able to run, the animals in the present study are in the early stages of dystrophy, when many children would still be able to exercise. It would also be of interest to assess if the association of passive exercise and AMPK/PPAR agonists have a comparable beneficial effect.


Accounts of participation in genetic research are dynamic and innately social. Participation is viewed not as an individualised act but as morally significant and involving relational responsibilities. Previous work has investigated individuals’ motivations for research participation in terms of such relational complexities. Here we explore the significance of relational responsibility in the context of participation in a familial cancer study where many family members are eligible for recruitment. We consider accounts of family (non)participation, (im)morality and (ir)responsibility. By looking at accounts we can explore the ways in which people use explanations to construct social reality. Accounts are more than disclosing internal attitudes, rather they provide claims of moral worth and participation in the social world. The accounts are taken from eight in depth interviews with participants from the kConFab Study, investigating familial breast cancer.Interviewees gave the juxtaposed accounts described elsewhere, with motivations for research participation framed in terms of self, family and society. In addition to accounts relating to themselves, all interviewees accounted for family members’ (non) participation. Accounts of self and relatives’ participation in the research were brief, with strongly altruistic framing and participation described as meeting expectations. Four participants knew of family members who had chosen not to participate. Accounts about this were longer and involved more elaborate moral character work. As research participation was framed as beneficial to the family and others, with minimal burden to the participant, excusing and justifying non-participation was required in order to explain it. This was done through descriptions of illness, disability, relatives’ feelings of guilt and logistics. In one instance a sister’s non-participation was not justified or excused but explained it in terms of her ‘selfishness’. Reports of non-participation by relatives involved negative feelings towards them, ranging from frustration to anger. Stronger emotional responses were described when the relative was perceived as having particular responsibilities towards the interviewee or their offspring, as a parent or sibling.The familial tensions described in the accounts are part of a broader narrative of pre-existing difficulties in family dynamics. The genetic research context became another field in which these difficulties became evident or entrenched. Our findings inform our understanding of how genetic research may be experienced within a family. With participation framed as acting responsibly towards others there may be relational ramifications if individuals decline to do so. This has implications for how consent is viewed and how studies may choose to recruit participants.


Pain relief is a major goal for palliative care in India so much that most palliative care interventions necessarily begin first with pain relief. Physical therapists play an important role in palliative care and they are regarded as highly proficient members of a multidisciplinary healthcare team towards management of chronic pain. Pain necessarily involves three different levels of classification–based upon pain symptoms, pain mechanisms and pain syndromes. Mechanism-based treatments are most likely to succeed compared to symptomatic treatments or diagnosis-based treatments. The objective of this clinical commentary is to update the physical therapists working in palliative care, on the mechanism-based classification of pain and its interpretation, with available therapeutic evidence for providing optimal patient care using physical therapy. The paper describes the evolution of mechanism-based classification of pain, the five mechanisms  are explained with recent evidence for physical therapy treatments for each of the mechanisms. Pain is the chief symptom reported by patients in a palliative care unit and most palliative care interventions begin first with pain relief. Since paPain is a common complaint among patients seeking physical therapy services. Physical therapists play an important role in palliative care and theyThe effects of physical therapy in pain management extend far beyond the physical aspect of symptom control, into the behavioral domain of quality of life during therapeutic care. PhysicalOne of the initial steps in a physical therapist’s evaluation of patients with chronic pain is to identify the basis for symptoms and their occurrence. PatientsWe are witnessing a paradigm shift in pain medicine towards a mechanism-based approach and in lSmart and Doody found amIn contrast to symptomatic and/or diagnosis-based treatments, mechanism-based treatments are most likely to succeed. The objeThe works of various authors14–16 insCentral sensitization/ central neurogenic mechanism/ central nociceptive mechanismPeripheral sensitization/ peripheral neurogenic mechanismPeripheral nociceptive mechanismSympathetically maintained pain/ sympathetically dependent pain mechanismCognitive-affective  mechanismet al.,[Central sensitization represents an enhancement in the function of neurons and circuits in nociceptive pathways caused by increases in membrane excitability and synaptic efficacy as well as by reduced inhibition and is a manifestation of the remarkable plasticity of the somatosensory nervous system in response to activity, inflammation, and neural injury. Nijs et et al., describeet al.,[Egloff et al., listed pet al.,Patient education Chronic pain patients mostly develop ‘pain behavior’ and treatments not addressed at their knowledge, attitudes, beliefs, and past experiences about pain are likely to be deleterious and very harmful. As any change in behavior originates from acquisition of knowledge, cognitive strategies such as education about pain works a large effect in these situations. Moseley proposed a pain neuromatrix approach[ approach to manag approach–26 found approachPain-relieving modalities such as transcutaneous electrical nerve stimulation-TENS (low frequency) through their operating ‘placebo effect’ would definitely facilitate descending inhibitory control over pain. There is a growing body of evidence for TENS in chronic pain states such as phantom limb and stump pain,[ump pain, chronic ump pain, and chroump pain, Similar ump pain,Relaxation as a treatment technique provides an excellent adjunct to other physical therapy treatments in chronic pain states, not only to manage stress and anxiety, and their deleterious effects on the sympathetic nervous system but also to induce increases in levels of endogenous opioids—endorphins and enkephalins.[ephalins. It operaephalins.Peripheral desensitization techniques help patients such as those with hyperalgesia and allodynia. Areas with hypersensitivity to light touch can be desensitized with deep pressure since the receptors and the afferent pathway for both are entirely different.[8, T1 dermatome), can be desensitized by application of cutaneous deep pressure C7-T1 spinal region .ifferent. DesensitBiofeedback facilitates closed-loop motor learning to relearn normal painless movement patterns and functional activities whilst inhibiting the features of central sensitization locally in that painful part.[ful part.Guided imagery/ mental imagery/ motor imagery[r imagery UtilizesMirror therapy[r therapy–36 Is ver therapy which inVirtual reality[l reality Through Manual therapy Through stimulation of wide dynamic range afferent neurons produces secondary hyperalgesia by diffuse noxious inhibitory control mechanism. The manual techniques also evoke changes in cortical pain representation and enhance the brain’s representation of body areas.[39dy areas.3940The International Association for the Study of Pain (IASP) defined peripheral neuropathic pain as ‘pain arising from or caused by dysfunction of peripheral nervous system’. This broPathophysiological and pathomechanical responses to nerve injury affect the vascular, connective tissue, and impulse-conducting tissue components of the nervous system and lead to the neurobiological mechanisms responsible for the positive and negative symptoms associated with musculoskeletal peripheral neuropathic pain.Nerve trunk pain typically presents as pain or abnormal sensations along the course of the peripheral nerve that can be clinically tested using the concept of neurodynamics. Neurodynamics is a concept based on the close interaction of the mechanics and physiology of the nervous system which is to be considered while assessing and treating patients via nervous system mobilization and manual therapy.Reproduction of a patient’s pain during neurodynamic testing and presence of mechanical allodynia on nerve trunk palpation were key diagnostic signs of neural tissue mechanosensitivity.46A recent systematic review of neuroHanai found thThis mechanism was the commonly recognized one among physiotherapists11 and itThe distribution of pain follows a characteristic anatomical pattern that corresponds to the tissue-at-fault. The pain is accompanied with other signs of inflammation such as tenderness, warmth, swelling and loss of function in the acute phases of injury. Specific aggravating and relieving factors which may be postures and/or movements will provoke symptoms. On testing active movements, passive movements and resisted isometric tests, a physical therapist can diagnose and differentiate injuries of contractile (muscle and tendon) tissues from those of inert (osseocapsuloligamentous) tissues. Manual palpation of the tissue provokes pain exhibiting as local tenderness that can be measured using pressure pain algometry.Treatments for nociceptive mechanism-induced pain may include different modalities as well as conditioning exercises for healing of injury and inflammation. Modalities like heat, cold, therapeutic ultrasound, TENS and interferential current; and conditioning exercises that include stretching and flexibility exercises, massage, myofascial release and joint mobilization are also very useful for relieving pain.Manual therapy was found to be effective for symptom relief in nociceptive/musculoskeletal pain in two reviews.53Cryotherapy or cold therapy increases pain thresholds and pain tolerance at the applied area which when combined with reduction in sensory nerve conduction velocity provides adequate pain relief.et al.,[et al.,[Feine et al., in theiret al., Rakel anet al., and Herb,[et al., found thThe hallmark of sympathetically maintained pain (SMP) is ongoing pain and allodynia, which is typically out of proportion to the injury. The causSymptoms of deep boring pain with altered intolerable sensations  mostly independent of positions and/or movements are a characteristic of SMP. Presence of autonomic signs like alterations in sweating, vasomotor function, thermoregulatory function, tissue permeability, skin trophic changes are a hallmark of diagnosis for SMP and complex regional pain syndromes. Diagnostic confirmation is only by means of a sympathetic block or sympathectomy, which mostly relieves symptoms of SMP.Thermal therapy can be used depending upon the local temperature of the affected region and thermal sensitivity. Local warmth may indicate cold therapy and vice versa. Use of thermal modalities like heat and cold are always done as an adjunct before beginning other therapy in patients with SMP.[with SMP.TENS has an inhibitory effect on the sympathetic nervous system so has been found effective in sympathetically maintained pain.[ned pain.Sympathetic slump mobilization is a neurodynamic technique under manual therapy that addresses neural tissues, especially the sympathetic trunk.[ic trunk. The techic trunk. and loweic trunk. and was ic trunk.Exercises can be given as a progression that includes passive range of motion, and isometric strengthening improves joint mobility and muscle strength, thereby providing alternate mechanical stimuli and graded exposure.et al.,[Casey et al., demonstret al., While coPatients’ education and cognitive strategies: . Education facilitates positive beliefs, problem-solving, enhanced patient functioning and self-management, and encourages active coping[ve coping with chrve coping Catastrove copingRelaxation techniques Hypnosis and operant conditioning usually provided by clinical psychologists help in inducing positive behavioral changes.[ changes.Distraction with videos or music helps reduce anxiety and depression associated with chronic pain.[nic pain.et al.,[Morley et al., in theiret al., numerouset al., as interet al., graded aet al., graded met al., and actiet al.,[This clinical commentary is the first of its kind to throw light on mechanism-based classification of pain with a physical therapy management perspective. Earlier studies utilized ‘pathomechanism-based classification (PMBC) system’ for low back-related neural leg pain which was proposed by Schafer et al., and studet al., and discet al., The PMBCet al.,[One of the mainstays in non-pharmacological interventions in pain and palliative care is physical therapy. Jones et al., describeet al., work-relet al., and worket al., MBC is dPatient-centered care should focus on individualizing treatments based on mechanisms and not on diagnosis. Such an Currently, there are no existent mechanism-based treatment guidelines or recommendations. The following suggestions are provided herewith, for planning future studies on MBC:Prevalence of MBC sub-groups in various pain conditions encountered in palliative care, Mechanism-based management using other treatments, and Effectiveness of educational and/or training programs on MBC.“An evidence-based approach to pain management is not always possible or beneficial to the patient. In the face of inconclusive evidence, a theory-based approach may help determine if the therapeutic effect of a given physical agent has the possibility of being a useful clinical tool in the context of treating a particular patient’s mechanism of pain generation.”-Allen. The review was intended to provide a detailed perspective of mechanism-based classification of pain for physical therapy management in palliative care settings. However, the use of this classification will have to be better validated in patient-based research in palliative physical therapeutic care.


Escherichia coli under conditions of limited glucose as a carbon source Twenty-five years and over 50,000 bacterial generations ago, one of the longest open-ended experiments in modern times began when the Lenski laboratory started passaging Laboratory passage has long been recognized as an effective means to modify the host range of pathogens, with successive passage of viruses in nonhuman hosts an early strategy for generating live attenuated vaccines Legionella pneumophila during its adaptation to mouse macrophages L. pneumophila normally replicates in freshwater amoebae Why do some pathogens specialize to one particular host while others remain generalists, able to infect a broad range of host species? One model is that exposure to diverse environmental hosts drives the expansion of host range and prevents specialization to any single host L. pneumophila. Whether due to less stringent selection under some conditions, disruptions in environmental host diversity, increased mutation, or population bottlenecking, transient changes in pathogen diversity might subsequently influence the outcome of human exposure to these bacteria.Intuitively, such host dynamics should also shape the genomes of vector-borne diseases, where pathogens must balance fitness in both their vector and their other hosts. This hypothesis has been extensively tested in arthropod-borne viruses, where host cycling between insect vector and mammalian host should prevent the accumulation of mutations that result in fitness costs in either host, perhaps holding such pathogens in a sort of evolutionary stasis Experimental evolution can also be used to modulate a pathogen's transmission between hosts. Usually, the conditions of serial passage (back dilution in growth media) in the laboratory do not recapitulate the nuances involved in natural transmission between hosts, selecting instead for mutations that increase within-host replication Pseudomonas aeruginosa in media designed to mimic the conditions encountered by bacteria in the lungs of cystic fibrosis patients selected for several mutations, including antibiotic resistance, that overlapped with known mutations previously identified in patient isolates Burkholderia cenocepacia, another frequent pathogen of cystic fibrosis patients In addition to transmission and host-range trade-offs, experimental evolution has also provided unique insight into other aspects of pathogenesis, including the fitness trade-offs between colonization and systemic infection Many of the concepts of experimental evolution are also relevant to the expanding field of genomic epidemiology, where using whole-genome sequencing to track the spread of disease through a population can also provide real-world information about how pathogens adapt to their human host. In particular, concepts such as parallel genome evolution (repeated selection for mutations in the same genes or pathways in independent clones) and fitness trade-offs between human infection and environmental persistence are as important for understanding outbreaks of disease as they are to laboratory adaptation.Burkholderia dolosaPseudomonas aeruginosagyrA (drug resistance) and global response regulators. In P. aeruginosa replicating for 200,000 generations, the observation of purifying negative selection suggested that these populations reached fitness peaks not previously observed during in vitro adaptation of the pathogen E. coli to the human host was monitored by sequencing a series of isolates taken from the bladders of six patients that were deliberately colonized with this strain The life-long colonization of cystic fibrosis patients with sequence first and ask questions later approach to understanding the laboratory evolution of pathogens. Indeed, complex population dynamics, including frequent clonal interference between competing clones within a population, are likely to be quite common during these experiments Like many fields, experimental evolution has not escaped the transformative effects of next-generation sequencing. By sequencing the genomes of laboratory-adapted strains, it is now possible to define the molecular basis of microbial adaptation to new environments or conditions. Given that, using current technologies—even large bacterial genomes currently cost less than $100 to sequence at >100× coverage—we favor what could best be described as a With the increased ability to link laboratory adaptations to specific genetic lesions via affordable, widely accessible sequencing, the application of experimental evolution to study pathogen adaptation has never held more potential . Future


When species shift their ranges to track climate change, they are almost certain to experience novel environments to which they are poorly adapted. Otaki and co-workers document an explosion of wing pattern variation accompanying range expansion in the pale grass blue butterfly. This pattern can be replicated in the laboratory using artificial selection on cold shocked pupae, at temperature extremes typical of recently colonized environments. We discuss how this phenotypic plasticity may be associated with successful colonization and how significant local adaptation is likely to re-establish developmental control. Integrating knowledge of trait plasticity into current genetic models of adaptation is central to our understanding of when and where a colonising population will be able to persist and adapt in novel surroundings. Species ranges are never fixed, but remain in continual flux in response to demographic, genetic, ecological and environmental variation. Colonization occurs at the range margin when populations spill over into new sites, typically followed by population extinction as environmental and other forces prevent persistence in these new habitats. As the climate has warmed, this turn-over has resulted in expansions that appear more permanent. Consistent northward range shifts have been documented for several vertebrate and invertebrate species in the northern hemisphere, but particularly Lepidoptera, whose historical distributions are usually well-known ,2. But wZizeeria maha). From 1990-2000, this species progressively marched more than 100 km up the west coast of Japan, towards the top of Honshu island - known in precise detail thanks to assiduous collecting of amateur Japanese lepidopterists. Similar northward movements are known in several other Japanese butterflies [Joji Otaki and co-workers  have chaAccompanying this colonisation, the pale grass blue has undergone an outbreak of wing pattern diversification. Three novel ventral wing patterns are recognizable with either inner spot elongation, outer spot elongation or reduction of both inner and outer spots Figure . Novel pPhenotypic plasticity has been proposed to be important in colonization and persistence in new environments -7. PlastAnother feature of the northern population is that the novel wing spot patterns appear in summer butterflies. These could not have been exposed to lower temperatures as pupae, so temperature can't be the sole explanation. To investigate this further, Otaki et al.  undertooFor this explanation to make sense, selection needs to have favoured novel spot patterns in colonizing butterflies. Little is known about natural or sexual selection on ventral spot patterns in the pale grass blue, so this remains somewhat hypothetical. However, recent theoretical work has shown that sexual selection can enhance the effect of random genetic drift and act to diversify mate preferences and sexual colour patterns . A changAnother, perhaps more plausible, explanation is that selection favours traits in the novel environment that are genetically correlated to developmental control of wing patterning. It seems likely that there has been local adaptation to the novel colder environmental conditions in the colonizing population. Could such traits be genetically correlated to wing pattern traits? Unfortunately it is not clear from Otaki et al.'s study whether artificial selection on wing spots caused correlated genetic change in adaptation to cold shock, which would be indicative of an underlying genetic correlation. Pupal survival rate under temperature shock increased through time in the selection line, as predicted under this hypothesis. But there was no appropriate control. We need to know the degree of adaptation  of a population held under the same conditions of cold shock but without artificial selection on wing spots. Moreover, there was only a single replicate of artificial selection in Otaki et al.'s study; several would be needed to exclude random effects.Certain types of phenotypic plasticity enable an organism to tolerate environmental variation without experiencing a major reduction in fitness in different environments. However environmental variation outside of the range usually experienced by an organism can generate novel phenotypic responses, which have not been previously shaped by selection , and mayJB, JRB and AP wrote the commentary. All authors read and approved the final manuscript.


GCK genes that appear to be compatible with function in their genomes. Glucokinase regulatory protein (GCKR) is the most important post-transcriptional regulator of GCK in the liver; it participates in the modulation of GCK activity and location depending upon changes in glucose levels. In experimental models, loss of GCKR has been shown to associate with reduced hepatic GCK protein levels and activity.Glucokinase (GCK) plays an important role in the regulation of carbohydrate metabolism. In the liver, phosphorylation of glucose to glucose-6-phosphate by GCK is the first step for both glycolysis and glycogen synthesis. However, some vertebrate species are deficient in GCK activity in the liver, despite containing GCKR genes and GCKR-like sequences were identified in the genomes of all vertebrate species with available genome sequences. The coding sequences of GCKR and GCKR-like genes were identified and aligned; base changes likely to disrupt coding potential or splicing were also identified.GCKR genes could not be found in the genomes of 9 vertebrate species, including all birds. In addition, in multiple mammalian genomes, whereas GCKR-like gene sequences could be identified, these genes could not predict a functional protein. Vertebrate species that were previously reported to be deficient in hepatic GCK activity were found to have deleted (birds and lizard) or mutated  GCKR genes. Our results suggest that mutation of the GCKR gene leads to hepatic GCK deficiency due to the loss of the stabilizing effect of GCKR. GCK expression or function in liver and pancreatic beta-cells are known to result in the maturity onset diabetes of the young 2 (MODY2) form of diabetes GCK causing elevated GCK activity in liver and pancreatic beta-cells are also known to result in persistent hyperinsulinemic hypoglycemia of infancy (HPPI), in which high insulin levels induce low blood glucose levels Glucose, a major source of energy for all tissues, is obtained from the diet and stored as glycogen in the liver and muscle when in excess. Storage and release of glucose is a tightly regulated process involving numerous enzymes and regulatory proteins, with glucokinase (GCK) being a key regulatory enzyme GCK gene: an upstream beta-cell specific promoter that is also used in gut and neuronal cells; and a downstream liver-specific promoter GCK expression is absolutely dependent upon the presence of insulin and is repressed by glucagon GCK expression is observed to increase after feeding or with insulin treatment, and expression is repressed when starved or with insulin deficiency GCK expression, potentially to protect phosphate homeostasis GCK gene expression in the liver, little change in GCK mRNA levels is seen with changes in blood glucose levels or insulin in pancreatic beta-cells, although some change in mRNA stability might occur GCK expression in these two major sites of expression are likely related to the tissue-specific functions: whereas the liver only needs GCK expression when there is excess blood glucose, pancreatic islet beta-cells requires constant expression of GCK as a sensor for measuring blood glucose levels A pair of tissue-specific promoters drives the expression of the GCK mRNA levels but decreased liver GCK protein concentration and activity GCKR in hepatocytes by adenoviral vectors increases both GCK protein and enzymatic activity In addition to regulation at the transcriptional level, GCK is also regulated in the liver at the post-transcriptional level by glucokinase regulatory protein (GCKR) GCK genes from diverse species, where intact GCK genes were found in the genomes of most vertebrates examined GCK genes were found to be missing their liver-specific first exon, which might prevent expression of GCK in the liver but not other sites The livers of some mammalian  and non-mammalian  vertebrate species have been reported to be deficient in GCK activity GCKR gene show a specific deficiency of GCK activity in their liver GCKR is the most important post-transcriptional regulator of GCK in most species and expressed almost exclusively in the liver of vertebrates that express GCK, including amphibians and reptiles GCKR genes, though, have only been identified or characterized in a few species GCKR sequences identified GCKR-like genes in mammals, amphibians, and fish, as well as in non-vertebrate species, and concluded that GCKR evolved from an N-acetylmuramate 6-phospate esterase by changing its binding specificity and losing its esterase activity GCKR genes from the genomes of diverse vertebrate species. Our results show that species deficient in liver GCK activity have mutated or deleted GCKR genes, supporting the hypothesis that the loss of GCKR, and its ability to stabilize GCK, explains the loss of liver-specific GCK activity.As indicated above, mice with a targeted disruption of the www.ensembl.org and pre.ensembl.org) in August 2012. Genomes from all available vertebrate species maintained in these databases were searched by gene name, gene symbol (GCKR), or by similarity searches with the tblastn algorithm www.ncbi.nlm.nih.gov) were used to complement the genomes gathered from the Ensembl database and aided in the identification of incomplete genes or coding region sequences. Sequences similar to GCKR were not found in several species. For these species, genomic sequences for genes in the predicted conserved genomic neighborhood were included in this analysis. Genomes were searched for orthologs of the genes that flank the GCKR genes in diverse species (see results for details) and the genomic sequences adjacent to these genes were searched for similarity to GCKR genes.Genomic sequences encoding GCKR-like protein sequences were downloaded from release 69 of the Ensembl and PreEnsembl databases  GCKR genes were used as masters for these alignments, and the locations of exons and coding regions for these genes was obtained from the Ensembl or NCBI databases. The identity and locations of repetitive elements in the human and mouse genomic sequences were identified using RepeatMasker (www.repeatmasker.org). Genomic alignments were used to refine the predicted potential coding regions of the genes. Potential pseudogenes were identified as sequences that failed to predict open reading frames due to the presence of base changes that either introduced stop codons, created frame shifts that disrupted the coding sequence, or disrupted splicing consensus sequences. Predicted open reading frames and protein sequences were aligned with the ClustalW algorithm Long genomic DNA sequences that included the GCKR genes, or inactivation due to frame shift and/or splice site mutations were inferred by parsimony on the consensus vertebrate phylogenetic tree from Ensembl (www.ensembl.org). Phylogenetic trees were generated from the aligned GCKR protein sequences  and NCBI databases for sequences that potentially encode GCKR. Our searches resulted in the identification of GCKR-like sequences in most species  missing , a reptile (anole lizard), all 5 available birds , and a fish (cod)  of the genomes is due to the loss of this gene. Gene loss can be due to either deletion of a sequence from the genome, or failure to find using the blast similarity search algorithm as the sequence became non-functional and degenerated into a pseudogene.While genomic sequences similar to sh (cod) . Since gGCKR genes to determine whether gene order is conserved in vertebrate genomes. The genes FNDC4 (fibronectin type III domain containing 4) and ITF172 (intraflagellar transport 172 homolog) are located 5′ to the human GCKR gene, while ZNF512 (zinc finger gene 512) gene and the predicted gene C2orf16 (chromosome 2 open reading frame 16) are located 3′  are found 5′ to the coelacanth GCKR gene, while genes similar to PTK6/SRMS  and BLK (B lymphoid tyrosine kinase) were found 3′  and the PTK6/SRMS gene was located 3′ to GCKR (as seen in the zebrafish and coelacanth) in the common ancestor of fish and tetrapods. The gene order near the GCKR gene in the other fish genomes examined differed from that of zebrafish, and they shared an order that was consistent with that shown for the takifugu  gene near the GCKR gene, albeit in different relative positions and in a different transcriptional orientation, suggesting that this genomic region has been reorganized within fish.The gene neighborhood of the zebrafish CKR gene . The zebh genome . These otakifugu . All of GCKR-like sequence. Since the ancestral tetrapod is predicted to have an FNDC4-like and a ZNF512-like gene flanking the GCKR gene, we used these sequences to search the mammalian genomes (shrew and platypus), reptilian (anole lizard) and avian  genomes where we failed to identify a GCKR gene with our similarity searches . This result is consistent with the low coverage and fragmented nature of these genome sequences, and thus we cannot determine whether the gene neighborhood is conserved, or if the GCKR gene was lost or is present in an unsequenced gap of these genome assemblies. In contrast, large genomic fragments were typically identified from the avian genomes, where the FNDC4 and ZNF512 genes were linked on the same genomic fragment, as shown for the chicken in FNDC4 and ZNF512-like genes from the bird genomes failed to identify any sequence with similarity to GCKR. These results strongly suggest that the GCKR gene was deleted from the avian genomes, and that this deletion event occurred before the divergence of the avian lineages represented by the genome sequences. The cod genome was also searched with genes that flank the GCKR gene in different fish genomes, and a genomic fragment with some similarity to that seen in coelacanth was found , but different from other ray-finned fish. This suggests that further reorganization has occurred (results not shown). Due to recombination, it is difficult to determine whether the cod GCKR gene was lost or is at a different location, such as an unsequenced gap, in this genome.Based on these gene maps , we usedches see . SearcheGCKR genes from many species, especially mammals, are poorly annotated  or not annotated at all in the Ensembl database. To determine why some GCKR genes are poorly annotated we used MultiPipMaker GCKR genes to the well-characterized human GCKR gene GCKR genes could be identified. Similar results were obtained when the mouse GCKR gene was used as the master sequence for the MultiPipMaker analysis (results not shown). Alignments of the GCKR gene exons allowed us to identify changes that have occurred in the GCKR gene in each species. Many of the exons that had not been annotated as exons of the GCKR genes in species with poorly annotated GCKR genes in the Ensembl database were found to have mutations that either introduced frame shifts (due to insertion or deletion of sequences) into the coding sequence, or would be predicted to prevent mRNA splicing  harbored mutations predicted to disrupt splicing or translation  genomic sequences do not align with the human, or any other mammalian, GCKR gene sequence used as a master sequence in MultiPipMaker (results not shown), due to the large amount of sequence divergence that has occurred since their common ancestor that existed at least 300 million years ago GCKR genes  tarsier, (2) ruminant artiodactyls, (3) cat, (4) bats, (5) hedgehog, and (6) common ancestor of Afrotheria and Xenarthra  where 4 parallel gene loss events are inferred to have occurred . However, the gene sequences from dog, panda, horse, and pig did not acquire these types of mutations, and thus retained intact open reading frames. Pseudogenes should acquire greater numbers of mutations compared to functional genes. Thus if all of the GCKR genes in Laurasiatheria are pseudogenes, then the intact GCKR sequences from dog, panda, horse and pig should have accumulated more mutations than those of other mammals that have functional GCKR genes, such as rodents and humans GCKR sequences may actually show less protein sequence evolution than other mammals, especially rodents, which is certainly not a pattern consistent with accelerated evolution that would be expected of pseudogenes. Similar conclusions are drawn from phylogenies generated using more distantly related outgroups, including fish, amphibians and reptiles, where the dog and elephant GCKR proteins sequences do not accumulate greater numbers of amino acid substitutions compared to human, mouse or Tasmanian devil Click here for additional data file.Table S1GCK genes.Genomic locations of non-mammalian (DOC)Click here for additional data file.