X Chromosome Inactivation in Carriers of Fabry Disease: Review and Meta-Analysis.

Anderson-Fabry disease is an X-linked inborn error of glycosphingolipid catabolism caused by a deficiency of α-galactosidase A. The incidence ranges between 1: 40,000 and 1:117,000 of live male births. In Italy, an estimate of incidence is available only for the north-western Italy, where it is of approximately 1:4000. Clinical symptoms include angiokeratomas, corneal dystrophy, and neurological, cardiac and kidney involvement. The prevalence of symptomatic female carriers is about 70%, and in some cases, they can exhibit a severe phenotype. Previous studies suggest a correlation between skewed X chromosome inactivation and symptoms in carriers of X-linked disease, including Fabry disease. In this review, we briefly summarize the disease, focusing on the clinical symptoms of carriers and analysis of the studies so far published in regards to X chromosome inactivation pattern, and manifesting Fabry carriers. Out of 151 records identified, only five reported the correlation between the analysis of XCI in leukocytes and the related phenotype in Fabry carriers, in particular evaluating the Mainz Severity Score Index or cardiac involvement. The meta-analysis did not show any correlation between MSSI or cardiac involvement and skewed XCI, likely because the analysis of XCI in leukocytes is not useful for predicting the phenotype in Fabry carriers.

X-Linked Emery-Dreifuss Muscular Dystrophy: Study Of X-Chromosome Inactivation and Its Relation with Clinical Phenotypes in Female Carriers.

X-linked Emery-Dreifuss muscular dystrophy (EDMD1) affects approximately 1:100,000 male births. Female carriers are usually asymptomatic but, in some cases, they may present clinical symptoms after age 50 at cardiac level, especially in the form of conduction tissue anomalies. The aim of this study was to evaluate the relation between heart involvement in symptomatic EDMD1 carriers and the X-chromosome inactivation (XCI) pattern. The XCI pattern was determined on the lymphocytes of 30 symptomatic and asymptomatic EDMD1 female carriers-25 familial and 5 sporadic cases-seeking genetic advice using the androgen receptor (AR) methylation-based assay. Carriers were subdivided according to whether they were above or below 50 years of age. A variance analysis was performed to compare the XCI pattern between symptomatic and asymptomatic carriers. The results show that 20% of EDMD1 carriers had cardiac symptoms, and that 50% of these were ≥50 years of age. The XCI pattern was similar in both symptomatic and asymptomatic carriers. Conclusions: Arrhythmias in EDMD1 carriers poorly correlate on lymphocytes to a skewed XCI, probably due to (a) the different embryological origin of cardiac conduction tissue compared to lymphocytes or (b) the preferential loss of atrial cells replaced by fibrous tissue.

Long Feeding High-Fat Diet Induces Hypothalamic Oxidative Stress and Inflammation, and Prolonged Hypothalamic AMPK Activation in Rat Animal Model.

Scope: The hypothalamus is a key brain region involved in the control of feeding and energy expenditure. Hypothalamic inflammation and oxidative stress are landmarks of both obesity and aging processes, although the molecular mechanisms are still unknown. Therefore, with the aim to understand the neurobiological mechanisms of energy homeostasis during aging, we evaluate the effects of long feeding high-fat diet (HFD) in rats, at different age, on modulation of hypothalamic molecular pathway, oxidative stress, and inflammation. Procedures: Male Wistar rats were divided into two groups: control group, receiving standard diet (CD), and treated group, receiving HFD. Both groups were treated with the appropriate diet for 1, 3, 6, 12, or 18 weeks. We investigated energy balance and body composition, as well as lipid profile, homeostatic model assessment index, and inflammatory state in serum. Furthermore, we also analyzed, at hypothalamic level, inflammation and oxidative stress, and adenosine monophosphate-dependent kinase (AMPK) and pAMPK expression levels. Results: Our data showed that aging and HFD induce increased energy intake and energy efficiency and decreased energy expenditure associated, at hypothalamic level, with inflammation and oxidative stress and activation of AMPK. Conclusion: Our results indicate that the age at which HFD feeding starts and the diet duration are critical in obesity development. The prolonged activation of hypothalamic AMPK may be related to the alterations in energy homeostasis.

Effects of an High-Fat Diet Enriched in Lard or in Fish Oil on the Hypothalamic Amp-Activated Protein Kinase and Inflammatory Mediators.

The high fat diet (HFD) rich in lard induces obesity, inflammation and oxidative stress, and the deregulation of hypothalamic nuclei plays an important role in this mechanism. One important factor involved in the food intake and inflammation is adenosine monophosphate-dependent kinase (AMPK), a serine/threonine kinase activated by phosphorylation. Omega (ω)3-polyunsaturated fatty acids (PUFA) are dietary compounds known to attenuate the obesity-related diseases, although the molecular mechanisms underlying their actions in the hypothalamus are not completely understood. We hypothesized that the beneficial effects of PUFA may be mediated by AMPK in the hypothalamus. To this aim, rats were fed a control diet (CD), or isocaloric HFD containing either fish oil (FD; rich in ω3-PUFA) or lard for 6 weeks, and the activation of AMPK, inflammatory state (IKKß, TNF-α) and oxidative stress were analyzed in the hypothalamus. In addition, we also studied serum lipid profile, homeostatic model assessment (HOMA) index, and pro-inflammatory parameters. Our results showed, at the hypothalamic level of LD-fed rats, an increase of AMPK activation, inflammation and oxidative stress, while no modifications were detected in FD-fed animals compared to CD. In addition body weight gain, serum lipid profile, pro-inflammatory parameters and insulin resistance were reduced in FD animals compared to LD. In conclusion, our data indicate that the substitution of saturated by unsaturated fatty acids in the diet has beneficial effects on modulation of hypothalamic inflammation and function in obesity, underlying, at hypothalamic level, the interaction among insulin and/or leptin resistance, AMPK activation and hyperphagia.

Kaledo, a board game for nutrition education of children and adolescents at school: cluster randomized controlled trial of healthy lifestyle promotion.

UNLABELLED: During childhood and adolescence, a game could be an effective educational tool to learn healthy eating habits. We developed Kaledo, a new board game, to promote nutrition education and to improve dietary behavior. A two-group design with one pre-treatment assessment and two post-treatment assessments was employed. A total of 3,110 subjects (9-19 years old) from 20 schools in Campania, Italy, were included in the trial. In the treated group, the game was introduced each week over 20 consecutive weeks. Control group did not receive any intervention. The primary outcomes were (i) score on the "Adolescent Food Habits Checklist" (AFHC), (ii) scores on a dietary questionnaire, and (iii) BMI z-score. At the first post-assessment (6 months), the treated group obtained significantly higher scores than the control group on the AFHC (14.4 (95 % confidence interval (CI) 14.0 to 14.8) vs 10.9 (95 % CI 10.6 to -11.2); F(1,20) = 72.677; p < 0.001) and on four sections of the dietary questionnaire: "nutrition knowledge" (6.5 (6.4 to 6.6) vs 4.6 (4.5 to 4.7); F(1,16) = 78.763; p < 0.001), "healthy and unhealthy diet and food" (11.2 (11.0 to 11.4) vs 10.4 (10.3 to 10.6); F(1,32) = 21.324; p < 0.001), "food habits" (32.4 (32.0 to 32.8) vs 27.64 (27.3 to 28.0); F(1,26) = 195.039; p < 0.001), and "physical activity" (13.4 (13.2 to 13.7) vs 12.0 (11.8 to 12.6); F(1,20) = 20.765; p < 0.001). Moreover, the treated group had significantly lower BMI z-score with respect to the controls at the first (0.44 (0.42 to 0.46) vs 0.58 (0.56 to 0.59), F(1,18) = 16.584, p = 0.001) and at the second (18 months) (0.34 (0.30 to 0.38) vs 0.58 (0.54 to 0.62), F(1,13) = 7.577; p = 0.017) post-assessments. CONCLUSION: Kaledo improved nutrition knowledge and dietary behavior over 6 months and had a sustained effect on the BMI z-score. Therefore, it may be used as an effective tool in childhood and adolescence obesity prevention programs.

Cortical spreading depression increases the phosphorylation of AMP-activated protein kinase in the cerebral cortex.

Cortical spreading depression (CSD) enhances ischemic tolerance to temporary focal ischemia. Although this effect most likely requires the expression or activation of neuroprotective factors, their identity remains relatively unknown. One important factor involved in neuroprotection is adenosine monophosphate-dependent kinase (AMPK), a serine/threonine kinase that is activated via phosphorylation. This activation occurs in response to brain ischemia, hypoxia, or glucose deprivation. Thus, to determine the potential mechanism of the neuroprotective effects of CSD, we tested whether AMPK becomes phosphorylated into phospho-AMP-activated protein kinase (pAMPK) after CSD. CSD was induced for 15 min in three groups of five rats. The animals were subsequently sacrificed after 2, 4 or 24 h. Western blot analyses were performed to determine the AMPKα and pAMPKα levels in the cortex (right and left hemispheres), and immunohistochemistry and immunofluorescence were performed to determine the localisation of AMPKα and pAMPKα in the cerebral cortex. These results demonstrated a significant increase in pAMPKα at 24 h (but not at 2 and 4 h) after CSD. In contrast, un-phosphorylated AMPK expression did not change. The increase in pAMPKα was confined to neurons (predominantly neurons located in the superficial layers of the cerebral cortex) and was not observed in astroglial cells. Taken together, these data indicate that AMPK is activated by CSD, and suggest that this activation may contribute to the neuroprotective effect of CSD.

Methionine-enriched diet decreases hippocampal antioxidant defences and impairs spontaneous behaviour and long-term potentiation in rats.

Diets high in methionine lead to elevation of plasma homocysteine levels which are possibly linked to neurodegenerative diseases and oxidative stress. In the present study, we investigated the effects of methionine-enriched diet on antioxidant defences, on rat spontaneous behaviour and on the ability to sustain long-term potentiation in the dentate gyrus (DG). Sprague-Dawley rats were fed either a standard laboratory diet or a methionine enriched-diet (1% or 5% methionine in drinking water) for 8 weeks. After the 8 weeks, the animals were tested for spontaneous motor activity and habituation in an open field maze, for anxiety-like behaviour in an elevated plus maze and for the ability to sustain long-term potentiation (LTP) induced in the dentate gyrus under urethane anaesthesia. The brains were then removed and histochemically stained for superoxide dismutase (SOD) activity. Rats fed on 5% methionine significantly reduced total distance travelled during the open field test and exhibited no habituation with respect to the other two groups. Rats fed on 5% methionine also showed a significant increase of the anxiety level. Moreover, in this group, the ability to induce LTP in DG was impaired. SOD activity was significantly increased in the cerebral cortex of the rats fed on 1% and 5% methionine with respect to the control group. In conclusion, 5% methionine in drinking water led to evident impairment of locomotor skills and of synaptic plasticity. SOD activity in the cortex was increased in both the groups fed on 1% and 5% methionine, thus suggesting that metabolic adjustments, triggered by the methionine-enriched diet, are likely mediated by reactive oxygen species.

Plasma glutamine decreases immediately after surgery and is related to incisiveness.

Glutamine (gln) is the most abundant free amino acid in the blood. It is involved in important metabolic and biochemical processes, like cell proliferation and oxidative stress. Previous studies have demonstrated that gln concentration in human plasma decreases in several conditions such as sepsis, ischemia-reperfusion, trauma, major surgery and burn. The aim of the present work was to compare the acute effects of different types of surgical interventions and of anesthetization on blood gln concentration. Plasma samples from 88 subjects (30 males and 58 females) were collected before and after major or minor surgery and the gln concentration was analyzed with high-performance liquid chromatography. The results showed that plasma gln concentration after surgery was lower than pre-surgery values and that in major surgery the decrease of gln was higher than in minor surgery. No significant effect was shown for sex or type of anesthesia. These results demonstrate the importance of a gln supplementation before a surgical intervention and show that the amount of gln supplementation should also be adjusted based on the type of surgery.

Pre-natal exposure of mice to bisphenol A elicits an endometriosis-like phenotype in female offspring.

Endometriosis is a chronic gynecological disease characterized by the growth of endometrial tissue outside the uterine cavity. Exposure to endocrine disruptors during critical period of development causes long-lasting effects, being the genital system one of the targets. This study describes the effects on female genital system caused by developmental exposure to the endocrine-disrupting chemical bisphenol A (BPA) during pre- and peri-natal development in mice. To this end, timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with BPA (100, or 1000 microg/kg/day). After delivery, pups were held for 3 months; then, pelvic organs were analyzed in their entirety and livers of both pups and moms were studied for the presence of BPA. We found in the adipose tissue surrounding the genital tracts of a consistent number of treated animals, endometriosis-like structure with the presence of both glands and stroma and expressing both estrogen receptor and HOXA-10. Moreover, cystic ovaries, adenomatous hyperplasia with cystic endometrial hyperplasia and atypical hyperplasia were significantly more frequent in treated animals respect to the controls. Finally, BPA was found in the livers of exposed moms and female offspring. In conclusion, we describe for the first time an endometriosis-like phenotype in mice, elicited by pre-natal exposition to BPA. This observation may induce to thoroughly reconsider the pathogenesis and treatment of endometriosis, considering the high incidence of endometriosis and the problems caused by associated infertility.

Persistent facial pain increases superoxide anion production in the spinal trigeminal nucleus.

Previous studies have demonstrated that there is an increase in oxidative stress in the cerebral cortex of rats after repeated painful stimulation and that long-lasting pain increases the production of superoxide ion (O(2) (-)), nitric oxide and peroxynitrite due to the activation of AMPA and NMDA receptors. The purpose of the present study was to evaluate the possible role of O(2) (-) in the transmission of oro-facial pain. Formaldehyde 1% was injected subcutaneously into one vibrissal pad of adult male Sprague-Dawley rats as a model of persistent pain, then O(2) (-) production and superoxide dismutase (SOD) activity were evaluated in the left and right spinal trigeminal nuclei. O(2) (-) production was revealed using dihidroetidium (DHE) injected at 10 or 45 min after the formalin injection in conscious or anaesthetized rats. A histochemical assay for SOD was performed to evaluate the activity of SOD at 10 min after the formalin injection. The results showed a significant increase in O(2) (-) production in the homolateral nucleus at 45 min. However, there was no significant difference between the two sides at 10 min after the formalin injection. No significant difference was observed in SOD activity between the two sides of the spinal trigeminal nucleus. This study demonstrated that there is an increased production of O(2) (-) in the second phase but not in the first phase of the formalin test; thus O(2) (-) is involved in pain induced by inflammation, but not in acute pain.

Smoking in adult attention-deficit/hyperactivity disorder: interaction between 15q13 nicotinic genes and Temperament Character Inventory scores.

Adults with ADHD smoke cigarettes at a higher rate than normal subjects. Nicotine has been shown to significantly improve clinical ADHD symptoms as measured by the Clinical Global Impression scale (CGI) as well as by measures of attention, vigour and arousal in ADHD subjects. In this study we hypothesized that the allele 113bp in D15S1360 marker at CHRNA7 and the 2 bp deletion allele at CHRFAM7A are associated with increased smoking in a sample of 90 DSM-IV patients affected by Adult ADHD. Temperament and Character Inventory (TCI) scores were included as covariates in the analysis to distinguish the contribution of personality traits from the contribution of the nicotinic genes under investigation. Smoking status was determined from the medical history questionnaire, and there were 35 current smokers and 55 non-smokers. Single marker associations and the CHRNA7-CHRFAM7A interaction were calculated by logistic regression, considering the 113 bp and the -2 bp deletion in a dominant model. No association of these genes with smoking was observed. Similarly, no significant interaction between the genes was observed in the logistic model. However, Persistence score of the TCI was significantly associated with smoking status. Further investigation on the hypothesis of the molecular interaction between CHRNA7 and CHRFAM7A genes is warranted.

Kalèdo, a new educational board-game, gives nutritional rudiments and encourages healthy eating in children: a pilot cluster randomized trial.

INTRODUCTION: Prevention of obesity and overweight is an important target for health promotion. Early prevention requires an intervention during childhood and adolescence. At these stages, the game could be an appropriate means to teach nutrition knowledge and to influence dietary behaviour. To this end, the authors developed Kalèdo, a new board-game. OBJECTIVE: The aim of the present study was to test the efficacy of Kalèdo on changes in nutrition knowledge and dietary behaviour in a pilot study conducted in three middle schools in Naples, Italy. MATERIALS AND METHODS: A simple two-group design (treatment and control) with pre- and post-assessment was employed. The classroom was the unit of recruitment and random assignment to groups. All students (307) in the participating schools were invited to participate. Data analysis was performed on 241 subjects. During 24 weeks, a group of 153 children from 8 classrooms (11-14 year old Caucasian subjects; 78 male, 75 female) was involved in 15-30 minute-long play sessions once a week. A questionnaire was given to the participants at the beginning and at the end of the study to evaluate nutrition knowledge (31 questions), physical activity (8 questions) and food intake (34 questions). Anthropometric measurements were also carried out. A second group of 88 children from 5 classrooms (same age and ethnicity; 55 male, 33 female) was investigated at the same times with the same questionnaire and anthropometric measures but they did not receive any play sessions with Kalèdo. OBSERVATION: Children playing Kalèdo showed a significant increase in nutrition knowledge (p<0.05) and in weekly vegetable intake (p<0.01) with respect to the control. CONCLUSION: The results suggest that Kalèdo could be an effective instrument to teach children about healthy diet. More research is needed to study the long term effect of this intervention.

Trigeminal pain transmission requires reactive oxygen species production.

Three experiments were conducted in order to investigate the possible involvement of the reactive oxygen species in the nociception within the subnucleus caudalis of the spinal trigeminal nucleus (Vc). In the first experiment the extracellular level of hydrogen peroxide was evaluated by microdialysis in the Vc of two groups of six rats before and after a formalin (group 1) or saline solution (group 2) injection into the upper lip. In the second experiment the formalin test was conducted in three groups of 6 rats after a microinjection of 2-methoxyestradiol (2-ME, a superoxide-dismutase inhibitor; group 1) or N-acetylcysteine (NAC, an oxygen intermediate scavenger; group 2) or saline solution (group 3) into the Vc. In the third experiment an histochemical assay for superoxide dismutase activity was performed on two groups of 4 rats each 2 h after a formalin (group 1) or saline solution (group 2) injection into the upper lip. The results showed that (1) the level of hydrogen peroxide increases into the Vc during facial pain (134% of baseline); (2) the inhibition of superoxide dismutase or the removal of oxygen intermediate within the Vc decreases the sensibility to facial pain stimuli; and (3) persistent facial pain stimuli decrease the superoxide activity into the Vc (90% of counter-lateral). These data indicate that reactive oxygen species are produced in the Vc during persistent facial pain and are necessary for the transmission of pain.