Intraspecific Interactions Decrease Stress Affecting Welfare in Shelter Dogs: A Comparison of Four Different Housing Conditions.

Shelters are stressful environments for domestic dogs (Canis familiaris). Evaluating dogs' welfare is crucial to improve their life condition and to promote a better management of shelters. We aimed at verifying which variables improved welfare in 10 shelter dogs ((hosted in the shelter "Centro cinofilo Caerite" in Bracciano (Rome)) by analysing their behavioural responses in different environmental conditions. Furthermore, faecal samples were taken to measure cortisol metabolites (CM), a non-invasive method to evaluate adrenocortical activity in dogs. Dogs were observed for a total of 400 h in 4 different cage conditions: (i) alone in a cage; ii) alone in an enriched cage; (iii) in cage with conspecifics; (iv) in cage with regular interaction with humans outside the cage. Alone in the cage situation showed highest frequencies of displacement activities (Friedman test: χ2 = 13.32; p = 0.004). In contrast, being in the cage with conspecifics seems to reduce displacement activity frequency, as well as the level of faecal cortisol metabolites (Friedman test: χ2 = 8.04; p = 0.045). Our results suggest that conspecifics' presence is the best way to reduce stress in shelter dogs. This research could provide some useful guidelines for managing shelters and improving dogs' life condition.

Spectrum of Genetic Variants in the Dystrophin Gene: A Single Centre Retrospective Analysis of 750 Duchenne and Becker Patients from Southern Italy.

Dystrophinopathies are X-linked recessive muscle disorders caused by mutations in the dystrophin (DMD) gene that include deletions, duplications, and point mutations. Correct diagnosis is important for providing adequate patient care and family planning, especially at this time when mutation-specific therapies are available. We report a large single-centre study on the spectrum of DMD gene variants observed in 750 patients analyzed for suspected Duchenne (DMD) or Becker (BMD) muscular dystrophy, over the past 30 years, at the Cardiomyology and Medical Genetics of the University of Campania. We found 534 (71.21%) large deletions, 73 (9.73%) large duplications, and 112 (14.93%) point mutations, of which 44 (5.9%) were small ins/del causing frame-shifts, 57 (7.6%) nonsense mutations, 8 (1.1%) splice site and 3 (0.4%) intronic mutations, and 31 (4.13%) non mutations. Moreover, we report the prevalence of the different types of mutations in patients with DMD and BMD according to their decade of birth, from 1930 to 2020, and correlate the data to the different techniques used over the years. In the most recent decades, we observed an apparent increase in the prevalence of point mutations, probably due to the use of Next-Generation Sequencing (NGS). In conclusion, in southern Italy, deletions are the most frequent variation observed in DMD and BMD patients followed by point mutations and duplications, as elsewhere in the world. NGS was useful to identify point mutations in cases of strong suspicion of DMD/BMD negative on deletions/duplications analyses. In the era of personalized medicine and availability of new causative therapies, a collective effort is necessary to enable DMD and BMD patients to have timely genetic diagnoses and avoid late implementation of standard of care and late initiation of appropriate treatment.

Molecular Research in Medical Genetics.

About 19,000-20,000 protein-coding genes in the human genome have been identified [...].

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.

Examination of a board game approach to children's involvement in family-based weight management vs. traditional family-based behavioral counseling in primary care.

The most effective intervention model for childhood obesity is known as family-based behavioral group treatments. There are also studies that investigate the effects of educational games for children to gain healthy eating and physical exercise habits. The aim of this study was to compare the efficacy of a family-based group treatment with an educational game (Kaledo) intervention in childhood obesity. Kaledo is a board game that was designed to improve nutritional knowledge and healthy life style habits. It is played with nutrition and activity cards that players can select from, and a total score is calculated in the end of the game according to energy intake and expenditure. Obese children between 9 and 12 ages were involved in this study. Participants randomly divided into behavioral and game intervention groups. Clinical evaluation was performed in the first and second counseling in both groups. Marmara University Family Medicine Department Obese Children and Adolescents Interview Form, Physical Activity Evaluation Form, and Three-day Food Record Form were used for this purpose. Strengths and Difficulties Questionnaire-Parent Report Version and Children's Depression Inventory were used for the assessment of psychiatric symptoms. After the clinical evaluation, an education session about healthy eating and physical activity was attended by both groups. After that, for the behavioral groups, parents and children were assigned to different groups, while for the game intervention group, parents were assigned to behavioral sessions and children were assigned to game (Kaledo) sessions. A total of six sessions with 1-h duration and 2-week interval were performed in both groups. Height and weight were measured in each session and analysis was performed on the data of the children who participated in all of the sessions. Although a total of 108 children were clinically evaluated, 52 children and their parents, 26 in the behavioral group and 26 in the game intervention group, participated in two or more sessions. Twenty-four participants, 12 in behavioral and 12 in the game intervention group, finished the study by participating in all of the six sessions. Thus, dropout rate was 74%. BMI and BMI z-scores decreased in both groups compared with the initial measures and these changes were statistically significant. For the behavioral group, these changes were - 1.01 (25.44 to 24.43, p = 0.03) and - 0.17 (2.07 to 1.90, p = 0.000) and for the game group, - 0.74 (26.98 to 26.24, p = 0.007) and - 0.09 (2.07 to 1.98, p = 0.003). There were no significant differences between behavioral and game intervention groups in point of BMI and BMI z-scores (p = 0.130 and p = 0.706). CONCLUSION: Family-based behavioral group treatment and game (Kaledo) intervention were found to be effective in childhood obesity management in this research. There was no significant difference between the two interventions. According to this study, these intervention models can be advised to primary care physicians to be used in the management of childhood obesity. What is Known: - Family-based behavioral group treatment is known as the most efficient model for childhood obesity management. What is New: - In this study, for the first time, a game (Kaledo) intervention was found to be effective in childhood obesity management. - Compared with family-based behavioral group treatment, there was no significant difference between the two interventions.

Healthy lifestyle promotion in primary schools through the board game Kaledo: a pilot cluster randomized trial.

The board game Kaledo was proven to be effective in improving nutrition knowledge and in modifying dietary behavior in students attending middle and high school. The present pilot study aims to reproduce these results in younger students (7-11 years old) attending primary school. A total of 1313 children from ten schools were recruited to participate in the present study. Participants were randomized into two groups: (1) the treatment group which consisted of playing Kaledo over 20 sessions and (2) the no intervention group. Anthropometric measures were carried out for both groups at baseline (prior to any treatment) and at two follow-up post-assessments (8 and 18 months). All the participants completed a questionnaire concerning physical activity and a 1-week food diary at each assessment. The primary outcomes were (i) BMI z-score, (ii) scores on physical activity, and (iii) scores on a dietary questionnaire. BMI z-score was significantly lower in the treated group compared to the control group at 8 months. Frequency and duration of self-reported physical activity were also significantly augmented in the treated group compared to the control group at both post-assessments. Moreover, a significant increase in the consumption of healthy food and a significant decrease in junk food intake were observed in the treated group. CONCLUSION: The present results confirm the efficacy of Kaledo in younger students in primary schools, and it can be used as a useful nutritional tool for obesity prevention programs in children. What is Known: • Kaledo is a new educational board game to improve nutrition knowledge and to promote a healthy lifestyle. • In two cluster randomized trials conducted in Campania region (Italy), we showed that Kaledo could improve nutrition knowledge and dietary behavior and have a positive effect on the BMI z-score in children with age ranging from 9 to 14 years old attending school. • Kaledo may be used as an effective tool for obesity prevention programs in middle and high school students. What is New: • Investigating the effects of Kaledo on younger primary school children (7-11 year olds), Kaledo could be an effective tool in obesity prevention programs for children as young as 7 years old.

Skewed X-chromosome inactivation plays a crucial role in the onset of symptoms in carriers of Becker muscular dystrophy.

BACKGROUND: Becker muscular dystrophy (BMD) is an X-linked recessive disorder affecting approximately 1: 18.000 male births. Female carriers are usually asymptomatic, although 2.5-18% may present muscle or heart symptoms. In the present study, the role of the X chromosome inactivation (XCI) on the onset of symptoms in BMD carriers was analysed and compared with the pattern observed in Duchenne muscular dystrophy (DMD) carriers. METHODS: XCI was determined on the lymphocytes of 36 BMD carriers (both symptomatic and not symptomatic) from 11 families requiring genetic advice at the Cardiomyology and Medical Genetics of the Second University of Naples, using the AR methylation-based assay. Carriers were subdivided into two groups, according to age above or below 50 years. Seven females from the same families known as noncarriers were used as controls. A Student's t-test for nonpaired data was performed to evaluate the differences observed in the XCI values between asymptomatic and symptomatic carriers, and carriers aged above or below 50 years. A Pearson correlation test was used to evaluate the inheritance of the XCI pattern in 19 mother-daughter pairs. RESULTS: The results showed that symptomatic BMD carriers had a skewed XCI with a preferential inactivation of the X chromosome carrying the normal allele, whereas the asymptomatic carriers and controls showed a random XCI. No concordance concerning the XCI pattern was observed between mothers and related daughters. CONCLUSIONS: The data obtained in the present study suggest that the onset of symptoms in BMD carriers is related to a skewed XCI, as observed in DMD carriers. Furthermore, they showed no concordance in the XCI pattern inheritance.

Osteopontin: Relation between Adipose Tissue and Bone Homeostasis.

Osteopontin (OPN) is a multifunctional protein mainly associated with bone metabolism and remodeling. Besides its physiological functions, OPN is implicated in the pathogenesis of a variety of disease states, such as obesity and osteoporosis. Importantly, during the last decades obesity and osteoporosis have become among the main threats to health worldwide. Because OPN is a protein principally expressed in cells with multifaceted effects on bone morphogenesis and remodeling and because it seems to be one of the most overexpressed genes in the adipose tissue of the obese contributing to osteoporosis, this mini review will highlight recent insights about relation between adipose tissue and bone homeostasis.

Role of the Orexin System on the Hypothalamus-Pituitary-Thyroid Axis.

Hypocretin/orexin (ORX) are two hypothalamic neuropeptides discovered in 1998. Since their discovery, they have been one of the most studied neuropeptide systems because of their projecting fields innervating various brain areas. The orexinergic system is tied to sleep-wakefulness cycle, and narcolepsy is a consequence of their system hypofunction. Orexinergic system is also involved in many other autonomic functions such as feeding, thermoregulation, cardiovascular and neuroendocrine regulation. The main aim of this mini review article is to investigate the relationship between ORX and thyroid system regulation. Although knowledge about the ORX system is evolving, its putative effects on hypothalamic-pituitary-thyroid (HPT) axis still appear unclear. We analyzed some studies about ORX control of HPT axis to know better the relationship between them. The studies that were analyzed suggest Hypocretin/ORX to modulate the thyroid regulation, but the nature (excitatory or inhibitory) of this possible interaction remains actually unclear and needs to be confirmed.

Clinical experience with N-carbamylglutamate in a single-centre cohort of patients with propionic and methylmalonic aciduria.

BACKGROUND: The effect of long-term N-carbamylglutamate (NCG) treatment on the rate and severity of decompensations due to propionic aciduria (PA) and methylmalonic aciduria (MMA) is unknown. This paper presents clinical experience from a single-centre cohort of patients with PA and MMA who received continuous long-term treatment with NCG. METHODS: The effect of oral NCG treatment (initial dose: 50 mg/kg/day) was investigated in patients with PA or MMA who were experiencing frequent progressive episodes of metabolic decompensation, who had pathological levels of ammonia, and who were referred to the Division of Metabolic Diseases, University Hospital of Padova between August 2014 and December 2015. Clinical and biochemical data, including the number of metabolic decompensations, lactic acid, uric acid and plasma ammonia levels, protein intake and body weight, were collected before and after the initiation of NCG treatment. RESULTS: Eight patients with PA (n = 4) and MMA (n = 4) aged 2-20 years were treated with NCG (50 mg/kg/day) for 7-16 months. Metabolic decompensation episodes decreased in number and severity, with three of the patients having no episodes (pre-treatment: 24 episodes; post-treatment: 9 episodes). After NCG treatment, all episodes were treated at home and none required hospitalisation, lactic acid values were 1.3-2.1 mmol/L and uric acid values were 0.21-0.36 mmol/L. Significant reductions in blood ammonia levels after NCG initiation were observed in five patients, whereas levels were reduced or maintained in the normal range in the remainder. Over the treatment period, patients had an increase in natural protein intake of 20-50% and gained 0-6.5 kg in bodyweight. CONCLUSION: These observations suggest that, in addition to short-term benefits for the acute treatment of hyperammonaemia, NCG may be effective and well tolerated as a long-term treatment in patients with severe PA and MMA, and that further prospective studies are warranted.

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.

Cortical spreading depression produces a neuroprotective effect activating mitochondrial uncoupling protein-5.

Depression of electrocorticogram propagating over the cortex surface results in cortical spreading depression (CSD), which is probably related to the pathophysiology of stroke, epilepsy, and migraine. However, preconditioning with CSD produces neuroprotection to subsequent ischemic episodes. Such effects require the expression or activation of several genes, including neuroprotective ones. Recently, it has been demonstrated that the expression of the uncoupling proteins (UCPs) 2 and 5 is amplified during brain ischemia and their expression exerts a long-term effect upon neuron protection. To evaluate the neuroprotective consequence of CSD, the expression of UCP-5 in the brain cortex was measured following CSD induction. CSD was evoked in four samples of rats, which were sacrificed after 2 hours, 4 hours, 6 hours, and 24 hours. Western blot analyses were carried out to measure UCP-5 concentrations in the prefrontal cortices of both hemispheres, and immunohistochemistry was performed to determine the localization of UCP-5 in the brain cortex. The results showed a significant elevation in UCP-5 expression at 24 hours in all cortical strata. Moreover, UCP-5 was triggered by CSD, indicating that UCP-5 production can have a neuroprotective effect.

Epigenetics and cortical spreading depression: changes of DNA methylation level at retrotransposon sequences.

Cortical spreading depression (CSD) is an evolutionarily conserved phenomenon that involves a slow and self-propagating depolarization wave associated with spontaneous depression of electrical neuronal activity. CSD plays a central role in the pathophysiology of several brain diseases and is considered to be able to promote "Preconditioning". This phenomenon consists of the brain protecting itself against future injury by adaptation. Understanding of the molecular mechanisms underlying Preconditioning has significant clinical implications. We have already proposed that the long-lasting effects of CSD could be related to silencing of retrotransposon sequences by histone methylation. We analyzed DNA methylation of two retrotransposon sequences, LINE1 and L1, and their corresponding expression pattern after CSD induction. Based on immunoprecipitation assay of the methylated DNA (meDIP), we demonstrated hypermethylation of both sequences in preconditioned rat brain cortex compared with a control 24 h after CSD induction. Using quantitative PCR, we also showed that CSD induction caused a decrease of the transcript level of both retrotransposon sequences. Our data are consistent with the hypothesis of epigenetic modifications in Preconditioning-dependent neuroprotection by increasing genome stability via the silencing of retrotransposon sequences.

Determining the role of skewed X-chromosome inactivation in developing muscle symptoms in carriers of Duchenne muscular dystrophy.

Duchenne and Becker dystrophinopathies (DMD and BMD) are X-linked recessive disorders caused by mutations in the dystrophin gene that lead to absent or reduced expression of dystrophin in both skeletal and heart muscles. DMD/BMD female carriers are usually asymptomatic, although about 8 % may exhibit muscle or cardiac symptoms. Several mechanisms leading to a reduced dystrophin have been hypothesized to explain the clinical manifestations and, in particular, the role of the skewed XCI is questioned. In this review, the mechanism of XCI and its involvement in the phenotype of BMD/DMD carriers with both a normal karyotype or with X;autosome translocations with breakpoints at Xp21 (locus of the DMD gene) will be analyzed. We have previously observed that DMD carriers with moderate/severe muscle involvement, exhibit a moderate or extremely skewed XCI, in particular if presenting with an early onset of symptoms, while DMD carriers with mild muscle involvement present a random XCI. Moreover, we found that among 87.1 % of the carriers with X;autosome translocations involving the locus Xp21 who developed signs and symptoms of dystrophinopathy such as proximal muscle weakness, difficulty to run, jump and climb stairs, 95.2 % had a skewed XCI pattern in lymphocytes. These data support the hypothesis that skewed XCI is involved in the onset of phenotype in DMD carriers, the X chromosome carrying the normal DMD gene being preferentially inactivated and leading to a moderate-severe muscle involvement.

Benefits of glucocorticoids in non-ambulant boys/men with Duchenne muscular dystrophy: A multicentric longitudinal study using the Performance of Upper Limb test.

The aim of this study was to establish the possible effect of glucocorticoid treatment on upper limb function in a cohort of 91 non-ambulant DMD boys and adults of age between 11 and 26 years. All 91 were assessed using the Performance of Upper Limb test. Forty-eight were still on glucocorticoid after loss of ambulation, 25 stopped steroids at the time they lost ambulation and 18 were GC naïve or had steroids while ambulant for less than a year. At baseline the total scores ranged between 0 and 74 (mean 41.20). The mean total scores were 47.92 in the glucocorticoid group, 36 in those who stopped at loss of ambulation and 30.5 in the naïve group (p < 0.001). The 12-month changes ranged between -20 and 4 (mean -4.4). The mean changes were -3.79 in the glucocorticoid group, -5.52 in those who stopped at loss of ambulation and -4.44 in the naïve group. This was more obvious in the patients between 12 and 18 years and at shoulder and elbow levels. Our findings suggest that continuing glucocorticoids throughout teenage years and adulthood after loss of ambulation appears to have a beneficial effect on upper limb function.

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.