Characterization of pre-mRNA Splicing Defects Caused by CLCN5 and OCRL Mutations and Identification of Novel Variants Associated with Dent Disease.

Dent disease (DD) is an X-linked renal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. Two-thirds of cases are associated with inactivating variants in the CLCN5 gene (Dent disease 1, DD1) and a few present variants in the OCRL gene (Dent disease 2, DD2). The aim of the present study was to test the effect on the pre-mRNA splicing process of DD variants, described here or in the literature, and describe the clinical and genotypic features of thirteen unrelated patients with suspected DD. All patients presented tubular proteinuria, ten presented hypercalciuria and five had nephrolithiasis or nephrocalcinosis. CLCN5 and OCRL genes were analyzed by Sanger sequencing. Nine patients showed variants in CLCN5 and four in OCRL; eight of these were new. Bioinformatics tools were used to select fifteen variants with a potential effect on pre-mRNA splicing from our patients' group and from the literature, and were experimentally tested using minigene assays. Results showed that three exonic missense mutations and two intronic variants affect the mRNA splicing process. Our findings widen the genotypic spectrum of DD and provide insight into the impact of variants causing DD.

Pathogenic Variants of SLC22A12 (URAT1) and SLC2A9 (GLUT9) in Spanish Patients with Renal Hypouricemia: Founder Effect of SLC2A9 Variant c.374C>T; p.(T125M).

Renal hypouricemia (RHUC) is a rare inherited disorder characterized by impaired urate reabsorption in the proximal tubule resulting in low urate serum levels and increased urate excretion. Some patients may present severe complications such as exercise-induced acute renal failure and nephrolithiasis. RHUC is caused by inactivating mutations in the SLC22A12 (RHUC type 1) or SLC2A9 (RHUC type 2) genes, which encode urate transporters URAT1 and GLUT9, respectively. In this study, our goal was to identify mutations associated with twenty-one new cases with RHUC through direct sequencing of SLC22A12 and SLC2A9 coding exons. Additionally, we carried out an SNPs-haplotype analysis to determine whether the rare SLC2A9 variant c.374C>T; p.(T125M), which is recurrent in Spanish families with RHUC type 2, had a common-linked haplotype. Six intragenic informative SNPs were analyzed using PCR amplification from genomic DNA and direct sequencing. Our results showed that ten patients carried the SLC22A12 mutation c.1400C>T; p.(T467M), ten presented the SLC2A9 mutation c.374C>T, and one carried a new SLC2A9 heterozygous mutation, c.593G>A; p.(R198H). Patients carrying the SLC2A9 mutation c.374C>T share a common-linked haplotype, confirming that it emerged due to a founder effect.

Sleep Extension Increases the Effect of Caloric Restriction Over Body Weight and Improves the Chronic Low-Grade Inflammation in Adolescents With Obesity.

PURPOSE: Diminution of sleep may be associated with obesity. However, evidence that extending sleep duration might favor weight loss is insufficient. The aim of this study was to compare the effect of dietary restriction with or without prescription of sleep extension on weight loss in adolescents with obesity. METHODS: A total of 52 adolescents with obesity (24 males and 28 females) received a diet with 500 calories restriction, randomly allocated to groups without (n = 27) and with sleep extension (n = 25) for 4 weeks. We collected data on anthropometry, caloric intake, and self-reported sleep diaries. Serum interleukin 6, tumor necrosis factor α, leptin, and insulin levels were quantified by enzyme-linked immunosorbent assay. Cortisol and 6-sulfatoxymelatonin excretions were measured in the first urine collection in the morning by liquid chromatography-mass spectrometry. Measurements were carried out at baseline and at the end of the intervention. RESULTS: After diet, weight decreased in both groups. Sleep extension, improved weight loss (p < .00001), and waist girth reduction (p = .00003), with diminution of insulin (p = .002) and interleukin 6 levels (p = .02). Caloric restriction was less effective in adolescent females. No differences in cortisol or 6-sulfatoxymelatonin excretion were found. CONCLUSIONS: A sleep extension favors weight loss in adolescents under caloric restriction and improves inflammation and metabolic conditions, thus supporting a possible additional benefit to diet in the treatment of obesity in adolescents.

The spatial cerebral damage caused by larger infarct and ß-amyloid toxicity is driven by the anatomical/functional connectivity.

Large cerebral infarctions are major predictors of death and severe disability from stroke. Conversely, data concerning these types of infarctions and the affected adjacent brain circuits are scarce. It remains to be determined if the co-morbid concurrence of large infarct and ß-amyloid (Aß) toxicity can precipitate the early development of dementia. Here, we described a dose-dependent effect of a unilateral striatal injection of vasoconstrictive endothelin-1 (ET-1) along with Aß toxicity on CNS pathogenesis; driven by the anatomical and functional networks within a brain circuit. After 21 days of treatment, a high dose (60 pmol) of ET-1 (E60) alone caused the greatest increase in neuroinflammation, mainly in the ipsilateral striatum and distant regions with synaptic links to the striatal lesion such as white matter (subcortical white matter, corpus callosum, internal capsule, anterior commissure), gray matter (globus pallidus, thalamus), and cortices (cingulate, motor, somatosensory, entorhinal). The combined E60 + Aß treatment also extended perturbation in the contralateral hemisphere of these rats, such as increased deposition of amyloid precursor protein fragments associated with the appearance of degenerating cells and the leakage of laminin from the basement membrane across a compromised blood-brain barrier. However, the cerebral damage induced by the 6 pmol ET-1 (E6), Aß and E6 + Aß rats was not detrimental enough to injure the complete network. The appreciation of the causal interactions among distinct anatomical units in the brain after ischemia and Aß toxicity will help in the design of effective and alternative therapeutics that may disassociate the synergistic or additive association between the infarcts and Aß toxicity.

Spatial Dynamics of Vascular and Biochemical Injury in Rat Hippocampus Following Striatal Injury and Aß Toxicity.

The hippocampus, a brain region vital for memory and learning, is sensitive to the damage caused by ischemic/hypoxic stroke and is one of the main regions affected by Alzheimer's disease. The pathological changes that might occur in the hippocampus and its connections, because of cerebral injury in a distant brain region, such as the striatum, have not been examined. Therefore, in the present study, we evaluated the combined effects of endothelin-1-induced ischemia (ET1) in the striatum and ß-amyloid (Aß) toxicity on hippocampal pathogenesis, dictated by the anatomical and functional intra- and inter-regional hippocampal connections to the striatum. The hippocampal pathogenesis induced by Aß or ET1 alone was not severe enough to significantly affect the entire circuit of the hippocampal network. However, the combination of the two pathological states (ET1 + Aß) led to an exacerbated increase in neuroinflammation, deposition of the amyloid precursor protein (APP) fragments with the associated appearance of degenerating cells, and blood-brain-barrier disruption. This was observed mainly in the hippocampal formation (CA2 and CA3 regions), the dentate gyrus as well as distinct regions with synaptic links to the hippocampus such as entorhinal cortex, thalamus, and basal forebrain. In addition, ET1 + Aß-treated rats also demonstrated protracted loss of AQP4 depolarization, dissolution of ß-dystroglycan, and basement membrane laminin with associated IgG and dysferlin leakage. Spatial dynamics of hippocampal injury in ET1 + Aß rats may provide a valuable model to study new targets for clinical therapeutic applications, specifically when areas remotely connected to hippocampus are damaged.

Eficacia de la "metodología vivencial de aprendizaje" en el nivel de conocimiento sobre enfermedades transmitidas por Aedes aegypti en un desastre natural / Efficacy of "Vivential Methodology of Learning in Adults" (MVAA) in the knowledge level about Aedes aegyptitransmitted diseases in a natural disaster

Objetivo: Determinar el efecto del uso de la MVAA en una intervención educativa, en el nivel de conocimientos sobre enfermedades transmitidas por Aedes aegypti en la región de Piura durante el mes de junio del 2017. Materiales y Métodos: Estudio cuasi-experimental, pre-post de un solo grupo. Se usó una intervención educativa basada en la MVAA en 955 personas de 19 empresas y/o instituciones educativas del departamento de Piura, a quienes se les realizó una medición de los conocimientos sobre medidas preventivas contra enfermedades transmitidas por Aedes aegypti en escala vigesimal. Resultados. Después de la intervención, el porcentaje de aprobados aumentó de 32.9% a 82.2% (Δ49.2%; IC95% 46.7%-51.0%, p<0.001). Se encontró una relación inversamente proporcional entre las variaciones de los puntajes y la edad (R2=0.200; p<0.001) en los intervenidos de sexo femenino. Se encontró la mayor diferencia entre puntajes pre-test y post-test en los intervenidos en instituciones educativas (Δ67.9%; IC95% 61.3%-69.4%). Conclusiones: La intervención educativa estudiada mostró incrementar el nivel de conocimientos sobre medidas preventivas para evitar enfermedades transmitidas por el mosquito Aedes aegypti en asegurados intervenidos en el departamento de Piura durante Junio 2017.

Chocolate, Air Pollution and Children's Neuroprotection: What Cognition Tools should be at Hand to Evaluate Interventions?

Millions of children across the world are exposed to multiple sources of indoor and outdoor air pollutants, including high concentrations of fine particulate matter (PM2.5) and ozone (O3). The established link between exposure to PM2.5, brain structural, volumetric and metabolic changes, severe cognitive deficits (1.5-2 SD from average IQ) in APOE 4 heterozygous females with >75 - < 94% BMI percentiles, and the presence of Alzheimer's disease (AD) hallmarks in urban children and young adults necessitates exploration of ways to protect these individuals from the deleterious neural effects of pollution exposure. Emerging research suggests that cocoa interventions may be a viable option for neuroprotection, with evidence suggesting that early cocoa interventions could limit the risk of cognitive and developmental concerns including: endothelial dysfunction, cerebral hypoperfusion, neuroinflammation, and metabolic detrimental brain effects. Currently, however, it is not clear how early we should implement consumption of cocoa to optimize its neuroprotective effects. Moreover, we have yet to identify suitable instruments for evaluating cognitive responses to these interventions in clinically healthy children, teens, and young adults. An approach to guide the selection of cognitive tools should take into account neuropsychological markers of cognitive declines in patients with Alzheimer's neuropathology, the distinct patterns of memory impairment between early and late onset AD, and the key literature associating white matter integrity and poor memory binding performance in cases of asymptomatic familial AD. We highlight potential systemic and neural benefits of cocoa consumption. We also highlight Working Memory Capacity (WMC) and attention control tasks as opened avenues for exploration in the air pollution scenario. Exposures to air pollutants during brain development have serious brain consequences in the short and long term and reliable cognition tools should be at hand to evaluate interventions.

Alterations in adhesion molecules, pro-inflammatory cytokines and cell-derived microparticles contribute to intima-media thickness and symptoms in postmenopausal women.

Menopause, the cessation of menses, occurs with estrogens decline, low-grade inflammation, and impaired endothelial function, contributing to atherosclerotic risk. Intima-media thickness (IMT) is an early subclinical biomarker of atherosclerosis. Inflammation may have a role on symptoms: hot flashes, anxiety, and depressive mood, which also are related to endothelial dysfunction, increased IMT and cardiovascular risk. In this study we compared several inflammatory markers in early vs. late postmenopausal women and studied the association of IMT and symptoms with these markers in the full sample. In a cross-sectional design including 60 women (53.1 ± 4.4 years old) at early and late postmenopause, we evaluated the expression of CD62L, ICAM-1, PSGL-1, CD11b, CD11c, and IL-8R on PBMC by flow cytometry. Serum soluble ICAM-1, sVCAM-1, sCD62E, sCD62P, CXCL8, IL-1ß, IL-6, and TNF-α levels were quantified by ELISA. Plasma levels of microparticles (MPs) were determined by FACS. Finally, carotid intima-media thickness (IMT) was measured by ultrasound. We observed that ICAM-1 expression by lymphocytes and serum sVCAM-1 levels were augmented at late postmenopause. Late postmenopause women with severe hot flashes had increased expression of CD62L and IL-8R on neutrophils. By multivariate analysis, the carotid IMT was strongly associated with membrane-bound TNF-α, CD11b expression, Annexin V(+) CD3(+) MPs, LPS-induced NO production, HDL-cholesterol and age. Depressive mood was associated negatively with PSGL-1 and positively with LPS-induced NO. Finally, Log(AMH) levels were associated with carotid IMT, IL-8R expression and time since menopause. IMT and depressive mood were the main clinical features related to vascular inflammation. Aging, hormonal changes and obesity were also related to endothelial dysfunction. These findings provide further evidence for a link between estrogen deficiency and low-grade inflammation in endothelial impairment in mature women.

Influence of heart rate variability and psychosocial factors on carotid stiffness, elasticity and impedance at menopause.

BACKGROUND AND AIMS: The risk for cardiovascular diseases (CVD) increases after menopause. Heart rate variability (HRV), a measure of autonomic control, is a strong predictor of CVD. We undertook this study to test the association of ultrasound indices of early carotid atherosclerosis with HRV, symptoms, hormonal conditions, metabolic state, indicators of stress, and psychosocial factors in women at peri- and postmenopause, registering ambulatory R-R interval monitoring. METHODS: In a cross-sectional design we studied 100 women at peri- and early postmenopause collecting anthropometry, symptoms, stress-related measurements, metabolic variables, cortisol, FSH and estradiol. We evaluated carotid ultrasonographic indices, and HRV was recorded for 4 h calculating time (SDNN, pNN50, rMSSD) and frequency domains (LF, HF, LF/HF) in women according to menopausal stage, estradiol levels, body mass index and waist circumference. RESULTS: Carotid indices were similar in peri- and postmenopausal women. For HRV measurements, SDNN was increased at postmenopause. Women with estradiol levels <109.2 pmol/L had increased intima-media thickness (IMT), resistive index, and systolic diameter. Using multivariate analysis, we found the associations of IMT positively with non-HDL-cholesterol, resistive index positively with LF-HRV, but negatively with effort/reward imbalance, carotid ß stiffness index inversely with estradiol, and arterial distensibility positively with HF-HRV and creatinine concentrations, but negatively with non-HDL-cholesterol. CONCLUSIONS: Carotid thickness was related mainly with lipid alterations. Indices of early carotid damage were related with various components of HRV as a manifestation of autonomic imbalance, indicating CVD risk. Other factors involved were time since last menses and psychological stress. Low creatinine was associated with diminished carotid distensibility. This suggests that estrogen, lifestyle, behavior and autonomic regulation participate in vascular damage.

Reproductive parameters in the critically endangered Blue-throated Macaw: limits to the recovery of a parrot under intensive management.

Rediscovered in the wild twenty years ago, the breeding biology of wild Blue-throated Macaws remains largely unexplored, yet is essential to its effective conservation and recovery. Here, we analyse reproductive parameters in an intensively managed wild population of Blue-throated Macaws, providing the first data on the breeding biology of this critically endangered species. During the six-year study period, 2007-2012, the number of active breeding pairs either remained constant or decreased, depending on the site, and no new breeding pairs were discovered despite extensive searching. We documented nesting attempts in natural cavities in dead palms or live hardwoods, and artificial nest boxes. Egg-laying was concentrated during the end of dry season and the beginning of the wet season, August through December. Hatching failure was the greatest cause of egg losses. Half of the breeding attempts of Blue-throated Macaws produced at least one fledging, on average two, after a 85 days nestling period. An average of 4.3 nestlings per year fledged from all known wild nests combined. Each pair lost roughly 65% of its initial reproductive investment at each nesting attempt. In most successful nesting attempts of individualized pairs, a new nesting attempt was not detected the following year. All monitored breeding pairs showed high nest site fidelity, reusing hardwood-tree cavities and nest boxes. Our findings will aid conservation efforts by refining current actions and prompting new approaches towards the conservation and recovery of the Blue-throated Macaw.

Relationship of sleep alterations with perimenopausal and postmenopausal symptoms.

OBJECTIVE: Sleep disturbance is an important change in menopause because it affects quality of life and can lead to other conditions such as depression. This study measured sleep alterations and explored associated physical, emotional, hormonal, and lifestyle factors during perimenopause and postmenopause. METHODS: This cross-sectional study enrolled 160 women who were classified as perimenopausal (n = 85) or postmenopausal (n = 75). Using diaries, we collected data on duration of sleep, time awake in bed, and sleep efficiency. Follicle-stimulating hormone, 17ß-estradiol, and cortisol levels were quantified by radioimmunoassay, and serum anti-Müllerian hormone levels were measured using enzyme-linked immunosorbent assay. Correlations between sleep measurements and symptoms were assessed using a generalized linear model. RESULTS: The reported duration of sleep was similar for both groups of women (close to 6.9 h), and sleep efficiency was 88%. We did not find any factor that was associated with duration of sleep. Sleep efficiency was negatively associated with age, perimenopause/postmenopause status, loss of sexual interest, hot flashes, and depressed mood. Time awake in bed was positively associated with depressed mood (P < 0.000001), cigarette smoking (P < 0.000041), menopause status (P < 0.00009), and age (P < 0.0009). These associations remained after controlling for exercise, alcohol consumption, and caffeine consumption as confounding variables. Finally, morning salivary cortisol was reduced in postmenopausal women. CONCLUSIONS: Time awake in bed shows the most significant associations. Depressed mood, age, and menopause status are the main factors associated with sleep disturbances.

Brain molecules and appetite: the case of oleoylethanolamide.

The neurobiological mechanisms of feeding involve the activity of several brain areas as well as the engagement of endogenous compounds such as ghrelin, melanin-concentrating hormone, orexin, neuropeptide Y, leptin, vasoactive intestinal peptide, cholecystokinin, among others. Furthermore, the family of food-intake modulators has been enlarged due to the inclusion of lipids such as N-arachidonoylethanolamide (anandamide), as well as oleoylethanolamide (OEA). In this regard, the food-intake suppressing properties of OEA have been described since pharmacological administration of this compound induces anorexia. It has been suggested that satiety induced by OEA may be through the activation of peroxisome proliferator-activated receptor-α (PPAR-α), a ligand-activated transcription factor that modulates several pathways of lipid metabolism. The mechanism of action of OEA remains unknown, it has been suggested that the ingestion of dietary fat stimulates epithelial cells of the small intestine and promotes the synthesis and release of OEA. Upon its release, this lipid acts within the gut engaging sensory fibers of the vagus nerve to diminish food-intake. Here, recent advances in our understanding of the neurobiological role of OEA in modulation of feeding will be reviewed. Also, we highlight the emerging molecular mechanism of anorexia induced by OEA.

A pilot trial of the microtubule-interacting peptide (NAP) in mice overexpressing alpha-synuclein shows improvement in motor function and reduction of alpha-synuclein inclusions.

Abnormal accumulation of α-synuclein is associated with several neurodegenerative disorders (synucleinopathies), including sporadic Parkinson's disease (PD). Genetic mutations and multiplication of α-synuclein cause familial forms of PD and polymorphisms in the α-synuclein gene are associated with PD risk. Overexpression of α-synuclein can impair essential functions within the cell such as microtubule-dependent transport, suggesting that compounds that act on the microtubule system may have therapeutic benefit for synucleinopathies. In this study, mice overexpressing human wildtype α-synuclein under the Thy1 promoter (Thy1-aSyn) and littermate wildtype control mice were administered daily the microtubule-interacting peptide NAPVSIPQ (NAP; also known as davunetide or AL-108) intranasally for 2 months starting at 1 month of age, in a regimen known to produce effective concentrations of the peptide in mouse brain. Motor performance, coordination, and activity were assessed at the end of treatment. Olfactory function, which is altered in PD, was measured 1 month later. Mice were sacrificed at 4.5 months of age, and their brains examined for proteinase K-resistant α-synuclein inclusions in the substantia nigra and olfactory bulb. NAP-treated Thy1-aSyn mice showed a 38% decrease in the number of errors per step in the challenging beam traversal test and a reduction in proteinase K-resistant α-synuclein inclusions in the substantia nigra compared to vehicle treated transgenics. The data indicate a significant behavioral benefit and a long lasting improvement of α-synuclein pathology following administration of a short term (2 months) NAP administration in a mouse model of synucleinopathy.

Cost-effectiveness analyses of docetaxel versus paclitaxel once weekly in patients with metastatic breast cancer in progression following anthracycline chemotherapy, in Spain.

OBJECTIVES: Our aim was to evaluate the cost-effectiveness of docetaxel versus weekly paclitaxel regimen in patients with metastatic breast cancer previously treated with anthracycline from the Spanish National Health Service (NHS) perspective. METHODS: A Markov model with a 21-day cycle duration was developed to estimate total treatment-related costs and clinical benefits over 5 years of docetaxel (100 mg/m²) and weekly paclitaxel (80 mg/m²). Patient data were obtained from the Randomized Phase III Study of Docetaxel Compared with Paclitaxel in Metastatic Breast Cancer (TAX- 311) and Anglo-Celtic IV trials. Utilities were obtained from literature, and unitary costs (€2009) from a Spanish health-cost database and the Catalogue of Medicines. Cost and benefits [life-years gained (LYG) and quality-adjusted life years (QALY)] were discounted at 3%. Sensitivity analyses were performed. RESULTS: Docetaxel yields higher health benefits (1.83 LYG; 1.08 QALY) than paclitaxel (1.46 LYG; 0.84 QALY). Global costs (treatment, concomitant medication, adverse events management, progression, best supportive care, and end of life phase) per patient were €20,052 and €9,982 with docetaxel and paclitaxel, respectively. Incremental cost-effectiveness ratio (ICER) of docetaxel versus paclitaxel was €190/LYG and €295/QALY. Based on a €30,000/QALY threshold, docetaxel has 99% probability of being cost-effective. ICER was mostly sensitive to hazard ratio (HR) (when varied from 1.46 to 1.09; €3,517/ QALY), discount over the ex-lab price of Taxol® (75%; €6,396/QALY) and granulocyte colony-stimulating factor (G-CSF) prophylactic treatment (when administered in 60% of cycles instead of 100%; cost saving). Variations in other inputs, such as time horizon (3-10 years), discount rate (0-5%), or adverse event cost (± 25%) were shown not to have relevant influence on the results. CONCLUSION: Compared to weekly paclitaxel, docetaxel therapy is cost effective for treating metastatic breast cancer patients.

Development of mitral cells and olfactory bulb layers in neonatally undernourished rats.

Cell alterations in the central nervous system are consistent consequences of early undernourishment. Because little is known about the effects of neonatal udernourishment upon the main olfactory bulb (OB) in Golgi-Cox stained material, we evaluated the total OB cross-sectional area, the area of individual OB layers, and the area of type II mitral cells perikarya and their dendritic processes in undernourished Wistar rats of 7, 14, and 21 days of age. Data showed that neonatal undernourishment reduced both the OB and the individual layers areas; minimal perikarya effects and significant reductions in the number and extension of MC dendrites. Although macroneurons are formed prenatally, neonatal undernourishment at critical periods may have long-lasting effects that interfere with the functional maturity of the OB. These findings may have relevant consequences for early odor discrimination of the offspring, since olfaction is a fundamental sensorial avenue for newborn adaptive responses and maternal care.

Organization of olfactory glomeruli in neonatally undernourished rats.

Newborn rats maintain mother-litter bonds by using olfactory signals. At birth, units in the olfactory glomeruli (OG) are immature and vulnerable to noxious epigenetic factors like undernutrition. Because little is known about the effects of neonatal undernutrition upon the OG morphological organization, different OG parameters were studied in undernourished Wistar rats at 7, 14 and 21 days of age. The issue was addressed by analyzing the olfactory bulb (OB) cross sectional area, the total number and area of OGs in the OB coronal sections, and the distribution of OG area in dorsal and ventral quadrants. Reductions in the OB and OG cross sectional areas were detected at 7 and 14 days posnatally. OG area comparisons by OB quadrants were reduced along the study in quadrants, with larger effects in medial than in lateral OB quadrants. Current OG cytoarchitectonic modifications may affect the newborn capabilities for odour discrimination by disrupting early mother-litter interactions.