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Bernstein D.L.,University of Pennsylvania | Le Lay J.E.,University of Pennsylvania | Ruano E.G.,August Pi i Sunyer Biomedical Research Institute IDIBAPS | Ruano E.G.,Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders | Kaestner K.H.,University of Pennsylvania
Journal of Clinical Investigation | Year: 2015

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator-like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics. © 2015, American Society for Clinical Investigation. All rights reserved. Source

Prats-Puig A.,Girona Institute for Biomedical Research | Osiniri I.,Pediatrics | Soriano-Rodriguez P.,Clinical Laboratory | Carreras-Badosa G.,Girona Institute for Biomedical Research | And 8 more authors.
Atherosclerosis | Year: 2014

Background: The undercarboxylated form of osteocalcin (ucOC) is an emerging marker of cardiovascular disease. It is unknown if ucOC in related to common cardiovascular risk markers in children. In offspring of families with and without metabolic syndrome (MetS+ and MetS- families), we assessed whether ucOC was related to a continuous metabolic syndrome score (MetS score) and to carotid intima-media thickness (cIMT). Methods: ucOC and total OC, MetS score and cIMT were assessed in 203 asymptomatic prepubertal children (age 7.6±0.1yr; 49% girls), of whom 99 were from MetS+ families. Results: In children from MetS+ families, percent ucOC was higher than in children from MetS- families (p<0.01). In offpring from MetS+ families, higher ucOC and especially higher percent ucOC was independently associated with both the MetS score and cIMT (both p≤0.01). Conclusions: The undercarboxylated form of OC is related to common cardiovascular risk markers in children at risk for cardiovascular disease. © 2014 Elsevier Ireland Ltd. Source

Escudero-Lopez B.,Pablo De Olavide University | Escudero-Lopez B.,University of Parma | Calani L.,University of Parma | Fernandez-Pachon M.-S.,Pablo De Olavide University | And 5 more authors.
BioFactors | Year: 2014

Two milliliters of a fermented, pasteurized orange juice containing ~1% alcohol and 2.3 μmol of (poly)phenolic compounds was fed to rats by gavage after which plasma and urine collected over a 36 h period were analyzed by UHPLC-mass spectrometry. The main constituents in the juice were hesperetin and naringenin-O-glycosides, apigenin-6,8-C-diglucoside, and ferulic acid-4′-O-glucoside. Plasma contained seven flavanone glucuronides, with the principal metabolites, naringenin-7-O-glucuronide, naringenin-4′-O-glucuronide, and an isosakuranetin-O-glucuronide, peaking 6 h after intake at concentrations of ~10 nmol/L. Urinary excretion of four hesperetin glucuronides was equivalent to 0.28% of intake while that of the two naringenin glucuronides was 2.8% of intake. The plasma and urine data suggest that while some absorption occurred in the small intestine, the main site of uptake was the colon. Urine also contained dihydroferulic acid-4'-O-glucuronide and dihydroferulic acid-4′-O-sulfate which were excreted in quantities corresponding to 48.2% of the ingested ferulic acid-4′-glucoside. This indicates that the hydroxycinnamate is much more bioavailable than the flavanones in the rat model. Conversion of the ferulic acid glucoside to the dihydroferulic acid metabolites involves the action of colonic microbial glycosidases and reductases/hydrogenases followed by postabsorption phase II metabolism before renal excretion. © 2013 International Union of Biochemistry and Molecular Biology, Inc. Source

Prats-Puig A.,Girona Institute for Biomedical Research | Grau-Cabrera P.,Pediatrics | Riera-Perez E.,Pediatrics | Cortes-Marina R.,Catalonian Institute of Health | And 7 more authors.
International Journal of Obesity | Year: 2013

Objective: Shorter sleep duration predisposes to obesity, but the mechanisms whereby sleep deprivation affects body weight are poorly understood. We tested whether this association is modulated by the obesity genes FTO, TMEM18 and NRXN3. Subjects: Body mass index (BMI), waist circumference, visceral fat (abdominal ultrasound), homeostasis model assessment for insulin resistance (HOMA-IR), systolic blood pressure (SBP) and sleep time per 24 h were assessed in 297 asymptomatic children (151 boys, 146 girls; age range 5-9 years; BMI s.d. score range-2.0-4.0). Associations between sleep duration and the abovementioned outcomes were tested for three common single-nucleotide polymorphisms (SNPs), namely FTO (rs9939609), TMEM 18 (rs4854344) and NRXN3 (rs10146997), as well as for their combination. Results: TT homozygotes (but not A * carriers) for the FTO SNP, exhibited nominal associations between decreasing sleep duration and increasing BMI, waist circumference, visceral fat and HOMA-IR (all P<0.05). Similar associations were observed in children with risk alleles (but not in those without risk alleles) for the TMEM18 and NRXN3 SNPs (P<0.05 to P<0.0001). The three SNPs had additive effects on the negative associations between sleep and, respectively, BMI (P<0.001), waist (P<0.005), visceral fat (P<0.001), HOMA-IR (P=0.010) and SBP (P<0.0005). The combined effects on obesity measures and SBP remained significant after correction for multiple testing. On average, 2 h of sleep less per night was associated with an increase in BMI of 1.0 s.d. (95% confidence interval 0.5-1.6 s.d.) and with 8.0 cm (95% confidence interval 3.6-12.2 cm) more waist circumference in genetically susceptible children. Conclusion: By age 7, common variations in FTO, TMEM18 and NRXN3 influence the vulnerability to metabolic complications of sleep deprivation. Further genetic studies are warranted to replicate these findings in other populations. © 2013 Macmillan Publishers Limited All rights reserved. Source

Cervantes S.,Institute dInvestigacions Biomediques August Pi i Sunyer IDIBAPS Hospital Clinic | Cervantes S.,Spanish Biomedical Research Center in Diabetes and Associated Metabolic Disorders
Histology and Histopathology | Year: 2013

The gastrointestinal tract carries out essential functions for the organism, including the digestion and absorption of nutrients. The cells lining the lumen of the gut tube derive from the endoderm, one of the three germ layers formed during gastrulation. The length of the intestinal tract determines its digestive and absorptive capacity, and so the intestine expands several times the length of the whole body to ensure an adequate absorptive area to meet nutritional demands. However, the endoderm starts out as a small sheet of cells spanning less than the whole length of the head-fold embryo. In order to achieve its final shape and size, the cells in the endoderm undergo extensive growth and profound morphogenetic changes, which are governed by embryonic signaling pathways and transcription factors. This review, based on mouse development, summarizes our current knowledge of the cellular and molecular mechanisms underlying the morphogenetic changes that participate in shaping the mature intestinal tract in vertebrates. Source

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