Macronutrient composition of the diet and prospective weight change in participants of the EPIC-PANACEA study.

BACKGROUND The effect of the macronutrient composition of the usual diet on long term weight maintenance remains controversial. METHODS 373,803 subjects aged 25-70 years were recruited in 10 European countries (1992-2000) in the PANACEA project of the EPIC cohort. Diet was assessed at baseline using country-specific validated questionnaires and weight and height were measured at baseline and self-reported at follow-up in most centers. The association between weight change after 5 years of follow-up and the iso-energetic replacement of 5% of energy from one macronutrient by 5% of energy from another macronutrient was assessed using multivariate linear mixed-models. The risk of becoming overweight or obese after 5 years was investigated using multivariate Poisson regressions stratified according to initial Body Mass Index. RESULTS A higher proportion of energy from fat at the expense of carbohydrates was not significantly associated with weight change after 5 years. However, a higher proportion of energy from protein at the expense of fat was positively associated with weight gain. A higher proportion of energy from protein at the expense of carbohydrates was also positively associated with weight gain, especially when carbohydrates were rich in fibre. The association between percentage of energy from protein and weight change was slightly stronger in overweight participants, former smokers, participants ≥60 years old, participants underreporting their energy intake and participants with a prudent dietary pattern. Compared to diets with no more than 14% of energy from protein, diets with more than 22% of energy from protein were associated with a 23-24% higher risk of becoming overweight or obese in normal weight and overweight subjects at baseline. CONCLUSION Our results show that participants consuming an amount of protein above the protein intake recommended by the American Diabetes Association may experience a higher risk of becoming overweight or obese during adult life.

Aptasensores impedimétricos para la detección de trombina

En el presente trabajo se ha desarrollado el primer aptasensor (biosensor de aptámero) en nuestro grupo de investigación, Grup de Sensors i Biosensors de la Universidad Autònoma de Barcelona. En concreto se han desarrollado dos aptasensores para la detección de la proteína trombina, uno basado en la inmovilización de aptámeros por adsorción física, y otro basado en la inmovilización de aptámeros por enlace covalente mediante la reacción EDAC-NHS. El aptasensor utiliza la afinidad específica de la cadena de DNA (aptámero) por la proteína con la que interacciona. Los cambios de carga y estéricos del complejo aptámero proteína alteran la capacidad y la resistencia de transferencia interfacial de electrones en la superficie del electrodo. El principio de detección se basa en la detección de cambios de estas propiedades de interfase del electrodo con el marcador redox [Fe(CN)6]3- / [Fe(CN)6]4-, utilizando mediciones de Espectroscopia Electroquímica de Impedancia. El aptasensor basado en adsorción física del aptámero mostró una respuesta lineal a trombina en el rango de 7.5 a 75 pM y un límite de detección de 5pM, después de optimizar todas las condiciones experimentales. Posteriormente se estudió la especificidad del sistema respecto proteínas potencialmente interferentes presentes en suero sanguíneo, obteniendo cierta interferencia por parte de fibrinógeno e inmunoglobulina G, pero no por parte de albúmina. El sensor demostró ser regenerable mediante la ruptura del complejo formado entre el aptámero y la trombina con una solución de NaCl 2.0 M, aumento de la temperatura y agitación. El segundo aptasensor, basado en enlace covalente del aptámero mostró una respuesta lineal a trombina y limite de detección mejor que el anterior sensor; de 2.5 a 100 pM y 1.5 pM respectivamente. Aunque cabe destacar que este aptasensor está siendo optimizado actualmente.

Evidence of new risk genetic factor to systemic lupus erythematosus: the UBASH3A gene

The ubiquitin associated and Src-homology 3 (SH3) domain containing A (UBASH3a) is a suppressor of T-cell receptor signaling, underscoring antigen presentation to T-cells as a critical shared mechanism of diseases pathogenesis. The aim of the present study was to determine whether the UBASH3a gene influence the susceptibility to systemic lupus erythematosus (SLE) in Caucasian populations. We evaluated five UBASH3a polymorphisms (rs2277798, rs2277800, rs9976767, rs13048049 and rs17114930), using TaqMan® allelic discrimination assays, in a discovery cohort that included 906 SLE patients and 1165 healthy controls from Spain. The SNPs that exhibit statistical significance difference were evaluated in a German replication cohort of 360 SLE patients and 379 healthy controls. The case-control analysis in the Spanish population showed a significant association between the rs9976767 and SLE (Pc = 9.9E-03 OR = 1.21 95%CI = 1.07-1.37) and a trend of association for the rs2277798 analysis (P = 0.09 OR = 0.9 95%CI = 0.79-1.02). The replication in a German cohort and the meta-analysis confirmed that the rs9976767 (Pc = 0.02; Pc = 2.4E-04, for German cohort and meta-analysis, respectively) and rs2277798 (Pc = 0.013; Pc = 4.7E-03, for German cohort and meta-analysis, respectively) UBASH3a variants are susceptibility factors for SLE. Finally, a conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs9976767 polymorphism. Our results suggest that UBASH3a gene plays a role in the susceptibility to SLE. Moreover, our study indicates that UBASH3a can be considered as a common genetic factor in autoimmune diseases.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Selected ABCB1, ABCB4 and ABCC2 Polymorphisms Do Not Enhance the Risk of Drug-Induced Hepatotoxicity in a Spanish Cohort.

BACKGROUND AND AIMS Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

Effects of Crypthecodiniumcohnii, Chlorela spp. e Isochrysisgalbana addition to milk replacer on goat kids and lamb growth.

The effects of Crypthecodinium cohnii (Cryp.), Chlorela spp. (Chlo.) and Isochrysis galbana (Iso.) addition to milk replacer on goat kids and lambs growth were evaluated. About 80 Majorera goat kids (males and females) and 80 Canarian sheep lambs were randomly assigned into four different groups (by specie) according to diet. Control groups were fed with a commercial milk replacer at 16% (w/w); Cryp. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of C. cohnii; Chlo. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of Chlorela spp.; Iso. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of I. galbana. After colostrum period, animals were individually bottle-fed twice daily (8 am and 8 pm) ad libitum with the corresponding diet until day 60 of life. Animals were weighted every week at 8 am and liquid diet intake was recorded weekly. No effects of microseaweed addition were observed, neither growth nor milk replacer intake.

Síntesis de nuevos catecoles como precursores de materiales adhesivos bioinspirados

En nuestro grupo de investigación y en colaboración con el Doctor Daniel Ruiz-Molina del Centro de Investigación en Nanociencia y Nanotecnología (CIN2) de este campus universitario, hemos iniciado un proyecto que se plantea lograr biomimetizar ciertas proteínas adhesivas de moluscos, cuya adherencia se debe a la presencia de la funcionalidad catecol. El objetivo es poder desarrollar nuevos materiales adhesivos basados en esa funcionalidad, siendo necesaria por lo tanto el desarrollar protocolos sintéticos para la obtención de compuestos tipo catecol convenientemente sustituidos. Concretamente, en el presente trabajo de Master en Experimentación Química se ha conseguido la síntesis de los nuevos catecoles 1 y 2, que incorporan en su estructura una cadena alquílica acabada con la funcionalidad tiol, con el fin de poder desarrollar más adelante superficies o nanopartículas de oro adhesivas. Para la síntesis del 4-(3’-mercaptopropil)catecol 1 se ha puesto a punto una ruta de obtención partiendo del producto comercial eugenol, consistente en 3 etapas sintéticas y con un rendimiento global cercano al 70%. También se ha desarrollado una ruta sintética para el 4-(7’-mercaptopropil)catecol 2 que parte del 3,4-dimetoxibenzaldehído, y consta de 6 etapas, presentando un rendimiento global del 25%.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

The effect of colostrum source (goat vs. sheep) and timing of the first colostrum feeding (2h vs. 14h after birth) on body weight and immune status of artificially reared newborn lambs.

Several factors can affect lamb body weight (BW) and immune status during the first days of life, including colostrum source and timing of the first colostrum feeding. The aim of this study was to evaluate the effects of colostrum source (goat or sheep) and timing of the first colostrum feeding (2 or 14h after birth) on lamb BW and immune status. In this study, 40 lambs were removed from their dams at birth and randomly assigned into 4 groups of 10 lambs each. Lambs were subsequently fed at 2 or 14h after birth with goat or sheep colostrum. Blood samples and BW recording were performed before feeding. Blood plasma was used to measure the immunoglobulin concentration (IgG and IgM), chitotriosidase activity, and complement system activity (total and alternative pathways). In general, no differences in any of the measured variables were observed among the 4 groups, indicating that neither colostrum source nor timing of the first colostrum feeding had an effect on these variables. These findings may improve management on lamb farms that raise animals under artificial conditions, because our results indicate that it is not necessary to feed colostrum to lambs immediately after birth and that goat colostrum may be used to feed newborn lambs.

Association of irisin with fat mass, resting energy expenditure, and daily activity in conditions of extreme body mass index.

FNDC5/irisin has been recently postulated as beneficial in the treatment of obesity and diabetes because it is induced in muscle by exercise, increasing energy expenditure. However, recent reports have shown that WAT also secretes irisin and that circulating irisin is elevated in obese subjects. The aim of this study was to evaluate irisin levels in conditions of extreme BMI and its correlation with basal metabolism and daily activity. The study involved 145 female patients, including 96 with extreme BMIs (30 anorexic (AN) and 66 obese (OB)) and 49 healthy normal weight (NW). The plasma irisin levels were significantly elevated in the OB patients compared with the AN and NW patients. Irisin also correlated positively with body weight, BMI, and fat mass. The OB patients exhibited the highest REE and higher daily physical activity compared with the AN patients but lower activity compared with the NW patients. The irisin levels were inversely correlated with daily physical activity and directly correlated with REE. Fat mass contributed to most of the variability of the irisin plasma levels independently of the other studied parameters. Conclusion. Irisin levels are influenced by energy expenditure independently of daily physical activity but fat mass is the main contributing factor.

Role of Sam68 in post-transcriptional gene regulation.

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

Regression of cardiac growth in kidney transplant recipients using anti-m-TOR drugs plus RAS blockers: a controlled longitudinal study.

BACKGROUND Left ventricular hypertrophy (LVH) is common in kidney transplant (KT) recipients. LVH is associated with a worse outcome, though m-TOR therapy may help to revert this complication. We therefore conducted a longitudinal study to assess morphological and functional echocardiographic changes after conversion from CNI to m-TOR inhibitor drugs in nondiabetic KT patients who had previously received RAS blockers during the follow-up. METHODS We undertook a 1-year nonrandomized controlled study in 30 non-diabetic KT patients who were converted from calcineurin inhibitor (CNI) to m-TOR therapy. A control group received immunosuppressive therapy based on CNIs. Two echocardiograms were done during the follow-up. RESULTS Nineteen patients were switched to SRL and 11 to EVL. The m-TOR group showed a significant reduction in LVMi after 1 year (from 62 ± 22 to 55 ± 20 g/m2.7; P=0.003, paired t-test). A higher proportion of patients showing LVMi reduction was observed in the m-TOR group (53.3 versus 29.3%, P=0.048) at the study end. In addition, only 56% of the m-TOR patients had LVH at the study end compared to 77% of the control group (P=0.047). A significant change from baseline in deceleration time in early diastole was observed in the m-TOR group compared with the control group (P=0.019). CONCLUSIONS Switching from CNI to m-TOR therapy in non-diabetic KT patients may regress LVH, independently of blood pressure changes and follow-up time. This suggests a direct non-hemodynamic effect of m-TOR drugs on cardiac mass.

Analysis of genes encoding penicillin-binding proteins in clinical isolates of Acinetobacter baumannii.

There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.