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Crocins are high-value soluble pigments that are used as colorants and supplements, their presence in nature is extremely limited and, consequently, the high cost of these metabolites hinders their use by other sectors, such as the pharmaceutical and cosmetic industries. The carotenoid cleavage dioxygenase 2L (CsCCD2L) is the key enzyme in the biosynthetic pathway of crocins in Crocus sativus. In this study, CsCCD2L was introduced into Nicotiana tabacum and Nicotiana glauca for the production of crocins. In addition, a chimeric construct containing the Brevundimonas sp. β-carotene hydroxylase (BrCrtZ), the Arabidopsis thaliana ORANGE mutant gene (AtOrMut), and CsCCD2L was also introduced into N. tabacum. Quantitative and qualitative studies on carotenoids and apocarotenoids in the transgenic plants expressing CsCCD2L alone showed higher crocin level accumulation in N. glauca transgenic plants, reaching almost 400 μg/g DW in leaves, while in N. tabacum 36 μg/g DW was obtained. In contrast, N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut accumulated, 3.5- fold compared to N. tabacum plants only expressing CsCCD2L. Crocins with three and four sugar molecules were the main molecular species in both host systems. Our results demonstrate that the production of saffron apocarotenoids is feasible in engineered Nicotiana species and establishes a basis for the development of strategies that may ultimately lead to the commercial exploitation of these valuable pigments for multiple applications. ; This study was supported by grants BIO2016-77000-R from the Spanish Ministerio de Ciencia; Innovación y Universidades and SBPLY/17/180501/000234 from the Junta de Comunidades de Castilla-La Mancha (co-financed European Union FEDER funds), the National Natural Science Foundation of China (31870278), the Spanish Ministry of Economy and Competitiveness (MINECO), Spain (RTI2018-097613-B-I00 and PGC2018-097655-B-I00). CZ and LG-G were participants of the European COST action CA15136 (EUROCAROTEN), and Programa Estatal de ...

14 p.-6 fig. ; Autophagy is a catabolic pathway by which cellular components are delivered to the lysosome for degradation and recycling. Autophagy serves as a crucial intracellular quality control and repair mechanism but is also involved in cell remodelling during development and cell differentiation. In addition, mitophagy, the process by which damaged mitochondria undergo autophagy, has emerged as key regulator of cell metabolism. In recent years, a number of studies have revealed roles for autophagy and mitophagy in the regulation of stem cells, which represent the origin for all tissues during embryonic and postnatal development, and contribute to tissue homeostasis and repair throughout adult life. Here, we review these studies, focussing on the latest evidence that supports the quality control, remodelling and metabolic functions of autophagy during the activation, self-renewal and differentiation of embryonic, adult and cancer stem cells. ; P.B. is supported by Spain's Ministerio de Economía y Competitividad [BFU2015-65623 (FEDER funding) and BFU2015-71869-REDT], European Cooperation in Science and Technology (COST) Action Transautophagy (CA15138) and the European Union Horizon 2020 Marie Skłodowska-Curie Innovative Training Networks (ITN-ETN) under grant agreement 765912. N.R.-M. is supported by a grant from the Muscular Dystrophy Association (MDA376743) and a Juan de la Cierva Incorporación fellowship from Spain's Ministerio de Economía y Competitividad. P.C. is supported by funding from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), and Université Paris Descartes-Sorbonne Paris Cité and grants from the Institut National Du Cancer (INCa) and the Agence Nationale de la Recherche (ANR). ; Peer reviewed

Over the recent years, Nicotiana benthamiana has gained great importance as a chassis for the production of high value, low volume pharmaceuticals and/or active pharmaceutical ingredients (APIs). The process involving infiltration of the N. benthamiana leaves with Agrobacterium spp, harbouring vectors with the gene of interest, facilitates transient expression. To date, little information is available on the effect of the agro-infiltration process on the metabolome of N. benthamiana, which is necessary to improve the process for large-scale, renewable manufacturing of high value compounds and medical products. Hence, the objective of the present study was to assess metabolic adaptation of N. benthamiana as a response to the presence of Agrobacterium. The present study elucidated changes of the steady-state metabolism in the agroinfiltrated leaf area, the area around the infection and the rest of the plant. Furthermore, the study discusses the phenotypic advantages of the N. benthamiana lab strain, optimised for agro-infiltration, compared to three other wild accessions. Results showed that the lab strain has a different metabolic composition and showed less alterations of the phenylpropanoid pathway and cell wall remodelling in the agroinfiltrated leaf areas, for example chlorogenic acid, cadaverine and C18:0–2-glycerol ester. In conclusion, both of these alterations present potential candidates to improve the phenotype of the N. benthamiana lab strain for a more efficient transient expression process. ; This work was funded by European Union Funding for Research and Innovation "Horizon 2020—NEWCOTIANA – Project no. 760331" and OPTICAR Project BB/P001742/1.

Yan Zhang,1,* Yuan Dong,1,* Zhenyu Xiong,1,* Zhengru Zhu,2 Fanya Gao,3 Tingting Wang,1 Wanrong Man,1 Dong Sun,1 Jie Lin,1 Tongbin Li,1 Congye Li,1 Zhijing Zhao,1 Min Shen,1 Dongdong Sun,1 Yanhong Fan1 1Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi'an, People's Republic of China; 2Department of Otolaryngology Head and Neck Surgery, First Hospital of Lanzhou University, Lanzhou, People's Republic of China; 3Department of Cardiology, Shaanxi Provincial People's Hospital, Xi'an Medical University, Xi'an, People's Republic of China*These authors contributed equally to this workCorrespondence: Dongdong Sun; Yanhong FanDepartment of Cardiology, Xijing Hospital, Fourth Military Medical University, 127 West Changle Road, Xi'an, Shaanxi 710032, People's Republic of ChinaTel +86 18691569930; +86 18829395402Email wintersun3@gmail.com; 18829395402@189.cnBackground: Endothelial-to-mesenchymal transition (EndMT) is an important source of myofibroblasts that directly affects cardiac function in diabetic cardiomyopathy (DCM) via an unknown underlying mechanism. Sirt6 is a member of the Sirtuin family of NAD(+)-dependent enzymes that plays an important role in glucose and fatty acid metabolism. In this study, we investigated whether Sirt6 participates in EndMT during the development of T2DM and the possible underlying regulatory mechanisms.Methods: Endothelium-specific Sirt6 knockout (Sirt6-KOEC) mice (C57BL/6 genetic background) were generated using the classic Cre/loxp gene recombination system. T2DM was induced in eight-week-old male mice by feeding with a high-fat diet for three weeks followed by i.p. injection with 30 mg/kg of streptozotocin. The weight, lipids profiles, insulin, food intake and water intake of experimental animals were measured on a weekly basis. Cardiac microvascular endothelial cells (CMECs) were obtained from adult male mice; the isolated cells were cultured with high glucose (HG; 33 mmol/L) and palmitic acid (PA; 500 μmol/L) in DMEM for 24 h, or with normal glucose (NG; 5 mmol/L) as the control.Results: Sirt6 expression is significantly downregulated in CMECs treated with HG+PA. Additionally, Sirt6-KOEC was found to worsen DCM, as indicated by aggravated perivascular fibrosis, cardiomyocyte hypertrophy, and decreased cardiac function. In vitro, Sirt6 knockdown exacerbated the proliferation, and migration of CMECs exposed to HG+PA. Mechanistically, Sirt6 knockdown significantly enhanced Notch1 activation in CMECs treated with HG+PA, whereas Notch1 adenoviral interference significantly blunted the effects of Sirt6 knockdown on CMECs.Conclusion: This study is the first to demonstrate that Sirt6 participates in EndMT via the Notch1 signaling pathway in CMECs stimulated with HG+PA. Therefore, the findings of this study suggest that Sirt6 could provide a potential treatment strategy for DCM.Keywords: diabetic cardiomyopathy, endothelial-to-mesenchymal transition, cardiac function, fibrosis, Sirt6

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) ; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ; Government of the Principality of Monaco ; CNPq: 573949/2008-5 ; CNPq: 462333/2014-0 ; CNPq: 406426/2012-0 ; The mercapturic acid pathway (MAP) is a major phase II detoxification route, comprising the conjugation of electrophilic substances to glutathione (GSH) in a reaction catalyzed by glutathione S-transferase (GST) enzymes. In mammals, GSH-conjugates are exported from cells, and the GSH-constituent amino acids (Glu/Gly) are subsequently removed by ectopeptidases. The resulting Cys-conjugates are reabsorbed and, finally, a mercapturic acid is generated through N-acetylation. This pathway, though very well characterized in mammals, is poorly studied in non-mammalian biological models, such as bivalve mollusks, which are key organisms in aquatic ecosystems, aquaculture activities and environmental studies. In the present work, the compound 1-chloro-2,4-dinitrobenzene (CDNB) was used as a model electrophile to study the MAP in Pacific oysters Crassostrea gigas. Animals were exposed to 10 NI CDNB and MAP metabolites were followed over 24 h in the seawater and in oyster tissues (gills, digestive gland and hemolymph). A rapid decay was detected for CDNB in the seawater (half-life 1.7 h), and MAP metabolites peaked in oyster tissues as soon as 15 min for the GSH-conjugate, 1 h for the Cys-conjugate, and 4 h for the final metabolite (mercapturic acid). Biokinetic modeling of the MAP supports the fast CDNB uptake and metabolism, and indicated that while gills are a key organ for absorption, initial biotransformation, and likely metabolite excretion, hemolymph is a possible milieu for metabolite transport along different tissues. CDNB-induced GSH depletion (4 h) was followed by increased GST activity (24 h) in the gills, but not in the digestive gland. Furthermore, the transcript levels of glutamate-cysteine ligase, coding for the rate limiting enzyme in GSH synthesis, and two phase II biotransformation genes (GSTpi and GSTo), presented a fast (4h) and robust (similar to 6-70 fold) increase in the gills. Waterborne exposure to electrophilic compounds affected gills, but not digestive gland, while intramuscular exposure was able to modulate biochemical parameters in both tissues. This study is the first evidence of a fully functional and interorgan MAP pathway in bivalves. Hemolymph was shown to be responsible for the metabolic interplay among tissues, and gills, acting as a powerful GSH-dependent metabolic barrier against waterborne electrophilic substances, possibly also participating in metabolite excretion into the sea water. Altogether, experimental and modeled data fully agree with the existence of a classical mechanism for phase II xenobiotic metabolism and excretion in bivalves. (C) 2016 Elsevier B.V. All rights reserved.

11 p.-5 fig.-3 tab. ; Background: Isobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms. Shimwellia blattae (p424IbPSO) is a robust strain resistant to high concentration of isobutanol that can achieve a high production rate of this alcohol. Nevertheless, this strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. Isobutanol production under anaerobic conditions leads to a depletion of NADPH, which is necessary for two enzymes in the metabolic pathway. In this work, two independent approaches have been studied to mitigate the co-substrates imbalance: (i) using a NADH-dependent alcohol dehydrogenase to reduce the NADPH dependence of the pathway and (ii) using a transhydrogenase to increase NADPH level. ; Results: The addition of the NADH-dependent alcohol dehydrogenase from Lactococcus lactis (AdhA) to S. blattae (p424IbPSO) resulted in a 19.3% higher isobutanol production. The recombinant strain S. blattae (p424IbPSO, pIZpntAB) harboring the PntAB transhydrogenase produced 39.0% more isobutanol than the original strain, reaching 5.98 g L−1 of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol. ; Conclusions: The isobutanol biosynthesis pathway in S. blattae (p424IbPSO) uses the endogenous NADPH-dependent alcohol dehydrogenase YqhD to complete the pathway. The addition of NADH-dependent AdhA leads to a reduction in the consumption of NADPH that is a bottleneck of the pathway. The higher consumption of NADH by AdhA reduces the availability of NADH required for the transformation of pyruvate into lactic acid and ethanol. On the other hand, the expression of PntAB from E. coli increases the availability of NADPH for IlvC and YqhD and at the same time reduces the availability of NADH and thus, the production of lactic acid and ethanol. In this work it is shown how the expression of AdhA and PntAB enzymes in Shimwellia blattae increases yield from 11.9% to 14.4% and 16.4%, respectively. ; This work was supported by grants from Biopolis S.L., Spanish Ministry of Science and Innovation BIOSOS CEN20091040 (CENIT-CDTI), RTI2018-095584-B-C44 and MINECO CTQ2017- 84963-C2-1-R and the Community of Madrid and the Structural Funds from European Union (Ref: S2018/BAA-4532 (ALGATEC-CM)). The grant for one of the authors (MGA) with reference BES-2014-068344 is gratefully recognized. ; Peer reviewed

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low. ; This research was partially supported by the grants [BIO2010-18239] from the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) and [IIM010491.RI1] from the Government of Navarra, and by Iden Biotechnology. This research was also supported by Scientific Research on Innovative Areas [22114507] and Grants-in-Aid for Scientific Research (B) [22380186] from the Ministry of Education, Culture, Sports, Science and Technology, Japan. ; Peer Reviewed

AbstractPolyhydroxyalkanoates have attracted great interest as a suitable alternative to petrochemical based plastics due to their outstanding properties such as biodegradability and biocompatibility. However, the biggest problem in the production of microbial polyhydroxyalkanoates is low cost-effectiveness. In this study, polyhydroxyalkanoate production was carried out using waste substrates with local isolates. Culture conditions were optimized to increase the polyhydroxyalkanoate production potential. The produced polyhydroxyalkanoate was characterized by FTIR analyses, and its metabolic pathway was determined by real-time PCR. According to the results, the best polyhydroxyalkanoate producer bacteria was characterized as Pseudomonas neustonica NGB15. The optimal culture conditions were detected as 30 g/L banana peel powder, 25 °C temperature, pH 8, and 4-day incubation time. Under the optimized conditions, 3.34 g/L PHA production was achieved. As a result of FTIR analyses, major peaks were obtained at 1723, 1277, 1261, 1097, 1054, and 993 cm−1. These peaks represent that the type of produced polyhydroxyalkanoate was poly-β-hydroxybutyrate. According to gene expression profile of NGB15, it was determined that Pseudomonas neustonica NGB15 produces PHA using the de novo fatty acid synthesis metabolic pathway. In conclusion, poly-β-hydroxybutyrate production by Pseudomonas neustonica NGB15 using a low-cost fermentation medium has been shown to be biotechnologically promising.

Summary. — 10-3 M gibberellic acid induces a significant increase of respiratory exchanges in tissue slices of tubers of Solanum tuberosum. The intervention of this hormone in the respiratory cycles is analysed both with the action of effectors and with the measure of specific activity of enzymes of this metabolic pathway. Enolase, hexokinase, aldolase and triose-phosphate isomerase enhance their activity. On the contrary there is few rise of peroxidase activity.

INTRODUCTION: Non-small cell lung cancer (NSCLC) patients diagnosed in early stage and surgically-treated have five-year mortality rate >20%. The identification of biomarkers able to predict progression and death may help to identify patients needing closer follow-up. METHODS: A retrospective cohort of early-stage surgically-treated NSCLC patients enrolled in the International Association for the Study of Lung Cancer (IASLC) Staging Project was created, and tissue Microarrays (TMAs) were constructed with tumor and non-tumor lung tissue. Pentose phosphate pathway (PPP) proteins (transketolase [TKT] and transketolase-like 1 [TKTL1]), inflammatory markers (cyclooxygenase-2 [COX-2], tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL1β], nuclear factor kappa-light-chain-enhancer of activated B cells [NFκB]-p65 and antigen Ki-67), and programmed death-ligand 1 (PDL1) were measured by immunohistochemistry. RESULTS: NSCLC patients with adenocarcinoma (ADC) or squamous cell carcinoma (SCC) were included in the study (n = 199). TKT and TKTL1 were significantly higher in ADC than in non-tumor tissue (p 30% above those of non-tumor tissue (p < 0.001). PDL1 analysis showed a higher percentage of positivity in ADC than in non-tumor tissue (p < 0.001). Multivariate Cox proportional hazards modeling confirmed that high IL1β level in tumor tissue was independently associated with 3-year mortality in NSCLC [HR = 2.05, 95% CI (1.1-3.7), p = 0.019], a relationship driven by ADC subtype. CONCLUSION: This study confirms an increase in metabolic activity and an inflammatory response in tumor tissue of early stage NSCLC, and a significant relationship between high levels of IL1β in the tumor and poor prognosis in ADC. ; This study was funded by PII Oncología Torácica from Sociedad Española de Neumología y Cirugía Torácica (SEPAR), FIS 12/02040, FIS 12/02534 and FIS 12/01310 from the Instituto de Salud Carlos III and Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES). CIBERES is an initiative of the Instituto de Salud Carlos III. This work was also supported by grants to E.M. and M.C. from Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR)—Generalitat de Catalunya, (2014 GRC-801/2017 GRC-716 and 2014 SGR-1017/2017 SGR-1033), ICREA Foundation (ICREA Academia) and MINECO from the Spanish Government and FEDER funds (SAF2014‐56059‐R, SAF2015‐70270‐REDT, SAF2017-89673-R, European Commission FEDER—Una manera de hacer Europa), and to C.B. from AGAUR-Generalitat de Catalunya (FI-DGR2014). M.C. also received support from CB17/04/00023 from the Instituto de Salud Carlos III and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERHD). CIBERHD is an initiative of the Instituto de Salud Carlos III. Financial support was also provided by the European Regional Development Fund (ERDF) and CERCA Programme / Generalitat de Catalunya.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease, especially nonalcoholic steatohepatitis (NASH), has become a major cause of liver transplantation and liver‐associated death. NASH is the hepatic manifestation of metabolic syndrome and is characterized by hepatic steatosis, inflammation, hepatocellular injury, and different degrees of fibrosis. However, there is no US Food and Drug Administration–approved medication to treat this devastating disease. Therapeutic activators of the AMP‐activated protein kinase (AMPK) have been proposed as a potential treatment for metabolic diseases such as NASH. Cordycepin, a natural product isolated from the traditional Chinese medicine Cordyceps militaris, has recently emerged as a promising drug candidate for metabolic diseases. APPROACH AND RESULTS: We evaluated the effects of cordycepin on lipid storage in hepatocytes, inflammation, and fibrosis development in mice with NASH. Cordycepin attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes subjected to metabolic stress. In addition, cordycepin treatment significantly and dose‐dependently decreased the elevated levels of serum aminotransferases in mice with diet‐induced NASH. Furthermore, cordycepin treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration, and hepatic fibrosis in mice. In vitro and in vivo mechanistic studies revealed that a key mechanism linking the protective effects of cordycepin were AMPK phosphorylation–dependent, as indicated by the finding that treatment with the AMPK inhibitor Compound C abrogated cordycepin‐induced hepatoprotection in hepatocytes and mice with NASH. CONCLUSION: Cordycepin exerts significant protective effects against hepatic steatosis, inflammation, liver injury, and fibrosis in mice under metabolic stress through activation of the AMPK signaling pathway. Cordycepin might be an AMPK activator that can be used for the treatment of NASH.

The alternative oxidase pathway (AOP) is associated with excess energy dissipation in leaves of terrestrial plants. To address whether this association is less important in palustrine plants, we compared the role of AOP in balancing energy and carbon metabolism in palustrine and terrestrial environments by identifying metabolic relationships between primary carbon metabolites and AOP in each habitat. We measured oxygen isotope discrimination during respiration, gas exchange, and metabolite profiles in aerial leaves of ten fern and angiosperm species belonging to five families organized as pairs of palustrine and terrestrial species. We performed a partial least square model combined with variable importance for projection to reveal relationships between the electron partitioning to the AOP (τa) and metabolite levels. Terrestrial plants showed higher values of net photosynthesis (AN) and τa, together with stronger metabolic relationships between τa and sugars, important for water conservation. Palustrine plants showed relationships between τa and metabolites related to the shikimate pathway and the GABA shunt, to be important for heterophylly. Excess energy dissipation via AOX is less crucial in palustrine environments than on land. The basis of this difference resides in the contrasting photosynthetic performance observed in each environment, thus reinforcing the importance of AOP for photosynthesis. ; This work was mainly supported by Ministerio de Economía y Competitividad (MINECO, Spain) (project PGC2018-093824-B-C41) and the ERDF (FEDER). Others funding were provided by FONDECYT No. 1191118 from National Agency for Research and Development (ANID) and the Chilean Scholarship Program/Becas de doctorado nacional/2017–21180329, the European Union's Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement no. 753301, the 'Ramon y Cajal' contract RYC2019-027244-I/AEI/10.13039/501100011033 and the European Social Fund. ; Peer reviewed

In this work, an untargeted metabolomic approach based on sensitive analysis by on-line solid-phase extraction capillary electrophoresis mass spectrometry (SPE-CE-MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild-type (wt) and HD (R6/1) mice of different ages (8, 12, and 30 weeks), were analyzed by C18-SPE-CE-MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR-ALS) was applied to the multiple full scan MS datasets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow-up the HD progression. The intracellular signaling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors, and changed expression of neurotransmitters. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. ; Laura Pont acknowledges the Spanish Ministry of Economy and Competitiveness for a FPI fellowship. This study was supported by a grant from the Spanish Ministry of Education and Science (CTQ2011-27130). Part of the study was supported by the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 320737. We also thank Josep Maria Marimón for the blood sample collection. ; Peer reviewed