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The brain is particularly sensitive to folate metabolic disturbances, because methyl groups are critical for brain functions. This study aimed to investigate the effects of different dietary levels of folic acid (FA) on postnatal cerebellar morphology, including the architecture and organisation of the various layers. A total of forty male OFA rats (a Sprague–Dawley strain), 5 weeks old, were classified into the following four dietary groups: FA deficient (0 mg/kg FA); FA supplemented (8 mg/kg FA); FA supra-supplemented (40 mg/kg FA); and control (2 mg/kg FA) (all n 10 per group). Rats were fed ad libitum for 30 d. The cerebellum was quickly removed and processed for histological and immunohistochemical analysis. Slides were immunostained for glial fibrillary acidic protein (to label Bergmann glia), calbindin (to label Purkinje cells) and NeuN (to label post-mitotic neurons). Microscopic analysis revealed two types of defect: partial disappearance of fissures and/or neuronal ectopia, primarily in supra-supplemented animals (incidence of 80 %, P≤0·01), but also in deficient and supplemented groups (incidence of 40 %, P≤0·05), compared with control animals. The primary fissure was predominantly affected, sometimes accompanied by defects in the secondary fissure. Our findings show that growing rats fed an FA-modified diet, including both deficient and supplemented diets, have an increased risk of disturbances in cerebellar corticogenesis. Defects caused by these diets may have functional consequences in later life. The present study is the first to demonstrate that cerebellar morphological defects can arise from deficient, as well as high, FA levels in the diet., Ministerio de Ciencia, Innovación y Universidades (España), Depto. de Anatomía y Embriología, Unidad Docente de Anatomía y Embriología, Fac. de Medicina, Fac. de Óptica y Optometría, TRUE, pub

En los últimos años, se han descrito numerosas experiencias que detallan los efectos beneficiosos del uso de La metodología flipped classroom (clase al revés, aula invertida o flipped learning model) en la enseñanza universitaria. Por ello, un grupo de profesores de la Facultad de Medicina (Universidad Complutense de Madrid, UCM) y un médico del Servicio de Medicina Intensiva del Hospital Clínico San Carlos diseñaron y utilizaron distintos mini-vídeos como material audiovisual en la docencia de algunos temas de la asignatura de Bioquímica Humana mediante el enfoque pedagógico de la clase invertida. Tras su uso en el aula, los estudiantes manifestaron un alto grado de satisfacción por la incorporación de esta metodología en los seminarios que se imparten en esta asignatura del Grado de Medicina.

Craniofacial development requires extremely fine-tuned developmental coordination of multiple specialized tissues. It has been evidenced that a folate deficiency (vitamin B9), or its synthetic form, folic acid (FA), in maternal diet could trigger multiple craniofacial malformations as oral clefts, tongue, or mandible abnormalities. In this study, a folic acid-deficient (FAD) diet was administered to eight-week-old C57/BL/6J female mouse for 2–16 weeks. The head symmetry, palate and nasal region were studied in 24 control and 260 experimental fetuses. Our results showed a significant reduction in the mean number of fetuses per litter according to maternal weeks on FAD diet (p < 0.01). Fetuses were affected by cleft palate (3.8%) as well as other severe congenital abnormalities, for the first time related to maternal FAD diet, as head asymmetries (4.6%), high arched palate (3.5%), nasal septum malformed (7.3%), nasopharynx duct shape (15%), and cilia and epithelium abnormalities (11.2% and 5.8%). Dysmorphologies of the nasal region were the most frequent, appearing at just four weeks following a maternal FAD diet. This is the first time that nasal region development is experimentally related to this vitamin deficiency. In conclusion, our report offers novel discoveries about the importance of maternal folate intake on midface craniofacial development of the embryos. Moreover, the longer the deficit lasts, the more serious the consequent effects appear to be., Instituto de Salud Carlos III (ISCIII), Ministerio de Sanidad, CEU-Banco Santander, Depto. de Anatomía y Embriología, Depto. de Medicina, Fac. de Medicina, TRUE, pub

Background: Epicardial adipose tissue (EAT) is a visceral fat depot with unique anatomic, biomolecular and genetic features. Due to its proximity to the coronary arteries and myocardium, dysfunctional EAT may contribute to the development and progression of cardiovascular and metabolic-related adiposity-based chronic diseases. The aim of this work was to describe, by morphological techniques, the early origin of EAT. Methods: EAT adipogenesis was studied in 41 embryos from 32 gestational days (GD) to 8 gestational weeks (GW) and in 23 fetuses until full term (from 9 to 36 GW). Results: This process comprises five stages. Stage 1 appears as mesenchyme at 33–35 GD. Stage 2 is characterized by angiogenesis at 42–45 GD. Stage 3 covers up to 34 GW with the appearance of small fibers in the extracellular matrix. Stage 4 is visible around the coronary arteries, as multilocular adipocytes in primitive fat lobules, and Stage 5 is present with unilocular adipocytes in the definitive fat lobules. EAT precursor tissue appears as early as the end of the first gestational month in the atrioventricular grooves. Unilocular adipocytes appear at the eighth gestational month. Conclusions: Due to its early origin, plasticity and clinical implications, factors such as maternal health and nutrition might influence EAT early development in consequence., Ministerio de Ciencia e Innovación (MICINN)/FEDER, Universidad Complutense de Madrid, Comunidad Autónoma de la Región de Murcia/Fundación Séneca, Depto. de Medicina, Sección Deptal. de Biología Celular (Medicina), Sección Deptal. de Bioquímica y Biología Molecular (Medicina), Fac. de Medicina, TRUE, pub

[Introduction]: Age-related hearing loss (ARHL) is a sensory impairment, with a dramatic increase in its incidence, which is caused by genetic and environmental factors such as noise and ototoxic drugs. Recent studies correlated ARHL to elevated plasma homocysteine (Hcy) by folate deficiency, suggesting that reduction of Hcy levels by folate supplementation could potentially ameliorate ARHL. Hyperhomocysteinemia (HHcy), a status that contributes to ARHL, may also arise from malfunction of Hcy remethylation by betaine homocysteine S-methyltransferases (BHMTs) and methionine synthase in the methionine cycle. The expression and/or activity of these enzymes may be altered by ototoxic drugs, including paracetamol (APAP)., [Objective]: To determine the effect of APAP in cochlear morphology and function of control and Bhmt-/- mice, and to analyze putative preventative effects of folic acid (FA) supplementation., [Materials and Methods]: Two-month-old Bhmt-/- mice (n=47), with greater dependence on folate metabolism for Hcy remethylation, and Bhmt+/+ mice (n=42) were fed control or FA supplemented diets for 30 days. The last day APAP (250 mg/kg) or placebo were injected intraperitoneally. Hearing was evaluated by recording auditory brainstem responses (ABR) at the beginning of the experiment and after treatments. Picrosirius red staining was used for evaluation of the cochlear lateral wall cytoarchitecture. Plasma and hepatic metabolite levels were determined by HPLC or on Spinlab 100® autoanalyzer., [Results]: Loss of Bhmt expression induced HHcy, but an impact on hearing acuity was not observed. Acute APAP administration did not induce ABR threshold shifts. However, following ototoxic treatment, changes of 5-17% in the areas of the stria vascularis and spiral ligament were detected between Bhmt-/- mice under different dietary treatments; cochlear structures of Bhmt-/- mice receiving APAP plus FA supplementation resemble those of the control group. APAP increases susceptibility to ototoxic damage in the presence of HHcy., [Discussion]: BHMT plays a central role in cochlear methionine metabolism. FA supplementation modulates Hcy levels, contributing to a proper remethylation status that prevents HL.

It is widely accepted that maternal folic acid (FA) deficiency during pregnancy is a risk factor for abnormal development. The tongue, with multiple genes working together in a coordinated cascade in time and place, has emerged as a target organ for testing the effect of FA during development. A FA-deficient (FAD) diet was administered to eight-week-old C57/BL/6J mouse females for 2–16 weeks. Pregnant dams were sacrificed at gestational day 17 (E17). The tongues and heads of 15 control and 210 experimental fetuses were studied. In the tongues, the maximum width, base width, height and area were compared with width, height and area of the head. All measurements decreased from 10% to 38% with increasing number of weeks on maternal FAD diet. Decreased head and tongue areas showed a harmonic reduction (Spearman nonparametric correlation, Rho = 0.802) with respect to weeks on a maternal FAD diet. Tongue congenital abnormalities showed a 10.9% prevalence, divided in aglossia (3.3%) and microglossia (7.6%), always accompanied by agnathia (5.6%) or micrognathia (5.2%). This is the first time that tongue alterations have been related experimentally to maternal FAD diet in mice. We propose that the tongue should be included in the list of FA-sensitive birth defect organs due to its relevance in several key food and nutrition processes., Ministerio de Sanidad, Instituto de Salud Carlos III/Ministerio de Ciencia e Innovación (MICCIN), Universidad Complutense de Madrid/Banco de Santander, Depto. de Anatomía y Embriología, Fac. de Medicina, TRUE, pub

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with D-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.

The methionine cycle has been mainly studied in the liver, an organ that presents with specific isoenzymes and proteins of this pathway. Classical knowledge ascribed enzymes of this cycle to the cytoplasm and suggested that metabolites could be transported to other subcellular locations as required. This hypothesis was reinforced by the discovery of S-adenosylmethionine (AdoMet) transporters in the mitochondria, but later challenged by the identification of glycine N-methyltransferase (GNMT), S-adenosylhomocysteine hydrolase (SAHH) and methionine adenosyltransferases (MATs) in the nucleus. As a result, new questions emerged regarding their role in that compartment and the putative link between anomalous subcellular localization and disease. In the last years, we have used several approaches trying to provide evidences that answer these questions, first by analyzing models of acute liver failure (D-galactosamine and acetaminophen intoxications), and second by studying the impact of changes in nutrient concentrations (methionine). The results obtained to date indicate that there is an opposite regulation for nuclear and cytoplasmic protein levels, the later correlating with expression changes. Methionine restriction induces expression of the three Mat genes in several cell lines, and cytoplasmic protein levels follow this expression pattern. Liver-specific genes (Mat1a, Bhmt, Gnmt) decrease their expression in hepatic intoxications, whereas the mRNA steady-state levels of the rest increase, the result being the well-known Mat1a/Mat2a switch. Total AdoMet levels decrease in hepatic intoxications, while nuclear accumulation of MAT¿1 (encoded by Mat1a), GNMT and SAHH is observed. This nucleo/cytoplasmic change in MAT¿1 localization correlates with enhanced nuclear levels of the tetrameric isoenzyme (MAT I with intermediate Vmax) and of specific epigenetic modifications involved in repression. Subcellular distribution is controlled by the ratio of glutathione species, acc

The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Methionine adenosyltransferase II is ubiquitously expressed, whereas isoenzymes I (homotetramer) and III (homodimer) are considered the hepatic enzymes. In this work, we identified methionine adenosyltransferase I/III in most rat tissues both in the cytoplasm and the nucleus. Nuclear localization was the preferred distribution observed in extrahepatic tissues, where the protein colocalizes with nuclear matrix markers. A battery of mutants used in several cell lines to decipher the determinants involved in methionine adenosyltransferase subcellular localization demonstrated, by confocal microscopy and subcellular fractionation, the presence of two partially overlapping areas at the C-terminal end of the protein involved both in cytoplasmic retention and nuclear localization. Immunoprecipitation of coexpressed FLAG and EGFP fusions and gel filtration chromatography allowed detection of tetramers and monomers in nuclear fractions that also exhibited S-adenosylmethionine synthesis. Neither nuclear localization nor matrix binding required activity, as demonstrated with the inactive F251D mutant. Nuclear accumulation of the active enzyme only correlated with histone H3K27 trimethylation among the epigenetic modifications evaluated, therefore pointing to the necessity of methionine adenosyltransferase I/III to guarantee the supply of S-adenosylmethionine for specific methylations. However, nuclear monomers may exhibit additional roles.