Identification and functional validation of genomic boundaries in mammals

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 30-06-2014Eukaryotic genomes are divided into expression domains, which contain DNA coding sequences together with all the regulatory elements needed for their correct spatio-­‐ temporal expression pattern. Genomic boundaries, also known as insulators, flank these domains preventing undesirable crosstalk between the regulatory elements of neighboring domains. They employ various mechanisms and thus, are functionally rather than structurally defined. For this reason, in an attempt to find boundaries in a genome-­‐ wide unbiased fashion in mammals, we focused on identifying those loci where the presence of boundary function would be required to satisfy a biological need. For example, we hypothesized that adjacent genes with opposite expression patterns would need to be separated by boundaries to maintain the independency of their different expression domains. Also, boundaries could be found partitioning the chromatin into inactive heterochromatic and active euchromatic domains, impeding the deleterious effects the spread of the former would have on the latter. Finally, boundaries could also bracket clusters of co-­‐expressed genes to ensure their co-­‐regulation and co-­‐expression. Different algorithms, based on the analysis of gene expression data, were developed in order to explore these scenarios. The resulting evolutionarily conserved non-­‐coding putative insulator sequences were functionally validated using a number of assays. Their enhancer-­‐ blocking properties were evaluated in vitro in human cells in culture, and then in vivo by using transgenic zebrafish. Additionally, one of the most powerful elements was further tested for its ability to protect from chromosomal position effects in transgenic mice. The description and characterization of new genomic boundaries would shed some light into the way mammalian genomes are organized, as well as expand the repertoire of genetic tools that can be incorporated in heterologous constructs to improve the gene transfer technologies by preventing chromosomal position effects.Los genomas de eucariotas están divididos en dominios de expresión, que se definen como aquellas porciones del genoma que contienen uno o varios genes y todos los elementos reguladores necesarios para que que se expresen de acuerdo con un patrón espacio-­‐temporal concreto. Los aisladores genómicos, también llamados insulators, flanquean estos dominios y los protegen de la influencia no deseada de los elementos reguladores contenidos en los dominios vecinos. Existen diversos mechanismos de aislamiento, por lo que los insulators no se definen por una secuencia de ADN concreta, sino porque comparten una misma función. Así, para encontrar aisladores en el genoma de mamíferos de una forma no sesgada, nos propusimos identificar aquellas posiciones del genoma donde se requiere la presencia de función aisladora para satisfacer un problema biológico. Por ejemplo, genes adyacentes con perfiles de expresión completamente distintos deberían estar separados por aisladores que mantuviesen dominios de expresión independientes. Asimismo, cabe esperar la presencia de aisladores entre dominios silentes de heterocromatina y dominios activos de eucromatina. Aquí, impedirían los efectos perjudiciales que el avance de los primeros tendrían sobre los segundos. Finalmente, también podrían encontrarse aisladores flanqueando grupos de genes co-­‐expresados para asegurar su co-­‐regulación y, por tanto, co-­‐expresión. Basándonos en estos escenarios, se desarrollaron diversos algoritmos que usaban datos de expresión génica para predecir la presencia de aisladores. Como resultado de estos algoritmos, se obtuvo una serie de secuencias conservadas evolutivamente y no codificantes que se validaron funcionalmente empleando varios tests. La capacidad de bloqueo de enhancers se evaluó mediante ensayos in vitro en células humanas en cultivo primero, y luego in vivo mediante el uso de peces cebra transgénicos. Además, se analizó la capacidad de uno de los elementos más potentes para proteger de efectos de posición cromosomales en ratones transgénicos. La descripción y caracterización de nuevos aisladores genómicos no sólo sirve para entender mejor cómo se organizan los genomas de mamíferos. También es útil para ampliar el abanico de herramientas disponibles que se pueden usar en construcciones heterólogas para bloquear los efectos de posición cromosomales que se dan comúnmente en experimentos de transferencia genética

Beverages based on second quality citrus fruits and maqui berry, a source of bioactive (Poly)phenols: Sorting out urine metabolites upon a longitudinal study

The intake of sugar-sweetened beverages has been associated with an augmented prevalence of metabolic diseases, namely, obesity, type II diabetes, and metabolic syndrome. On the other hand, nowadays, it is broadly accepted that foods and beverages rich in (poly)phenols could contribute to reducing the incidence of these pathologies. In this sense, the objective of the work was to revalue second quality citrus fruits for the development of new beverages, rich in anthocyanins and flavanones (maqui berry and second qualities citrus-based), and evaluate the influence of alternative sweeteners (sucralose, sucrose, or stevia), regarding the bioaccessibility and bioavailability of these bioactive compounds in the frame of a chronic (longitudinal) intervention. To fulfill this objective, a longitudinal study of the urinary excretion of anthocyanins and flavanones, after 2-months of ingestion of the developed maqui-citrus beverage, by 138 volunteers (n = 46 per beverage) and the analysis of the resulting phenolic metabolites by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-ESI-QqQ-MS/MS) was carried out. As major results, the bioavailable metabolites of caffeic acid (CA), catechol (CAT), 3,4-di-hydroxyphenylacetic acid (DHPAA), eriodictyol (E), homoeriodictyol (HE), hippuric acid (HA), naringenin (N), trans-ferulic acid (TFA), 2,4,6-tri-hydroxybenzaldehyde (THBA), trans-isoferulic acid (TIFA), and vanillic acid (VA) were detected. Accordingly, significantly different bioavailability was dependent on the sweetener used, allowing proposing stevia and, to a lower extent, sucralose, as valuable alternatives to sucrose.This research was funded by the Spanish MINECO, grant number (AGL2016-75332-C2-1-R AEI/FEDER UE and PID2019-104212RB-I00/AEI/10.13039/501100011033). VA was supported by a FPI grant (BES-2017-082424)

Sweetener influences plasma concentration of flavonoids in humans after an acute intake of a new (poly)phenol-rich beverage

The overconsumption of sucrose is closely related to sugar-sweetened beverages and one of the main factors associated with the increase of metabolic diseases, such as type 2 diabetes, obesity, and insulin resistance. So, the addition of alternative sweeteners to new fruit-based drinks could contribute to minimizing the incidence or severity of these pathologies. Nevertheless, current knowledge on the influence of these additives on the bioactive compounds present in these beverages is still scarce.new-onset hypertension, but few data were published in Asian. We aimed to investigate the association of lipid profiles with new-onset hypertension in a Chinese community-based non-hypertensive cohort without lipid-lowering treatment (n = 1802). Hence, to contribute to the understanding of this issue, the plasma concentration of phenolic compounds (anthocyanins and flavanones), after the ingestion of a new maqui-citrus-based beverage, supplemented with sucrose (natural high caloric), stevia (natural non-caloric), or sucralose (artificial non-caloric), was evaluated as evidence of their intestinal absorption and metabolism previous to renal excretion. The beverages were ingested by volunteers (n = 20) and the resulting phenolic metabolites in plasma were analyzed by UHPLC-ESI-MS/MS. A total of 13 metabolites were detected: caffeic acid sulfate, caffeic acid glucuronide, 3,4-dihydroxyfenylacetic, 3,4-dihydroxyfenylacetic sulfate. 3,4-dihydroxyfenylacetic acid di-sulfate, 3,4-dihydroxyfenylacetic di-glucuronide, 3,4-dihydroxyfenylacetic glucuronide-sulfate, trans-ferulic acid glucuronide, naringenin glucuronide, vanillic acid, vanillic acid sulfate, vanillic acid glucuronide-sulfate, and vanillic acid di-glucuronide, being recorded their maximum concentration after 30–60 min. In general, sucralose provided the greatest absorption value for most of these metabolites, followed by stevia. Due to this, the present study proposes sucralose and stevia (non-caloric sweeteners) as valuable alternatives to sucrose (high caloric sweetener), to avoid the augmented risk of several metabolic disorders.This work was funded by the Spanish MINECO through Research Project AGL2016-75332-C2-1-R (AEI/FEDER, UE). V.A. was supported by FPI (BES-2017-079754) grant of the Fellowship Program from the Spanish Ministry of Science, Innovation, and Universities (MICIU)

The use of alternative sweeteners (sucralose and stevia) in healthy soft-drink beverages, enhances the bioavailability of polyphenols relative to the classical caloric sucrose

The comparison of non-caloric sweeteners (stevia and sucralose) and sucrose, on the plasma concentration and cumulative effects of phenolic compounds, was achieved. A long-term intervention, consisting of the daily intake of 330 mL of healthy citrus-maqui soft drinks, for 60 days, by 138 healthy overweight adults, was followed. A total of 24 bioavailable metabolites derived from caffeic acid, 3,4-di-hydroxyphenylacetic acid, eriodictyol, homoeriodictyol, hippuric acid, naringenin, 2,4,6-tri-hydroxybenzaldehyde, and vanillic acid were detected in peripheral blood plasma. A similar augment of bioactive compounds in plasma concentrations were found for the three beverages, in the range 12.3% (day 0)- 85.3% (day 60), depending on the analyte considered. Due to this, the present study highlights sucralose and stevia as valuable alternatives to sucrose, providing and non-significantly different plasma concentration and cumulative effect in the plasma, thus contributing to prevent a diversity of metabolic disorders and health constraints.This research was funded by the Spanish MINECO, grant number (AGL2016-75332-C2-1-R AEI / FEDER UE and PID2019-104212RB-I00/AEI/ 10.13039 / 501100011033). VA was supported by an FPI grant (BES-2017-082424). Informed consent was obtained from all subjects involved in the study. The authors declare no conflict of interest

Effect of a new rich in bioactive compounds beverage for energetic metabolism modulation in overweight adults

[SPA] En las últimas décadas se ha demostrado una relación positiva entre el consumo de bebidas azucaradas y el riesgo de obesidad, diabetes y enfermedades cardiovasculares. Para reducir el contenido en azúcares de los alimentos manteniendo un sabor agradable, se han propuesto nuevas opciones usando edulcorantes, aunque se cuestiona si dichos aditivos contribuyen a una mayor ingesta calórica o si producen efectos negativos a largo plazo. Además, para fomentar el consumo de frutas y hortalizas se están desarrollando nuevas formulaciones que aumenten la vida útil de las frutas frescas, preserven los nutrientes y reduzcan el contenido energético de los zumos de frutas. La investigación comenzará con el desarrollo de bebidas a base cítricos y maqui, ricas en compuestos bioactivos (poli) fenólicos, que incluyan diferentes edulcorantes no nutritivos tanto de tipo natural (stevia) como artificial (sucralosa). El proyecto abarcará desde el desarrollo tecnológico alimentario hasta la nutrición y prevención de enfermedades ya que, realizando estudios de intervención con voluntarios sanos con sobrepeso, se analizarán los efectos de estas bebidas en los mecanismos subyacentes en la hiperglicemia postprandial y se determinarán los metabolitos en circulación responsables de la evidencia observada. [ENG] In recent decades, a positive relationship has been demonstrated between the consumption of sugary drinks and the risk of obesity, diabetes and cardiovascular diseases. To reduce the sugar content of foods while maintaining a pleasant taste, new options have been proposed using sweeteners, although it is questioned whether these additives contribute to a higher caloric intake or if they produce negative long-term effects. In addition, to promote the consumption of fruits and vegetables, new formulations that increase the shelf life of fresh fruits, preserve nutrients and reduce the energy content of fruit juices are being developed. The research will begin with the development of citrus-based and maqui-based beverages, rich in bioactive (poly) phenolic compounds, which include different non-nutritive sweeteners, both natural (stevia) and artificial (sucralose). The project will cover everything from food technological development to nutrition and disease prevention. The effects of these drinks on the underlying mechanisms in postprandial hyperglycemia will be analyzed and metabolites in circulation will be determined responsible for the observed evidence by intervention studies with healthy overweight volunteers.Colaboración y asesoramiento técnico a escala semi-industrial de la Dra Martí (UMH) y financiación del proyecto coordinado AGL2016-75332-C2-1-R y AGL2016-75332-C2-2

Anthocyanin metabolites in human urine after the intake of new functional beverages

Sugar intake abuse is directly related with the increase of metabolic diseases such as type 2 diabetes, obesity, and insulin resistance. Along this line, the development of new beverages using alternative sweeteners could help with combatting the pathophysiological disorders associated to the consumption of sugar. To provide evidence on this issue, in the present work, the bioavailability of anthocyanins was evaluated after the acute ingestion of a new maqui-citrus-based functional beverage rich in polyphenols, and supplemented with a range of sweeteners including sucrose (natural high caloric), stevia (natural non-caloric), and sucralose (artificial non-caloric), as an approach that would allow reducing the intake of sugars while providing bioactive phenolic compounds (anthocyanins). This approach allowed the evaluation of the maximum absorption and the diversity of metabolites excreted through urine. The beverages created were ingested by volunteers (n = 20) and the resulting anthocyanin metabolites in their urine were analyzed by UHPLC-ESI-MS/MS. A total of 29 degradation metabolites were detected: Caffeic acid, catechol, 3,4-dihidroxifenilacetic acid, hippuric acid, trans-ferulic acid, 2,4,6-trihydroxybenzaldehyde, trans-isoferulic acid, and vanillic acid derivatives, where peak concentrations were attained at 3.5 h after beverage intake. Sucralose was the sweetener that provided a higher bioavailability for most compounds, followed by stevia. Sucrose did not provide a remarkably higher bioavailability of any compounds in comparison with sucralose or stevia. The results propose two sweetener alternatives (sucralose and stevia) to sucrose, an overused high calorie sweetener that promotes some metabolic diseasesThis research was funded by the Spanish MINECO, grant number AGL2016-75332-C2-1-R and AGL-2016-75332-C2-2-R. VA was funded by a FPI grant (BES-2017-079754) of the Spanish MINECO. The APC was funded by the projects AGL2016-75332-C2-1-

Stevia vs. Sucrose: Influence on the phytochemical content of a citrus–maqui beverage—A shelf life stud

The consumption of sugar-sweetened beverages has been related with the risk of cardiovascular diseases and other pathophysiological situations, such as obesity or diabetes mellitus. Given the increasing awareness on this fact, food industries are developing new products to reduce the amount of added sugar in development of food products development. Accordingly, in the present work, new functional beverages, constituting a dietary source of bioactive phenolics and supplemented with stevia or sucrose, were designed in order to study the influence of the sweetener during processing and shelf-life. This study is of critical for the informed selection of the sweetener based on its e_ect on the final phytochemical profile of beverages, especially taking into consideration that there are no previous studies on Stevia rebaudiana. Physicochemical features and phytochemical composition, as well as stability of the di_erent beverages concerning these parameters, were evaluated for 90 days during storage under di_erent conditions (refrigeration (4 _C) and room temperatura (25 _C) under light or darkness conditions). Physicochemical parameters (pH, titratable acidity, total soluble solids, and color) did not display statistically significant di_erences between beverages. Storage temperature was the greatest determinant a_ecting the stability of all the analyzed bioactive compounds (vitamin C, anthocyanins, and flavanones). The main di_erence between sweeteners was observed in flavanones, which exhibited a higher loss during storage under day light conditions when stevia was added instead of sucrose. In addition, the juices’ colors were rather stable, keeping a reddish coloration and natural appearance throughout the shelf life. Hence, stevia could be considered as an alternative sweetener by the beverage industry.This work was funded by the Spanish MINECO through Research Project AGL2016-75332-C2-1-R (AEI/FEDER, UE). F.J.S. and V.A. were supported by FPU (FPU18/00332) and FPI (BES-2017-079754) grant of the Fellowship Program from the Spanish Ministry of Science, Innovation, and Universities (MICIU), respectively. We also thank Nuria Martí for the technical advice and processing at semi-industrial scale

Unravelling the function of Myf5 in the developing limb musculature

Motivation: Myogenesis is a complex process controlled by different networks, depending on the origin of different muscles, and that’s why there are different types of muscle distrophies. However, the myogenic cascade is always regulated by the same Myogenic Regulatory Factors (MRFs): Myf5, Mrf4, MyoD and MyoG. These transcription factors bind DNA and activate the expression of specific genes in particular progenitor cells that will give rise to the different muscles in the adult body. The MRFs’ cascade is initiated by Myf5, the first MRF to be expressed in the embryo. We and others have extensively studied the complex transcriptional regulation of the Myf5/Mrf4 locus using transgenic mice. Nowadays, we know that there are more than 25 regulatory elements controlling the expression of Mrf4 and Myf5 in a specific time frame and in particular embryonic progenitors.The limb enhancer is located 57 kb upstream of the Myf5 transcriptional start site. This enhancer controls Myf5 expression in limbs during development. While the mechanisms involved in the spatiotemporal regulation of Myf5 have been extensively studied at single-enhancer and global regulatory levels, the function of Myf5 in different subpopulations of muscle progenitor cells is still not clear. This project focuses on the characterization of a new allele in which the limb enhancer has been removed from the genome.Methods: To unravel the function of Myf5 in the developing limb musculature we have generated a new knock-out (KO) allele with CRISPR/Cas9 in which the limb enhancer has been targeted. Then, we have prepared RNA probes for In Situ Hybridisation (ISH) of genes that are expressed in limbs and are potential targets of Myf5 in other muscle progenitors (López-Mayorga et al., unpublished data), to test if the expression patters of these genes are modified in the KO allele. We are going to study three different embryonic stages: 10.5, 11.5 and 12.5 days post-coitum (dpc) by ISH using KO and wild type (WT) embryos. This time window was chosen to maximise the probability of detecting any pattern changes before the phenotype is rescued by the activity of MyoD, as previously shown. We are also preparing total RNA of fore-limbs from KO and WT embryos at 11.5 dpc to perform microarrays, which will give us some information about the genes regulated by Myf5 direct or indirectly. Finally, we will validate the results from microarrays by ISH and qPCR

Uso de la barrera de látex en lesbianas: ¿relación con la compulsividad sexual?

Las mujeres que tienen sexo con mujeres (MSM) constituyen un grupo heterogéneo al que se ha prestado muy poca atención. Si bien representan un sector de la población de dimensiones considerables, desconocemos el tipo de prácticas sexuales que habitualmente realizan. Así como el uso que hacen de los distintos métodos de prevención –en concreto la barrera de látex. Éste era precisamente el objetivo con que nos planteamos la evaluación de 40 MSM . Resultados: A través de la aplicación de diferentes cuestionarios, constatamos una alta frecuencia de diferentes prácticas sexuales (sobre todo cunnilingus y tribadismo) tanto con parejas estables como esporádicas. Además, observamos una muy baja frecuencia de uso de la barrera de látex. Discusión: Estos resultados sugieren la necesidad de seguir investigando en un sector de la población con riesgo de contraer ciertas ITS o el VIH. Particularmente en cuanto al diseño de estrategias eficaces y adaptadas para la prevención de la actividad sexual de riesgo en este colectivo.Introduction: Women who have sex with women (WSW) are a heterogeneous group who have received minor attention. While they represent a considerable sector of population, we do not know the type of sexual behaviors that they usually practice, as well as the use of several prevention methods –in particular, latex barrier. This was the main purpose to evaluate 40 wsw. Results: Through the application of different questionnaires, we find a high frequency of different sexual practices (especially cunnilingus and tribadism) for both steady and casual partners. Moreover, we observe a lower frequency of latex barrier use. Discussion: These results support the need for further research in a sector of the population who have risk of contracting some stis or hiv. In particular, to design effective and adjusted strategies to prevent the risk sexual activity of this group.Este trabajo ha sido financiado por el proyecto de investigación del Ministerio de Ciencia e Innovación PSI 2011-27992/11 I 384 y de la Universitat Jaume I de Castellón P1.B2012-49