Estudio de la cinética de digestión de la grasa de la leche bajo distintas condiciones gastrointestinales simuladas

[ES] La insuficiencia pancreática es una manifestación clínica que se caracteriza por la incapacidad del páncreas de liberar suficiente enzima pancreática al intestino delgado, necesaria para digerir los nutrientes intraluminares. La falta de enzimas digestivas lleva a la dificultad en la absorción de nutrientes, que conduce a la desnutrición y la falta de crecimiento y desarrollo, debido a la pérdida de calorías. El tratamiento habitual en personas que padecen esta enfermedad consiste en la administración por vía oral de enzimas pancreáticas que presentan actividades proteasa, lipasa y amilasa. No obstante, existen una serie de factores relativos al alimento (matriz, tipo de grasa, etc.), así al entorno digestivo (pH intestinal, concentración biliar entre otros), que pueden influir en la digestibilidad de los nutrientes. Por ello y debido a las limitaciones de este tratamiento en términos de eficacia, se propuso el presente trabajo con la finalidad de profundizar en el conocimiento del funcionamiento de las enzimas pancreáticas en diferentes condiciones intestinales simuladas. En un primer momento se procedió a evaluar la actividad lipolítica del suplemento enzimático de pancreatina (SEP), utilizado habitualmente en el tratamiento de la insuficiencia pancreática, en diferentes condiciones intestinales de pH (6, 7 y 8) y concentración biliar (1, 10 y 20). Los resultados obtenidos se compararon con los resultados de lipólisis obtenidos a partir de disoluciones de pancreatina porcina y lipasa: colipasa (10:1). Posteriormente, se utilizó un modelo de digestión in vitro para estudiar y caracterizar la cinética de la reacción de lipólisis de la grasa de leche. Para ello se simularon diferentes condiciones de pH (6, 7 y 8) y concentración biliar (1, 5 y 10 mM) empleando una concentración conocida de suplemento de pancreatina (8,27 UL/mL). De esta forma, a partir de la información de las constantes cinéticas calculadas, se obtuvo la relación enzima: sustrato que maximiza la eficiencia catalítica de la enzima. Los resultados mostraron la influencia del efecto del pH y la concentración biliar en la actividad de las disoluciones enzimáticas (SEP, pancreatina porcina y lipasa: colipasa). La actividad aumentó tanto al incrementar el pH de 6 a 8, como al aumentar la concentración de bilis alcanzando un máximo a 10 mM. Por otro lado, los resultados de cinética mostraron que el aumento del pH y concentración biliar tiende a aumentar la velocidad de reacción (aumenta rm) así como la afinidad de la enzima por el sustrato (disminuye km). Finalmente, las relaciones E/S que saturan a la enzima mostraron que para aquellas condiciones duodenales favorables (pH 7-8 y 10 mM), la enzima es capaz de alcanzar valores máximos de velocidad con bajas cantidades de sustrato. De esta forma, conociendo la relación E/S para la cual se alcanza la velocidad máxima, será posible calcular la dosis óptima de enzima para lograr la máxima eficiencia lipólítica.[EN] Pancreatic insufficiency is a clinical manifestation characterized by the in-ability of the pancreas to release enough pancreatic enzyme into the small intestine, necessary to digest intraluminal nutrients. The lack of digestive enzymes leads to the difficulty to absorb nutrients, which drives to malnutrition and lack of growth and development, due to the loss of calories. The habitual treatment of these patients involves oral administration of pancreatic enzymes with protease, lipase and amylase activities. However, there are a number of food related factors (matrix, type of fat, etc.) and digestive environment (intestinal pH, bile concentration, among others), which will influence the digestibility of nutrients. Therefore, and due to the limitations of this treatment in terms of efficacy, this study was proposed in order to deepen the understanding of the functioning of pancreatic enzymes under different simulated intestinal conditions. At first study was addressed to evaluate the lipolytic activity of the pancreatic enzyme supplement (PES), commonly used in the treatment of pancreatic insufficiency, under different intestinal conditions of pH (6, 7 and 8) and bile concentrations (1, 10 and 20 mM). The obtained results were compared to those obtained with solutions of porcine pancreatin and lipase: colipase (10: 1). Subsequently, an in vitro digestion model was used to study and characterize the kinetics of the lipolysis reaction of milkfat. For this, different pH conditions (6, 7 and 8) and bile concentrations (1, 5 and 10 mM) were simulated, using a fixed concentration pancreatic supplement (8.27 UL / mL). Thus, from the information of the calculated kinetic constants, it was possible to obtain the enzyme: substrate ratio that maximizes the catalytic efficiency of the enzyme. The results illustrate the influence of the pH and bile concentration in the activity of the enzyme solutions (SEP, porcine pancreatin and lipase: colipase). The activity increased by increasing both the pH from 6 to 8, as well as by increasing the bile concentration, from 1 to 10 mM. Moreover, the results showed that an increase in pH and bile concentration tends to increase the reaction rate (increases rm) as well as the affinity of the enzyme for the substrate (decreases km). Finally, relations E/S saturating the enzyme showed that, at favorable duodenal conditions (pH 7, 8 and 10 mM) the enzyme is able to reach a maximum speed with low amounts of substrate. Thus, knowing the E/S at which the maximum speed is reached, it will be possible to calculate the optimal enzyme dose to achieve the maximum lipolytic efficiency.Villa Consuegra, S. (2016). Estudio de la cinética de digestión de la grasa de la leche bajo distintas condiciones gastrointestinales simuladas. http://hdl.handle.net/10251/68953.TFG

On the "CRISPR" of the wave: Development of a new CRISPR-Cas9 application.

The recent emergence of the prokaryotic CRISPR-Cas9 system provides the opportunity for efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA (1). Apart from its use in genome editing, CRISPR-Cas9 system can be used as a programmable RNA-dependent DNA-binding protein by using a catalytically inactive Cas9 protein (dCas9) (2). Precisely recruiting of dCas9 to desired regions of the genome offers numerous applications such as fusion of dCas9 to transcriptional effector or repressor domains, upregulating or downregulating the transcription of endogenous target genes (3). On the other hand, dCas9 can be fused to a fluorescent protein allowing the visualization of endogenous genomic elements in living cells (4). Recently, this system has been implemented in the fission yeast Schizosaccharomyces pombe, a potent genetic model organism. Genome editing in S. pombe with CRISPR/Cas9 involves the manual identification of unique PAM and spacer sequences (sgRNA) and cloning of these sequences into an expression plasmid (5).This project proposes the validation of a novel idea that can represent a valuable tool in the study of the location and dynamics of genomic regions of interest. To achieve that, the new-fangled system combines the Cas9 nuclease with the bi-molecular fluorescent complementation technique (BiMFC) in the yeast Schizosaccharomyces pombe. The catalytically inactive Cas9 nuclease (dCas9 from S. pyogenes harbouring D10A and A840A substitutions; Addgene #46920) is fused to the yellow fluorescent protein (YFP), both the whole protein and the N-terminal and C-terminal ends. The resulting protein is directed to the centromeric regions of S. pombe through the introduction of sgRNAs specific to those regions into plasmids derivate from the S. pombe plasmid pMZ376 (Addgene #74213). On the other hand, the protein Cnp1, a variant of the H3 histone that binds to the centromeres, is fused to the cherry protein and the two halves of the YFP. As a result, we will be able to determine if there is a colocalization between the YFP signal of dCas9 and the red signal of the cherry protein fused to Cnp1. In the case of YFP halves, the colocalization of the dCas9 and Cnp1 will allow to recover the full structure of the yellow fluorescent protein, resulting in the emission of yellow fluorescence. The development of this system allows an accurate study of the DNA-protein interactions at cell level

Aurora B kinase erases monopolar microtubule-kinetochore arrays at the meiosis I-II transition

Summary: During meiosis, faithful chromosome segregation requires monopolar spindle microtubule-kinetochore arrays in MI to segregate homologous chromosomes, but bipolar in MII to segregate sister chromatids. Using fission yeasts, we found that the universal Aurora B kinase localizes to kinetochores in metaphase I and in the mid-spindle during anaphase I, as in mitosis; but in the absence of an intervening S phase, the importin α Imp1 propitiates its release from the spindle midzone to re-localize at kinetochores during meiotic interkinesis. We show that “error-correction” activity of kinetochore re-localized Aurora B becomes essential to erase monopolar arrangements from anaphase I, a prerequisite to satisfy the spindle assembly checkpoint (SAC) and to generate proper bipolar arrays at the onset of MII. This microtubule-kinetochore resetting activity of Aurora B at the MI-MII transition is required to prevent chromosome missegregation in meiosis II, a type of error often associated with birth defects and infertility in humans

Mutational analysis of N-Ethyl-N-Nitrosourea (ENU) in the fission yeast Schizosaccharomyces pombe

Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe. As opposed to many other alkylating agents, ENU only induces an SN1-type reaction with a low s constant (s = 0.26), attacking preferentially O2 and O4 in thymine and O6 deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields.This work was supported by Grupos Emergentes UPO grant APP2D1011 to V.A.T. and BFU2016-77297 grant from the Spanish MINECO to J.J. R.H.M. is supported by FPU grant FPU16/04283 from the Spanish MECD. S.V.C is supported by FPU grant FPU18/04507 from the Spanish MEFP.Peer reviewe