Role of microRNAs in Duchenne Muscular Dystrophy and in muscle differentiation

Duchenne Muscular Dystrophy (DMD), caused by mutations in the dystrophin gene, is one of the most severe myopathies. Among different therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional mRNA. Making use of exon skipping strategy we demonstrated that in DMD, the absence of dystrophin at the sarcolemma delocalizes and downregulates Nitric Oxide Synthase (nNOS); this alters HDAC2 S-nitrosylation and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes crucial in DMD physiopathology. Namely, we identified miR-1 as regulator of the redox state of the cell through modulation of the G6PD enzyme while miR-29 controls the fibrotic process targeting extracellular matrix proteins. We also show that, at variance with other myomiRs, miR-206 and miR-31 escape from the dystrophin-nNOS control being expressed in activated satellite cells before dystrophin expression. In these cells, miR-206 contributes to muscle regeneration through repression of the satellite specific factor Pax7, while miR-31 inhibits the early expression of dystrophin by directly repressing its mRNA. Finally, in human DMD myoblasts treated with exon skipping molecules, we demonstrate that miR-31 inhibition increases dystrophin rescue representing a new approach to coadiuvate the existing therapeutic strategies. We conclude that: - the pathway activated by dystrophin/nNOS controls key miRNA circuitries increasing the robustness of the muscle differentiation programme. - specific miRNAs are induced during muscle regeneration controlling the timing of mRNA expression during myoblasts differentiation. - the inhibition of specific miRNA extression enhances dystrophin rescue obtained through exon skipping treatments

Role of microRNAs in Duchenne Muscular Dystrophy and in muscle differentiation

Duchenne Muscular Dystrophy (DMD), caused by mutations in the dystrophin gene, is one of the most severe myopathies. Among different therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional mRNA. Making use of exon skipping strategy we demonstrated that in DMD, the absence of dystrophin at the sarcolemma delocalizes and downregulates Nitric Oxide Synthase (nNOS); this alters HDAC2 S-nitrosylation and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes crucial in DMD physiopathology. Namely, we identified miR-1 as regulator of the redox state of the cell through modulation of the G6PD enzyme while miR-29 controls the fibrotic process targeting extracellular matrix proteins. We also show that, at variance with other myomiRs, miR-206 and miR-31 escape from the dystrophin-nNOS control being expressed in activated satellite cells before dystrophin expression. In these cells, miR-206 contributes to muscle regeneration through repression of the satellite specific factor Pax7, while miR-31 inhibits the early expression of dystrophin by directly repressing its mRNA. Finally, in human DMD myoblasts treated with exon skipping molecules, we demonstrate that miR-31 inhibition increases dystrophin rescue representing a new approach to coadiuvate the existing therapeutic strategies. We conclude that: - the pathway activated by dystrophin/nNOS controls key miRNA circuitries increasing the robustness of the muscle differentiation programme. - specific miRNAs are induced during muscle regeneration controlling the timing of mRNA expression during myoblasts differentiation. - the inhibition of specific miRNA extression enhances dystrophin rescue obtained through exon skipping treatments

Strengthening Farmers’ Bargaining Power in the New CAP

The food supply chain plays a substantial role in the European economy. Agricultural markets are highly interdependent and competition at different stages of the supply chain matters for the overall functioning of the entire food sector. Welfare and distributional implications of competition at any stage of the food supply chain concern several ‘weak subjects’ such as small farmers and consumers who negotiate with “strong subjects” operating in more concentrated (industrial and retailing) sectors. In order to balance power across the EU agro-food supply chain and achieve a more sustainable distribution of value-added, the main instruments featuring the new paradigm of market organisation proposed by the CAP reform are Producer Organisation, Association Producer Organisation and Inter-Branch Organisation. We employ a simple bargaining model aimed to identify the determinants of the bargaining power in the food supply chain; the model is used to assess the capability of POs to strengthen farmers’ bargaining power. The results show that, under certain conditions, the joint selling and production planning can be effective tools for rebalancing power in the agro-food system. We also concluded that requiring a minimum size for POs may improve their effectiveness. Such requirement should be calibrated on the structure of the downstream (upstream) relevant market: more consolidated buyer (seller) industries call for larger POs

Estabilidad estructural y P en fracciones de agregados en la cuenca del Aº El Divisorio (Coronel Pringles, BA)

En los últimos años la calidad del agua en el embalse Paso de las Piedras se ha visto afectada por la eutrofización, perjudicando la provisión para la población de Bahía Blanca y alrededores. Se ha señalado a la actividad agropecuaria como originaria del aumento de la concentración de fósforo (P) en el arroyo El Divisorio, que desemboca en el embalse. El objetivo de este estudio fue cuantificar las relaciones entre algunas propiedades relacionadas con el potencial de contaminación de estos suelos: la estabilidad estructural, la distribución de tamaños de agregados y las concentraciones de las formas de P en los diferentes agregados. Se seleccionaron 16 transectas en lotes de productores a lo largo del curso del arroyo. Se tomaron muestras de suelo (0-5 cm) en las posiciones loma (L), media loma (M) y bajo (B). Se determinaron los diámetros medios ponderado en seco (DMPs) y en húmedo (DMPh), calculándose el cambio en el diámetro medio ponderado (CDMP). Se determinaron los contenidos de P orgánico (Po) e inorgánico (Pi) en cada una de las posiciones y tamaños de agregados. Si bien los suelos de la cuenca mostraron buenos valores de estabilidad estructural, las fuerzas físicas provenientes de la actividad agrícola favorecieron la destrucción de los agregados menos estables encontrándose gran cantidad de material fino (<1mm). Esta fracción presentó una importante concentración de P, representando cerca del 30% del total en el suelo. La concentración del Pi fue semejante en todos los tamaños de agregados en las posiciones L y M (200-240 mg kg-1), mientras que fue significativamente superior (360-400 mg kg-1) en los bajos. Estas diferencias podrían estar originadas en texturas distintas y la menor estabilidad de los agregados en los bajos. En cuanto al Po las diferencias fueron menos marcadas, aunque evidenciaron valores más altos en la mayoría de los tamaños de agregados en la posición B. De acuerdo con estos resultados, las pérdidas podrían ser importantes aún con tormentas de poca capacidad erosiva que arrastren materiales menores, ya que estos contribuyen con sustanciales cantidades de P. El mayor cuidado deberá tenerse en los bajos, ya que tienen mayor contenido de P en todas las fracciones y menor estabilidad de los agregados.In the last years, water quality in the Paso de las Piedras reservoir has been affected by eutrophication, leading to problems in the provision of potable water to the population of Bahía Blanca city and surrounding areas. Agricultural activity has been pointed out as the possible non-point source of P contaminating the El Divisorio stream, which flows directly into the reservoir. The objective of this work was to assess some selected edaphic properties associated with the contamination potential of soils such as structural stability, aggregate distribution and concentration of organic (Po) and inorganic (Pi) P in the aggregates. Sixteen locations were selected along the stream in farmers’ fields. Soil samples from the surface 5 cm-layer were collected in the upper, intermediate and lower positions (L, M and B respectively) on each location. Structural stability was determined by the difference between dry and wet mean weight diameters. Soil Po and Pi were measured in each wet aggregate fraction. The values of structural stability were high; however, a great amount of fine material (&lt;1 mm) was present, probably as a consequence of the destruction of less stable aggregates due to agricultural mechanic forces. A large concentration of P was present in this fraction, representing around 30% of total soil P. Pi concentration was similar among all fractions in L and M (200-240 mg P kg-1) while it was significantly higher in B (360-400 mg P kg-1). These differences may be attributed to finer texture and weaker stability in the B positions. Differences among landscape positions were less evident for Po, although greater concentrations were encountered in B in most aggregate fractions. According to these results, P losses could be of significant magnitudes even at low storm intensities, because the small soil particles that are carried in runoff water contribute with great amounts of P. Management practices tending to reduce the impact of agricultural P on water quality should be aimed to reduce the risk related to B positions due to their higher P content and lower structural stability

Identification of agronomic interesting QTL in the segregating generations of a tomato second cycle hybrid

El objetivo fue detectar QTL de interés agronómico en las generaciones segregantes (F2 y retrocruzas) de un híbrido de segundo ciclo (HSC) de tomate (Solanum lycopersicum L.), obtenido entre dos líneas endocriadas recombinantes derivadas del cruzamiento inter específico S. lycopersicum cv. Caimanta x S. pimpinellifolium LA 722. La caracterización molecular se hizo por marcadores AFLP mientras que los caracteres morfológicos analizados fueron peso, diámetro, altura, contenido en sólidos solubles, acidez, color, pH, forma, dureza y vida poscosecha de los frutos. Para identificar QTL, la asociación entre bandas polimórficas y caracteres cuantitativos con variancia genética significativa se realizó por el análisis de único punto. Con seis combinaciones de cebadores, seleccionadas por detectar elevado porcentaje de polimorfismo, se obtuvo 221 bandas de las cuales 135 (61,1%) fueron polimórficas. En la F2, 29 fragmentos polimórficos siguieron la distribución mendeliana esperada, identificándose un total de 28 QTL para todos los caracteres analizados. En las retrocruzas, 15 fragmentos polimórficos siguieron una segregación mendeliana 1:1 (detectándose en 12 de ellas un comportamiento de novo) y se identificó en total 13 QTL para los caracteres contenido en sólidos solubles, altura, peso y forma del fruto. Los AFLP permitieron identificar QTL de importancia agronómica en las generaciones segregantes del HSC de tomate.The objective was to detect QTL of agronomic interest in the segregating generations F2 and backcrosses of a tomato (Solanum lycopersicum L.) second cycle hybrid (SCH) obtained between two recombinant inbred lines derived from the interspecific cross S. lycopersicum cv. Caimanta x S. pimpinellifolium LA 722. Molecular characterization was achieved by AFLP markers and agronomic traits under study were fruit weight, diameter, height, soluble solids content, acidity, color, pH, shape, firmness and postharvest life. Single point analysis was applied to detect QTL, assessing the association of AFLP polymorphic bands and quantitative traits with significant genetic variance. Amplifications with six primers combinations, selected because of detecting high polymorphism percentage, 221 AFLP bands were obtained, and 135 (61.1%) of them were polymorphic in the tomato populations analyzed. In the F2 population, 29 polymorphic fragments adjusted to the expected mendelian segregation and a total of 28 QTL were detected for all evaluated traits. In the backcross population, 15 polymorphic fragments adjusted to the expected mendelian proportion 1:1 (12 of these fragments showed a de novo pattern) and a total of 13 QTL were identified for the traits soluble solids content, height, weight and diameter. AFLP allowed to identify QTL of agronomic interest in the segregating generations of a tomato SCH.Fil: Cabodevila, Victoria Guadalupe. Consejo Nacional de Investigaciones Científicas y TécnicasFil: Cacchiarelli, Paolo. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Genética y Mejoramiento Vegetal.Fil: Pratta, Guillermo Raúl. Consejo Nacional de Investigaciones Científicas y Técnica

Sport e Fenomenologia: una teoresi pedagogica

Abstract: La fenomenologia ha determinato un ripensamento critico del rapporto tra scienza e il proprio oggetto di studio. La pedagogia, come altre scienze umane, volendo emanciparsi da un approccio tecnicistico, ha adottato gli assunti metodologici della fenomenologia per una valida rifondazione epistemologica. Si sono così delineate le basi per quella che è definita pedagogia fenomenologica. Obiettivo di questo lavoro è mostrare la validità del metodo fenomenologico in pedagogia e l’auspicabilità di sviluppare una pedagogia fenomenologica dello sport capace di comprendere le implicazioni sportive ed educative nel contesto sportivo. Abstract: Phenomenology implicated a critical reconsideration of the relationship between sciences and their own research objects. As other human sciences, pedagogy adopted methodological assumptions of phenomenology aiming to a sound epistemological refounding, willing to emancipate from a mere technical approach, In doing so, the basis for a so-called phenomenological pedagogy were defined. The aim of this paper is to show the phenomenological method effectiveness in pedagogy and to foster the possibilities to develop a phenomenological pedagogy of sport able to understand both sport and educational implications within sport environment. Parole chiave: educazione, fenomenologia, pedagogia, sport Keywords: education, pedagogy, phenomenology, spor

Single-Cell RNA Sequencing Analysis: A Step-by-Step Overview

Thanks to innovative sample-preparation and sequencing technologies, gene expression in individual cells can now be measured for thousands of cells in a single experiment. Since its introduction, single-cell RNA sequencing (scRNA-seq) approaches have revolutionized the genomics field as they created unprecedented opportunities for resolving cell heterogeneity by exploring gene expression profiles at a single-cell resolution. However, the rapidly evolving field of scRNA-seq invoked the emergence of various analytics approaches aimed to maximize the full potential of this novel strategy. Unlike population-based RNA sequencing approaches, scRNA seq necessitates comprehensive computational tools to address high data complexity and keep up with the emerging single-cell associated challenges. Despite the vast number of analytical methods, a universal standardization is lacking. While this reflects the fields’ immaturity, it may also encumber a newcomer to blend in. In this review, we aim to bridge over the abovementioned hurdle and propose four ready-to-use pipelines for scRNA-seq analysis easily accessible by a newcomer, that could fit various biological data types. Here we provide an overview of the currently available single-cell technologies for cell isolation and library preparation and a step by step guide that covers the entire canonical analytic workflow to analyse scRNA-seq data including read mapping, quality controls, gene expression quantification, normalization, feature selection, dimensionality reduction, and cell clustering useful for trajectory inference and differential expression. Such workflow guidelines will escort novices as well as expert users in the analysis of complex scRNA-seq datasets, thus further expanding the research potential of single-cell approaches in basic science, and envisaging its future implementation as best practice in the field

Duplication of the dystroglycan gene in most branches of teleost fish

<p>Abstract</p> <p>Background</p> <p>The dystroglycan (DG) complex is a major non-integrin cell adhesion system whose multiple biological roles involve, among others, skeletal muscle stability, embryonic development and synapse maturation. DG is composed of two subunits: α-DG, extracellular and highly glycosylated, and the transmembrane β-DG, linking the cytoskeleton to the surrounding basement membrane in a wide variety of tissues. A single copy of the DG gene (<it>DAG1</it>) has been identified so far in humans and other mammals, encoding for a precursor protein which is post-translationally cleaved to liberate the two DG subunits. Similarly, <it>D. rerio </it>(zebrafish) seems to have a single copy of <it>DAG1</it>, whose removal was shown to cause a severe dystrophic phenotype in adult animals, although it is known that during evolution, due to a whole genome duplication (WGD) event, many teleost fish acquired multiple copies of several genes (paralogues).</p> <p>Results</p> <p>Data mining of pufferfish (<it>T. nigroviridis </it>and <it>T. rubripes</it>) and other teleost fish (<it>O. latipes </it>and <it>G. aculeatus</it>) available nucleotide sequences revealed the presence of two functional paralogous DG sequences. RT-PCR analysis proved that both the DG sequences are transcribed in <it>T. nigroviridis</it>. One of the two DG sequences harbours an additional mini-intronic sequence, 137 bp long, interrupting the uncomplicated exon-intron-exon pattern displayed by <it>DAG1 </it>in mammals and <it>D. rerio</it>. A similar scenario emerged also in <it>D. labrax </it>(sea bass), from whose genome we have cloned and sequenced a new DG sequence that also harbours a shorter additional intronic sequence of 116 bp. Western blot analysis confirmed the presence of DG protein products in all the species analysed including two teleost Antarctic species (<it>T. bernacchii </it>and <it>C. hamatus</it>).</p> <p>Conclusion</p> <p>Our evolutionary analysis has shown that the whole-genome duplication event in the Class Actinopterygii (ray-finned fish) involved also <it>DAG1</it>. We unravelled new important molecular genetic details about fish orthologous DGs, which might help to increase the current knowledge on DG expression, maturation and targeting and on its physiopathological role in higher organisms.</p