Julia Rueda

Skeletal muscle specific genes networks in cattle

While physiological differences across skeletal muscles have been described, the differential gene expression underlying them and the discovery of how they interact to perform specific biological processes are largely to be elucidated. The purpose of the present study was, firstly, to profile by cDNA microarrays the differential gene expression between two skeletal muscle types, Psoas major (PM) and Flexor digitorum (FD), in beef cattle and then to interpret the results in the context of a bovine gene coexpression network, detecting possible changes in connectivity across the skeletal muscle system. Eighty four genes were differentially expressed (DE) between muscles. Approximately 54% encoded metabolic enzymes and structural-contractile proteins. DE genes were involved in similar processes and functions, but the proportion of genes in each category varied within each muscle. A correlation matrix was obtained for 61 out of the 84 DE genes from a gene coexpression network. Different groups of coexpression were observed, the largest one having 28 metabolic and contractile genes, up-regulated in PM, and mainly encoding fast-glycolytic fibre structural components and glycolytic enzymes. In FD, genes related to cell support seemed to constitute its identity feature and did not positively correlate to the rest of DE genes in FD. Moreover, changes in connectivity for some DE genes were observed in the different gene ontologies. Our results confirm the existence of a muscle dependent transcription and coexpression pattern and suggest the necessity of integrating different muscle types to perform comprehensive networks for the transcriptional landscape of bovine skeletal muscle.

A comprehensive characterisation of the fibre composition and properties of a limb ( Flexor digitorum superficialis, membri thoraci ) and a trunk ( Psoas major ) muscle in cattle

BackgroundThe fibre type attributes and the relationships among their properties play an important role in the differences in muscle capabilities and features. Comprehensive characterisation of the skeletal muscles should study the degree of association between them and their involvement in muscle functionality. The purposes of the present study were to characterise the fibre type composition of a trunk (Psoas major, PM) and a limb (Flexor digitorum, membri thoraci, FD) muscle in the bovine species and to study the degree of coordination among contractile, metabolic and histological properties of fibre types. Immunohistochemical, histochemical and histological techniques were used.ResultsThe fibre type composition was delineated immunohistochemically in calf muscle samples, identifying three pure (I, IIA, and IIX) and two hybrid type fibres (I+IIA, and IIAX). Most of the fibres in FD were types I and IIA, while pure IIX were absent. All fibre types were found in PM, the IIX type being the most frequent. Compared to other species, small populations of hybrid fibres were detected. The five fibre types, previously identified, were ascribed to three different acid and alkaline mATPase activity patterns. Type I fibres had the highest oxidative capacity and the lowest glycolytic capacity. The reverse was true for the IIX fibres, whereas the type IIA fibres showed intermediate properties. Regarding the histological properties, type I fibres tended to be more capillarised than the II types. Correlations among contractile, metabolic and histological features on individual fibres were significantly different from zero (r values varied between -0.31 and 0.78). Hybrid fibre values were positioned between their corresponding pure types, and their positions were different regarding their metabolic and contractile properties.ConclusionCoordination among the contractile, metabolic and histological properties of fibres has been observed. However, the magnitude of the correlation among them is always below 0.8, suggesting that the properties of muscles are not fully explained by the fibre composition. These results support the concept that, to some extent, muscle plasticity can be explained by the fibre type composition, and by the properties derived from their metabolic and histological profiles.

Shoot regeneration in four Begonia genotypes

In vitro regeneration of four Begonia genotypes, B. semperflorens, B. rex, B.×elatior, and hybrid of Begonia with unknown parents ‘Tiger’ was carried out starting from leaf and petiole segments as explants. Five Murashige and Skoogs derived media were tested, three of them supplemented with α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), and the other two with NAA and kinetin (KIN) in different concentrations. Shoot regeneration was preferentially induced on the BA containing media, quantitative differences being observed among explants and genotypes.

Genetic and epigenetic relationship in rye, Secale cereale L., somaclonal variation within somatic embryo-derived plants

In vitro regenerated plants of rye, Secale cereale L., Ailés and Merced cultivars, were studied to verify if genetic and/or epigenetic changes were promoted by in vitro conditions. Inter-simple sequence repeat (ISSR) fingerprints on HpaII/MspI-digested and uncut DNA were generated. DNA digested with methylation-sensitive isoschizomers revealed epigenetic modifications, while modification of ISSR patterns obtained with undigested DNA indicated genetic changes. With this technique, it was possible to study both genetic and/or epigenetic changes within the same DNA sequences. The frequency of plants with at least one variation was high: 73% and 30% of rye plants showed at least one genetic change, and 50% and 73% carried at least one methylation change, in the Ailés and Merced cultivars, respectively. Further analyses revealed that a considerable number of variable markers showed both types of modifications, indicative of both genetic and epigenetic changes. Moreover, genetic variation was related to the presence of the CCGG target in the analyzed bands. These results indicate the possible existence of a common mechanism connecting both types of variation.

Quantification of DNA

Quantification of DNA is a very important step in many procedures where it is necessary to know the amount of DNA that is present when carrying out restriction digests or performing different techniques such as PCR and RAPDs. There are several methods for quantifying DNA, the most widespread being: (i) the comparison of an aliquot of the extracted sample with standard DNAs of known concentration using gel electrophoresis and (ii) spectrophotometric determination. With both methods additional information is gained concerning the quality and purity of the extracted sample obtained. Normally both methods are used, but if the amount of DNA available is very small the gel electrophoresis technique alone may be employed. If there is no limitation on DNA amount, however, spectrophotometric measures should also be taken.

Thermal processing effects on the IgE-reactivity of cashew and pistachio

Thermal processing can modify the structure and function of food proteins and may alter their allergenicity. This work aimed to elucidate the influence of moist thermal treatments on the IgE-reactivity of cashew and pistachio. IgE-western blot and IgE-ELISA were complemented by Skin Prick Testing (SPT) and mediator release assay to determine the IgE cross-linking capability of treated and untreated samples. Moist thermal processing diminished the IgE-binding properties of both nuts, especially after heat/pressure treatment. The wheal size in SPT was importantly reduced after application of thermally-treated samples. For cashew, heat/pressure treated-samples still retain some capacity to cross-link IgE and degranulate basophils, however, this capacity was diminished when compared with untreated cashew. For pistachio, the degranulation of basophils after challenge with the harshest heat/pressure treatment was highly decreased. Boiling produced more variable results, however this treatment applied to both nuts for 60 min, led to an important decrease of basophil degranulation.

Muscle-specific gene expression is underscored by differential stressor responses and coexpression changes

Variations on the transcriptome from one skeletal muscle type to another still remain unknown. The reliable identification of stable gene coexpression networks is essential to unravel gene functions and define biological processes. The differential expression of two distinct muscles, M. flexor digitorum (FD) and M. psoas major (PM), was studied using microarrays in cattle to illustrate muscle-specific transcription patterns and to quantify changes in connectivity regarding the expected gene coexpression pattern. A total of 206 genes were differentially expressed (DE), 94 upregulated in PM and 112 in FD. The distribution of DE genes in pathways and biological functions was explored in the context of system biology. Global interactomes for genes of interest were predicted. Fast/slow twitch genes, genes coding for extracellular matrix, ribosomal and heat shock proteins, and fatty acid uptake centred the specific gene expression patterns per muscle. Genes involved in repairing mechanisms, such as ribosomal and heat shock proteins, suggested a differential ability of muscles to react to similar stressing factors, acting preferentially in slow twitch muscles. Muscle attributes do not seem to be completely explained by the muscle fibre composition. Changes in connectivity accounted for 24% of significant correlations between DE genes. Genes changing their connectivity mostly seem to contribute to the main differential attributes that characterize each specific muscle type. These results underscore the unique flexibility of skeletal muscle where a substantial set of genes are able to change their behavior depending on the circumstances.

Estudio de asociación con caracteres de calidad de carne y marcadores SNP en regiones relacionadas con genes diferencialmente expresador en dos músculos en la raza bovina avileña-negra ibérica

Con el objetivo de identificar regiones del genoma asociadas a algunos caracteres de calidad de carne en la raza Avilena Negra-Iberica (ANI) en dos musculos, se han tenido en cuenta la posicion en el genoma de 205 genes diferencialmente expresados (DE) entre el Psoas major y Flexor digitorum en esta misma raza. Se dispuso de genotipos de 397 terneros de raza Avilena-Negra Iberica obtenidos con la plataforma Illumina Bovine SNP50. Los marcadores con un “call rate” menor del 98% o fijados fueron excluidos del analisis. Los caracteres incorporados en el analisis fueron dos caracteres organolepticos (Flavor y Terneza) y dos caracteres laboratoriales (Grasa intramuscular y Terneza instrumental). Para el analisis de asociacion se utilizo un total de 3110 marcadores que bien estaban contenidos en los genes DE (104 marcadores) o en las zonas flanqueantes a dichos genes en una ventana de 500kb. El analisis estadistico se realizo con QXPak 5.0. Se encontraron en total veinte marcadores asociados a las caracteristicas estudiadas, de los cuales 12 se asociaron a grasa intramuscular , cinco a terneza organoleptica y tres a terneza instrumental. Estos marcadores corresponden a 23 genes DE distribuidos en las zonas flanqueantes (22) y contenidos dentro de genes DE (1). No se encontraron marcadores asociados a flavor. Tampoco se encontraron coincidencias de marcadores entre los caracteres ni entre musculos.

Plant Total DNA Extraction

Several methods for the extraction of plant DNA have been reported. The protocol described here is a modification of the method developed by Dellaporta et al. (1). This method is generally applicable and has been successfully used on a broad range of tissues, fresh or dried, including calli from many species. The DNA produced is of a moderately high molecular weight and can be used as a satisfactory substrate for some restriction endonucleases as well as for genomic blot analysis. However, the DNA obtained following the Dellaporta method is not of sufficient quality to apply PCR techniques, such as RAPDs (see Chapter 8), where variable DNA quality may lead to difficulties in reproducing results. In addition, DNA of poor quality cannot be restricted with certain enzymes.

Combinando información de expresión diferencial y genotipado a genoma completo para redefinir regiones QTLs conocidas por su relación con calidad de carne en vacuno.

Con el objetivo de profundizar en los mecanismos que afectan a las diferencias de calidad de carne entre musculos se han combinado diferentes fuentes de informacion: resultados de expresion diferencial (DE) con microarrays en los que se encontraron 204 genes diferencialmente expresados entre el musculo M. flexor digitorum (morcillo) y M. psoas major (solomillo), genotipado con chips de alta densidad de SNPs de 397 terneros de raza Avilena- Negra Iberica y 958 regiones QTL encontradas hasta ahora para caracteres de calidad de carne. El 78% de los SNPs en el chip estaban inicialmente contenidos en las regiones QTL. Cuando se consideraban regiones QTL que contenian genes DE, el numero de regiones involucradas disminuyo a 276. Se han identificado 173 genes DE como candidatos posicionales a explicar las diferencias en calidad de carne. Finalmente, al considerar las regiones genomicas asociadas a QTLs de calidad que contenian los genes DE entre musculos se pudo reducir notablemente el numero de SNPs implicados. Como ejemplo, para terneza, flavor y jugosidad, tres caracteres identificados como prioritarios por los consumidores, se seleccionaron 1092, 3046 y 2103 SNPs asociados, respectivamente.