Peroxidase expression in a cereal cyst nematode (Heterodera avenae) resistant hexaploid wheat line.

The incompatible interaction between plant and pathogen is often determined by the hypersensitive reaction (HR). This response is associated with accumulation of reactive oxygen species (ROS), which results in adverse growth conditions for pathogens. Two major mechanisms involving either NADPH oxidases or peroxidases have been proposed for generation of ROS. Peroxidases (PER, EC 1.11.1.7), present in all land plants, are members of a large multigenic family with high number of isoforms involved in a broad range of physiological processes. PER genes, which are expressed in nematode feeding sites, have been identified in several plant species (Zacheo et al. 1997). A strong correlation between HR and PER activities at four and seven days post nematode infection, was detected in roots of wheat lines carrying Cre2, Cre5 (from Ae. ventricosa) or Cre7 (from Ae. triuncialis) Heterodera avenae resistance genes (Andrés et al. 2001; Montes et al. 2003, 2004). We have studied changes in root of peroxidase mRNAs levels after infection by H. avenae of a wheat/Ae. ven¬tricosa introgression line (H-93-8) carrying Cre2 (Delibes et al. 1993). We also report and classify the predicted protein sequences derived from complete peroxidase transcripts

Familial imbalance in 16p13.11 leads to a dosage compensation rearrangement in an unaffected carrier

Background: We and others have previously reported that familial cytogenetic studies in apparently de novo genomic imbalances may reveal complex or uncommon inheritance mechanisms. Methods: A familial, combined genomic and cytogenetic approach was systematically applied to the parents of all patients with unbalanced genome copy number changes. Results: Discordant array-CGH and FISH results in the mother of a child with a prenatally detected 16p13.11 interstitial microduplication disclosed a balanced uncommon rearrangement in this chromosomal region. Further dosage and haplotype familial studies revealed that both the maternal grandfather and uncle had also the same 16p duplication as the proband. Genomic compensation observed in the mother probably occurred as a consequence of interchromosomal postzygotic nonallelic homologous recombination. Conclusions: We emphasize that such a dualistic strategy is essential for the full characterization of genomic rearrangements as well as for appropriate genetic counselingFISH and aCGH materials were supported by grant 08/PI1207 from Fondo de Investigaciones Sanitarias (FIS) and research project ENDOSCREEN (S2011/BMD-2396) from Comunidad de Madri

Metodología aplicada en el estudio del desarrollo del lenguaje en niños con detección temprana de la hipoacusia neonatal

Producción CientíficaDado que el potencial discapacitante que causa la deficiencia auditiva neonatal disminuye con una detección e intervención tempranas, hemos elaborado un proyecto de investigación, con el que nos proponemos conocer el grado de desarrollo del lenguaje de los niños y niñas que procedentes del cribado auditivo universal, han sido diagnosticados/as por nosotros de hipoacusia prelingual en estos últimos 15 años y analizar las variables determinantes y las que son modificables. El objeto de esta comunicación consistirá en presentar la metodología que vamos a utilizar. Método: Partimos de los datos almacenados en nuestro Servicio de ORL, que comprende a 282 niños con hipoacusia. Hemos tenido la oportunidad de crear un grupo de investigación en el que coincidimos especialistas de la audición infantil y del lenguaje, por lo que contamos con medios suficientes para el estudio. Resultados: Describimos los elementos que configuran este proyecto, en relación al equipo de trabajo y a su desarrollo. Tras aplicar unos criterios de exclusión/inclusión, hemos seleccionado a un grupo de 45 niños entre 3 y 15 años, definiendo sus características auditivas. Mediante pruebas específicas, adaptadas a la edad, estudiaremos los diferentes aspectos del lenguaje; y a través Rev. ORL, 20xx, x x de una entrevista estructurada realizada a los padres, intentaremos determinar las variables que influyen en el proceso re-habilitador. Finalmente, los datos serán analizados estadísticamente. Discusión: La variabilidad y la escasa prevalencia de la hipoacusia infantil, dificultan la realización de estudios con población suficiente para obtener resultados estadísticamente significativos. Sin embargo, creemos que el grupo de niños seleccionado y la metodología utilizada nos permitirán conocer mejor las variables influyentes en el desarrollo del lenguaje. Conclusiones: El programa de cribado auditivo universal ha permitido una intervención más precoz, lo que debería mejorar los niveles de lenguaje de los niños detectados/as con hipoacusia. Aunque el desarrollo normalizado de la comunicación depende de otros factores difíciles de determinar, a través del protocolo presentado pretendemos equiparar estos resultados, validando el proceso de cribado/diagnóstico e intervención de nuestro medio.Universidad de Valladoli

A Single Argonaute Gene Participates in Exogenous and Endogenous RNAi and Controls Cellular Functions in the Basal Fungus Mucor circinelloides

The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1(-) M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi

Valoración de una nueva estrategia docente en las consultas externas para la enseñanza de la Cirugía General

La disminución de casos ingresados que provoca la cirugía ambulatoria repercute negativamente en la enseñanza de pregrado. Para enfrentar este problema se contextualizó el programa de Cirugía General en el Hospital Carlos J. Finlay, incrementándoles la cantidad de consultas externas a los alumnos de cuarto año durante la rotación por cirugía general de 4 a 21, pasando además las dos primeras semanas en la Terapia Intermedia de Cirugía. Las dos primeras rotaciones del curso 2002-03 realizaron más de 20 exámenes físicos entre los del cuello, mamas, abdomen y regiones inguinocrurales, aunque no así con el tórax. Al comparar los casos vistos en la consulta con los ingresados en el mismo período, se aprecia que casi la totalidad de los enfermos fueron atendidos de forma ambulatoria. Las consultas externasbrindan la posibilidad de que los educandos de pregrado adquieran las habilidades mínimas que se requieren en cirugía general.Palabras Clave: Estrategia docente, consultas externas, cirugía general.</p

Conectividad ecológica horizontal y vertical

Se ha estudiado el funcionamiento del paisaje con perspectiva sistémica y expresándolo en términos cartesianos.El objetivo de fondo es la conservación de la naturaleza, asumiéndose que quizá no debiera seguir considerándose la idea con el objetivo básico de delimitar espacios naturalísticamente valiosos, pues, reconociéndose el importante logro que representa disponer de 'espacios naturales protegidos', el concepto de sistema debe llevarse a la práctica de una vez. Tanto la gestión de la 'trama ecológica natural', que nunca queda limitada a los límites de un espacio protegido, como la consideración de las poblaciones humanas deben entrar a formar parte decididamente de los objetivos de la conservación. La conectividad se concibe como un proceso 'horizontal' y 'vertical'. El primero lo constituye un conjunto de fenómenos físicos y biológicos que generan conexiones espaciales a manera de trama o tejido territorial. En la conectividad se han incluido también procesos 'verticales', derivados de la interacción entre la estructura del paisaje y la socioeconómica, asumiéndose que buena parte del funcionamiento de los hoy llamados 'sistemas socioecológicos' deriva de la interdependencia entre ambas estructuras. Se ha llevado a cabo una descripción de estos procesos de manera simple, mediante modelos cartográficos previamente ensayados, ecuaciones sencillas de regresión, superficies de tendencia y productos de matrices de datos. En el estudio se ha considerado la relevancia de la ampliación de la red de carreteras 2000-2025 del territorio español, que constituye un ejemplo de interferencia entre las citadas infraestructuras naturales y rurales y esta red artificial, identificándose puntos de 'tensión' entre ambas, modelizándose y cartografiándose los costes ambientales o 'impactos' derivados de esta interferencia. Se han analizado también lagunas legales detectadas al tratar de aplicar estas ideas a la conservación de la naturaleza y a la planificación ambiental, indicándose las posibilidades de adaptación y aplicación de leyes y normativas a estos nuevos objetivos

A non-canonical RNA silencing pathway promotes mRNA degradation in basal fungi

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing

RNAi-Based Functional Genomics Identifies New Virulence Determinants in Mucormycosis

Mucorales are an emerging group of human pathogens that are responsible for the lethal disease mucormycosis. Unfortunately, functional studies on the genetic factors behind the virulence of these organisms are hampered by their limited genetic tractability, since they are reluctant to classical genetic tools like transposable elements or gene mapping. Here, we describe an RNAi-based functional genomic platform that allows the identification of new virulence factors through a forward genetic approach firstly described in Mucorales. This platform contains a whole-genome collection of Mucor circinelloides silenced transformants that presented a broad assortment of phenotypes related to the main physiological processes in fungi, including virulence, hyphae morphology, mycelial and yeast growth, carotenogenesis and asexual sporulation. Selection of transformants with reduced virulence allowed the identification of mcplD, which encodes a Phospholipase D, and mcmyo5, encoding a probably essential cargo transporter of the Myosin V family, as required for a fully virulent phenotype of M. circinelloides. Knock-out mutants for those genes showed reduced virulence in both Galleria mellonella and Mus musculus models, probably due to a delayed germination and polarized growth within macrophages. This study provides a robust approach to study virulence in Mucorales and as a proof of concept identified new virulence determinants in M. circinelloides that could represent promising targets for future antifungal therapies

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

BACKGROUND: RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.  RESULTS: Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.  CONCLUSION: This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model

Seasonal differences in leaf-level physiology give lianas a competitive advantage over trees in a tropical seasonal forest

Lianas are an important component of most tropical forests, where they vary in abundance from high in seasonal forests to low in aseasonal forests. We tested the hypothesis that the physiological ability of lianas to fix carbon (and thus grow) during seasonal drought may confer a distinct advantage in seasonal tropical forests, which may explain pan-tropical liana distributions. We compared a range of leaf-level physiological attributes of 18 co-occurring liana and 16 tree species during the wet and dry seasons in a tropical seasonal forest in Xishuangbanna, China. We found that, during the wet season, lianas had significantly higher CO2 assimilation per unit mass (Amass), nitrogen concentration (Nmass), and δ13C values, and lower leaf mass per unit area (LMA) than trees, indicating that lianas have higher assimilation rates per unit leaf mass and higher integrated water-use efficiency (WUE), but lower leaf structural investments. Seasonal variation in CO2 assimilation per unit area (Aarea), phosphorus concentration per unit mass (Pmass), and photosynthetic N-use efficiency (PNUE), however, was significantly lower in lianas than in trees. For instance, mean tree Aarea decreased by 30.1% from wet to dry season, compared with only 12.8% for lianas. In contrast, from the wet to dry season mean liana δ13C increased four times more than tree δ13C, with no reduction in PNUE, whereas trees had a significant reduction in PNUE. Lianas had higher Amass than trees throughout the year, regardless of season. Collectively, our findings indicate that lianas fix more carbon and use water and nitrogen more efficiently than trees, particularly during seasonal drought, which may confer a competitive advantage to lianas during the dry season, and thus may explain their high relative abundance in seasonal tropical forests