Development of expression vectors based on pepino mosaic virus

<p>Abstract</p> <p>Background</p> <p>Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus <it>Potexvirus</it>, family <it>Flexiviridae</it>), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.</p> <p>Results</p> <p>Agroinfectious clones were produced from two different PepMV genotypes (European and Chilean), and these were able to initiate typical PepMV infections. We explored several strategies for vector development including coat protein (CP) replacement, duplication of the CP subgenomic promoter (SGP) and the creation of a fusion protein using the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. We found that CP replacement vectors were unable to move systemically and that vectors with duplicated SGPs (even heterologous SGPs) suffered from significant transgene instability. The fusion protein incorporating the FMDV 2A catalytic peptide gave by far the best results, maintaining stability through serial passages and allowing the accumulation of GFP to 0.2-0.4 g per kg of leaf tissue. The possible use of PepMV as a virus-induced gene silencing vector to study gene function was also demonstrated. Protocols for the use of this vector are described.</p> <p>Conclusions</p> <p>A stable PepMV vector was generated by expressing the transgene as a CP fusion using the sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide to separate them. We have generated a novel tool for the expression of recombinant proteins in plants and for the functional analysis of virus and plant genes. Our experiments have also highlighted virus requirements for replication in single cells as well as intercellular and long-distance movement.</p

Experimental study of concentration polarization in a crossflow reverse osmosis system using Digital Holographic Interferometry

Digital Holographic Interferometry (DHI) has been used to visualize the polarization concentration layer during crossflow RO. This technique is based on the fact that changes in the concentration of the solution produce changes in its refractive index. Therefore, the concentration profile formed due to the polarization phenomenon can be visualized as interference fringes. Experiments with Na2SO4 and NaCl solutions have been carried out. Three variables of the process were studied: crossflow velocity, initial concentration and pressure applied. In each experiment, crossflow velocity was changed every 30min, in an increasing or decreasing sequence. Few minutes after changing the crossflow velocity the steady-state was reached. Interference fringe patterns of the polarization layer and their corresponding concentration profiles, as well as the permeate flux in different experimental conditions, are presented. The major experimental result is the visualization for the first time in situ and in real-time of the polarization layer in a process of crossflow by a non-invasive method. Experimental results show a close relationship among crossflow velocity, permeate flux and polarization layer. Furthermore, experimental maximum concentration values reached at the membrane surface were compared with values calculated by using the film theory approach and a good agreement was obtained.This research has been sponsored by the Plan Nacional de I+D+I CTQ2006-14904 (Ministerio de Ciencia e Innovación) and Generalitat Valenciana, Consellería de Educación (ACOMP/2009/366)

Determination of the Secondary Structure of an RNA fragment in Solution: Selective 2`- Hydroxyl Acylation Analyzed by Primer Extension Assay (SHAPE)

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RN

Guía de propuestas para mejorar la inserción laboral de las personas con discapacidad en el sector turístico

Esta guía es el resultado del proyecto de investigación: “Propuestas para mejorar la inserción laboral de las personas con discapacidad en el sector turístico de la provincia de Alicante”, cuyo objetivo era averiguar cuáles son las barreras para la inserción laboral de personas con diversidad funcional en las empresas y entidades turísticas de la provincia de Alicante y qué se debería hacer para superarlas. Para alcanzar este objetivo, se entrevistó a las personas, entidades y empresas antes mencionadas y después los resultados obtenidos se validaron en una jornada abierta y participativa en la que medio centenar de personas interesadas en el tema aportaron sus ideas.Cátedra Aguas de Alicante de Inclusión Social de la Universidad de Alicante

La implantació de la figura del tècnic d’Intervenció Sociocomunitària (TISOC) als centres d’Ensenyament Secundari de les Illes Balears: cap a un nou model d’intervenció

&#091;cat&#093; Després del primer any d’implantació de la figura del TISOC als centres d’Ensenyament Secundari de les Illes Balears, els resultats d’aquesta experiència constitueixen l’objecte d’aquest treball. Les memòries dels TISOC del curs 2017-18, deixen un balanç positiu d’aquest primer curs d’implantació del programa, que ha servit perquè el professorat i personal no docent dels centres educatius coneguin les possibilitats de treball que pot tenir la figura de l’educadora social dins l’àmbit formal, definir funcions i establir estratègies d’intervenció conjuntes de cara als propers cursos. Així mateix, encara hi ha tota una feina a fer per tal de millorar-ne la coordinació i la posada en marxa d’intervencions que permetin millorar el funcionament dels centres.&#091;spa&#093; Tras el primer año de implantación de la figura del TISOC en los centros de Enseñanza Secundaria de las Islas Baleares, los resultados de esta experiencia constituyen el objeto de este trabajo. Las memorias de los TISOC del curso 2017-18 dejan un balance positivo de este primer curso de implantación del programa, que ha servido para que el profesorado y personal no docente de los centros educativos conozca las posibilidades de trabajo que puede tener la figura de la educadora social en el ámbito formal, definir funciones y establecer estrategias de intervención conjuntas de cara a los próximos cursos. Así mismo, todavía hay un trabajo por hacer para mejorar la coordinación y la puesta en marcha de intervenciones que permitan mejorar el funcionamiento de los centros

Density dependence in marine protected populations: a review

The cessation or reduction of fishing in marine protected areas (MPAs) should promote an increase in abundance and mean size and age of previously exploited populations. Thus density-dependent changes in life-history characteristics should occur when populations are allowed to recover in MPAs. In this review, we synthesize the existing information on resource limitation in marine ecosystems, density-dependent changes in life-history traits of exploited populations and evidence for biomass export from MPAs. Most evidence for compensatory changes in biological variables has been derived from observations on populations depleted by high fishing mortality or on strong year classes, but these changes are more evident in juveniles than in adults and in freshwater rather than in marine systems. It is unclear if adults of exploited marine populations are resource limited. This may suggest that exploited populations are controlled mainly by density-independent processes, which could be a consequence of the depleted state of most exploited populations. MPAs could be a useful tool for testing these hypotheses. If we assume that resources become limiting inside MPAs, it is plausible that, if suitable habitats exist, mobile species will search for resources outside of the MPAs, leading to export of biomass to areas which are fished. However, it is not possible to establish from the available data whether this export will be a response to resource limitation inside the MPAs, the result of random movements across MPA boundaries or both. We discuss the implications of this process for the use of MPAs as fisheries management tools.Financial support was provided by the EU CEC DGXII – MAST III contract number: MAS3-ct97-0155

Hacia un Campus Inclusivo: El Proyecto Campus Accesible, Campus Igualitario de la Universidad de Alicante

El Campus de la Universidad de Alicante es un espacio de una enorme calidad tanto por sus arquitecturas construidas, la mayoría notables y algunas de ellas magníficas, como por sus espacios libres, zonas verdes e hitos que lo dotan de identidad y llenan de significados varios. Pero, además, es el espacio de trabajo, de relación y encuentro, de vida en definitiva, de toda la comunidad universitaria, razón por la cual, el proyecto institucional Campus Accesible, Campus Igualitario, promovido por el Vicerrectorado de Campus y Sostenibilidad en colaboración con la Unidad de Igualdad y el Vicerrectorado de Estudiantes, se plantea como objetivo primordial alcanzar la misma excelencia como espacio modelo de integración de todas las personas, sea cual fuere su situación o condición. Para ello atiende a dos aspectos básicos: uno, la consecución de accesibilidad universal y, dos, la incorporación de la perspectiva de género a su construcción, mantenimiento y gestión como generadora de nuevas miradas que enriquecen los espacios y la vida desde la bienvenida a los mismos de la complejidad y la diversidad. El presente trabajo recoge tanto las reflexiones como las acciones que, en esta dirección y con carácter transversal y pionero, se han desarrollado

Stable and Broad Spectrum Cross-Protection Against Pepino Mosaic Virus Attained by Mixed Infection

While recent pepino mosaic virus (PepMV; species Pepino mosaic virus, genus Potexvirus, family Alphaflexiviridae) epidemics seem to be predominantly caused by isolates of the CH2 strain, PepMV epidemics in intensive tomato crops in Spain are caused by both CH2 and EU isolates that co-circulate, representing a challenge in terms of control, including cross-protection. In this work, we hypothesized that mixed infections with two mild isolates of the EU and CH2 strains (PepMV-Sp13 and -PS5, respectively) may be useful in PepMV cross-protection in Spanish epidemics, providing protection against a broad range of aggressive isolates. Thus, we performed a range of field trials and an experimental evolution assay to determine the phenotypic and genetic stability of PepMV-Sp13 and -PS5 mixed infections, as well as their cross-protective efficiency. Our results showed that: (i) the phenotype of PepMV-Sp13 and -PS5 mixed infections was mild and did not change significantly when infecting different tomato cultivars or under different environmental conditions in Spain, (ii) PepMV-Sp13 and -PS5 mixed infections provided more efficient protection against two aggressive EU and CH2 isolates than single infections, and (iii) PepMV-Sp13 and -PS5, either in single or in mixed infections, were less variable than other two PepMV isolates occurring naturally in PepMV epidemics in Spain