Estado de micorrización de árboles truferos en función de su carácter productivo y su edad. Primeros resultados del Subproyecto 1 del Plan de Desarrollo Integral de la Truficultura de Teruel

La truficultura es una actividad con clara dimensión forestal, entre otras razones porque las especies implicadas, tanto el hongo como su árbol simbionte, son de gran valor silvícola. Además, la truficultura y la selvicultura trufera son prácticas que contribuyen a la recuperación de masas forestales y zonas potencialmente boscosas, con el valor añadido de la obtención de las trufas, de modo, en la mayor parte de las ocasiones, totalmente ecológico. El Plan Específico para Teruel (PET) integra varias actuaciones promovidas por el Gobierno Español entre las que se encuentra la ejecución de un Proyecto de Investigación denominado “Desarrollo Integral de la Truficultura de Teruel”, financiado por INIA, en el que se pretende apoyar al sector trufero turolense mediante el conocimiento y la mejora de sus técnicas de cultivo, producción y comercialización. La presente comunicación resume los primeros resultados obtenidos en uno de los 7 subproyectos que lo integran y que afecta al estudio del cortejo micorrícico de los árboles de las plantaciones, productoras o no, y su relación con la producción. La composición micorrícica de los árboles truferos es un factor esencial para realizar predicciones sobre la producción futura. Tanto la ausencia de micorrizas de trufa negra como la detección de otras, capaces de desplazar a la primera de las raíces de los árboles, puede permitir el diagnóstico precoz de fracasos de plantaciones y por lo tanto el ahorro de muchos años de cuidados a la espera de las primeras recolecciones. Para el estudio, se han seleccionado 3 grupos de plantaciones por edad, con 2 localizaciones distintas. Se está desarrollando un inventario periódico de las micorrizas presentes en un total de 48 árboles. Se propone una clasificación de las mismas en función de su capacidad para comprometer la producción de trufa negra, factor que se ha determinado en base al carácter productor o no productor de cada árbol

A reliable protocol for the stable transformation of non-embryogenic cells cultures of grapevine (Vitis vinifera L.) and Taxus x media

One of the major intent of metabolic engineering in cell culture systems is to increase yields of secondary metabolites. Efficient transformation methods are a priority to successfully apply metabolic engineering to cell cultures of plants that produce bioactive or therapeutic compounds, such as Vitis vinifera and Taxus x media. The aim of this study was to establish a reliable method to transform non-embryogenic cell cultures of these species. The V. vinifera cv. Gamay/cv. Monastrell cell lines and Taxus x media were used for Agrobacterium-mediated transformation using the Gateway-compatible Agrobacterium sp. binary vector system for fast reliable DNA cloning. The Taxus x media and Vitis cell lines were maintained in culture for more than 4 and 15 months, respectively, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernible effect on cell growth, or led to extracellular accumulation of phytoalexin trans-Resveratrol (t-R) in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA) in the grapevine transgenic cell lines compared to the parental control. The method described herein provides an excellent tool to exploit exponentially growing genomic resources to enhance, optimize or diversify the production of bioactive compounds generated by grapevine and yew cell cultures, and offers a better understanding of many grapevine and yew biology areas.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-01, BIO2011-29856-C02-02 and BIO2014-51861-R), European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). J.M.C. holds a postdoctoral grant from SENESCYT-GOVERNMENT OF ECUADOR (006-IECESMG5-GPLR-2012)

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrol-converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-01, BIO2011-29856-C02-02 and BIO2014-51861-R), Generalitat de Catalunya (2014SGR215) and European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). J.M.C. acknowledges a postdoctoral and research grants from SENESCYT GOVERNMENT OF ECUADOR (006-IECE-SMG5-GPLR-2012 and Programa1-Senescyt-2014) and a grant from UTEQ (UTEQAmbiental-9-FCAmb-IFOR-2014-FOCICYT002)

The impact of staff training on the knowledge of support staff in relation to bereavement and people with an intellectual disability

This study aimed to investigate whether a 1-day training course improved support staff knowledge about bereavement and grief in people with a learning disability. A questionnaire based, mixed design was used. Forty-eight participants were randomly assigned to one of two equal groups. A staggered design allowed for group 2 to act both as a control group and to receive training. Within- and between-group comparisons were made. Training significantly improved staff knowledge in all the areas measured. An analysis of staff knowledge at 1 month after training was not possible due to a low response rate. A short training course was successful in significantly increasing the knowledge of paid carers about issues relating to bereavement for people with a learning disability. The clinical and ethical implications of the study are discussed along with limitations and suggestions for further research

Cu2Se and Cu Nanocrystals as Local Sources of Copper in Thermally Activated in Situ Cation Exchange

Among the different synthesis approaches to colloidal nanocrystals, a recently developed toolkit is represented by cation exchange reactions, where the use of template nanocrystals gives access to materials that would be hardly attainable via direct synthesis. Besides, postsynthetic treatments, such as thermally activated solid-state reactions, represent a further flourishing route to promote finely controlled cation exchange. Here, we report that, upon in situ heating in a transmission electron microscope, Cu2Se or Cu nanocrystals deposited on an amorphous solid substrate undergo partial loss of Cu atoms, which are then engaged in local cation exchange reactions with Cu “acceptor” phases represented by rod- and wire-shaped CdSe nanocrystals. This thermal treatment slowly transforms the initial CdSe nanocrystals into Cu2−xSe nanocrystals, through the complete sublimation of Cd and the partial sublimation of Se atoms. Both Cu “donor” and “acceptor” particles were not always in direct contact with each other; hence, the gradual transfer of Cu species from Cu2Se or metallic Cu to CdSe nanocrystals was mediated by the substrate and depended on the distance between the donor and acceptor nanostructures. Differently from what happens in the comparably faster cation exchange reactions performed in liquid solution, this study shows that slow cation exchange reactions can be performed at the solid state and helps to shed light on the intermediate steps involved in such reactions

The Factor Inhibiting HIF Asparaginyl Hydroxylase Regulates Oxidative Metabolism and Accelerates Metabolic Adaptation to Hypoxia.

Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia

Intensive pharmacological immunosuppression allows for repetitive liver gene transfer with recombinant adenovirus in nonhuman primates

Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector