Expresión del gen chit42 de Trichoderma harzianum en plantas de fresa: obtención de plantas de fresa con mayor resistencia a la infección por Colletrotrichum acutatum

La antracnosis, causada por diferentes especies del hongo Colletotrichum, origina importantes pérdidas de producción en el cultivo de la fresa, un cultivo de gran interés agrícola en Andalucía, que aporta el 92.01% de la producción nacional y que, a su vez, genera más del 10% de la producción mundial total. C. acutatum está considerado un organismo de cuarentena en Europa (Directiva EC 77/93, Real Decreto 2071/1993). No se conocen variedades de fresa totalmente resistentes a C. acutatum y pese a la importancia agronómica de este cultivo en el mundo, existen muy pocos estudios moleculares de los mecanismos relacionados con la defensa de la planta de fresa a Colletotrichum spp. Esta falta de conocimiento molecular afecta negativamente a programas de mejora genética de la resistencia de la fresa a patógenos que, ya en sí, se ven notablemente mermados por el hecho de que la fresa es un cultivo de propagación clonal. Nuestro grupo PAI CVI-278 lleva más de 10 años investigando en fresa a nivel de biología molecular los mecanismos relacionados con diversos procesos bioquímicos, como la maduración del fruto y la defensa y resistencia de la planta a patógenos. Para incrementar la resistencia de la planta a C. acutatum, nuestro grupo está produciendo líneas transgénicas de fresa que expresan genes, procedentes de especies del hongo Trichoderma, que codifican enzimas que actúan sobre las paredes celulares de hongos ascomicetos y basidiomicetos, y que tienen, sustancialmente, una mayor actividad antifúngica que las propias enzimas de la planta y son activas frente a un rango más amplio de fitopatógeno

Down-regulation of a pectin acetylesterase gene modifies strawberry fruit cell wall pectin stracture and increases fruit firmness

Antisense-mediated down-regulation of several fruit-specific genes has previously demonstrated how the cell wall disassembly in strawberry fruit is mediated by a series of enzymes that act sequentially (Posé et al. 2011). An interesting example, the silencing of the polygalacturonase gene FaPG1, was also related with a significant increase of the post-harvest strawberry fruit firmness (Posé et al. 2013). Our research group has isolated a pectin acetylesterase gene, FaPAE1, which expression is enhanced during strawberry ripening. The main goal of this work was to elucidate the role of the degree of acetylation in cell wall integrity and fruit firmness through the antisense-mediated down-regulation of FaPAE1 in strawberry plants. Several transgenics lines were generated and 5 of them produced fruits 5-15% firmer than controls. Cell wall from ripe fruits was isolated from two independent transgenic lines and a control line, and sequentially extracted with different solvents (PAW, H2O, CDTA, Na2CO3). Modifications in fraction yield, its sugar composition and the degree of acetylation in each fraction were determined. Higher amounts of CDTA and Na2CO3 fractions were obtained in transgenic fruits, suggesting a decreased pectin solubilization as results of FaPAE1 silencing. Accordingly, the degree of acetylation of the Na2CO3-soluble pectins was greater in the transgenic lines than the control, but the opposite result was found in pectins from the CDTA fraction. These results suggest that PAE is preferentially active in pectis that are tightly bound to the cellulose-hemicellulose network and its activity could reduce the complexity of the cell wall structure, allowing that other hydrolytic enzymes could access the pectin chains. Thus, the increased fruit firmness observed in the transgenic FaPAE1 lines could be attributed to the direct effect of the silencing of the PAE enzyme and also to the indirect effect that the increase of the degree of acetylation of pectins has on the activity of other enzymes involved in the cell wall degradation. * Posé et al. (2011). Genes, Genomes and Genomics, 5 (Special Issue 1):40-48 * Posé et al. (2013). Plant Physiology, 150: 1022-1032 We acknowledge support from the Spanish Ministry of Economy and competitivity and Feder EU Funds (grant reference AGL2011-24814), FPI fellowships support for SP (BES-2006-13626) and CP (BES-2009027985), and grant "Ramón y Cajal" support for AJMA (RYC-2011-08839).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Lavado de manos: prevención de infecciones nosocomiales en una clínica de podología

The transfer of microorganisms via the hands of medical staff has been identified as the most important factor in the transmission of infections in hospitals or Health Clinics. Health ministers, technical experts and the World Health Organization (WHO) suggest some measures to combat hospital - acquired infections, also known as nosocomial infections. One of the main measures to avoid these infections is hand washing. For the importance of this topic an inquiry was conducted in a university clinic of podiatry, to identify knowledge of the people working there, or doing practices on prevention of these infections, and to determine the frequency with which they perform hand washing and if they performed correctlyLa transferencia de microorganismos a través de las manos del personal sanitario ha sido identificada como el factor más importante en la transmisión de infecciones en medios hospitalarios. Ministros de salud y funcionarios superiores, expertos técnicos y la Organización Mundial de la Salud (OMS) sugieren una serie de medidas fundamentales para luchar contra las infecciones asociadas a la atención sanitaria, también conocidas como infecciones nosocomiales, que ponen en peligro los avances obtenidos con grandes esfuerzos en los ámbitos de la salud y la esperanza de vida. Una de las medidas primordiales para evitar estas infecciones, es el lavado de manos. Debido a su repercusión y la importancia de este tema se realiza una encuesta en una Clínica Universitaria de Podología, para identificar los conocimientos del personal que trabaja o hace practicas en esta clínica sobre la prevención de este tipo de infecciones, así como para conocer la frecuencia con la que realizan el lavado de manos y si se realiza de manera correcta

A Comprehensive Study of the WRKY Transcription Factor Family in Strawberry

WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry

The Intragenesis and Synthetic Biology Approach towards Accelerating Genetic Gains on Strawberry: Development of New Tools to Improve Fruit Quality and Resistance to Pathogens

Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines

Contribution of the single photon emission computed tomography with 99mTc red blood cells in splenosis

The term splenosis refers to the presence of auto-transplanted splenic tissue in a heterotopic location. These foci can be localized to the liver simulating a malignant lesion. Sometimes these lesions are difficult to identify using conventional imaging techniques (ultrasound, CT and MR). Then, a scan with denatured erythrocytes marked with 99mTc has proven to be an effective technique to confirm the diagnosis of splenosis and to establish its extension. The incorporation of hybrid imaging techniques (SPECT-CT) into usual clinical practice has increased the precision of the localization of these foci of splenosis. We hereby report the cases of two patients diagnosed with splenosis, the first by laparotomy and the second after performing scintigraphy with red blood cells labeled with 99mTc. In the first case, the laparotomy revealed numerous reticulated nodules on the diaphragmatic peritoneal surface, the transverse colon and the right kidney. Finally, the anatomopathological diagnosis confirmed a case of splenosis. In the second case, the results of the 99mTc marked red blood cell gammagraphy and SPECT-CT were consistent with the diagnosis of splenosis in the patient. To obtain correct information in cases of lesions highly suspicious of splenosis, 99mTc marked red blood cell gammagraphy should be performed due to the high sensitivity and specificity of the test. Combined diagnostic imaging (SPECT-CT), have increased the specificity of this test due to improvements in the characterization of lesions. We believe that the use of this technique will help avoid unnecessary surgical procedures

Benefits of cryopreservation as long-term storage method of encapsulated cardiosphere-derived cells for cardiac therapy: A biomechanical analysis

[EN]Cardiosphere-derived cells (CDCs) encapsulated within alginate-poly-L-lysine-alginate (APA) microcapsules present a promising treatment alternative for myocardial infarction. However, clinical translatability of encapsulated CDCs requires robust long-term preservation of microcapsule and cell stability, since cell culture at 37 degrees C for long periods prior to patient implantation involve high resource, space and manpower costs, sometimes unaffordable for clinical facilities. Cryopreservation in liquid nitrogen is a well-established procedure to easily store cells with good recovery rate, but its effects on encapsulated cells are understudied. In this work, we assess both the biological response of CDCs and the mechanical stability of microcapsules after long-term (i.e., 60 days) cryopreservation and compare them to encapsulated CDCs cultured at 37 degrees C. We investigate for the first time the effects of cryopreservation on stiffness and topographical features of microcapsules for cell therapy. Our results show that functionality of encapsulated CDCs is optimum during 7 days at 37 degrees C, while cryopreservation seems to better guarantee the stability of both CDCs and APA microcapsules properties during longer storage than 15 days. These results point out cryopreservation as a suitable technique for long-term storage of encapsulated cells to be translated from the bench to the clinic.This work has been supported by the European Union's H2020 Framework Program (H2020/2014-2020) and National Authorities through the Electronic Components and Systems for European Leadership Joint Undertaking (ECSEL JU) program under grant agreement Ecsel-78132-Position-II-2017-IA. The regional Government of Aragon provided L.P. studentship. This research was partially funded by Instituto de Salud Carlos III (PI20/00247) and Agencia Estatal de Investigacion (PID2019-107329RA-C22), cofunded by European Regional Development Fund "A way to make Europe.

Identification of soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) as a possible biomarker of subclinical atherosclerosis

OBJECTIVES: Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to identify proteins differentially released by healthy versus atherosclerotic arterial walls, which could be found in plasma and serve as markers of atherosclerosis. METHODS AND RESULTS: We have analyzed supernatants obtained from cultured human carotid plaques and healthy arteries by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry ProteinChip System. Surface-enhanced laser-desorption/ionization analysis unveiled an 18.4-kDa peak released in lower amount by carotid plaques than normal endarteries. This protein was identified as soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). To confirm that sTWEAK was the protein of interest, Western blot and enzyme-linked immunosorbent assay were performed. Both techniques confirmed that sTWEAK levels were decreased in carotid plaque supernatants. Subsequent measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis (N=30) compared with healthy subjects matched by sex and age (N=28) (P<0.001). Furthermore, in a test population of 106 asymptomatic subjects, we showed that sTWEAK concentrations negatively correlated with the carotid intima-media thickness (r=-0.4; P<0.001), an index of subclinical atherosclerosis. CONCLUSIONS: These results suggest that sTWEAK could be a potential biomarker of atherosclerosis

A major role of TWEAK/Fn14 axis as a therapeutic target for post-angioplasty restenosis

Background: Tumor necrosis factor-like weak inducer of apoptosis (Tnfsf12; TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Tnfrsf12a; Fn14) participate in the inflammatory response associated with vascular remodeling.However, the functional effect ofTWEAK on vascular smoothmuscle cells (VSMCs) is not completely elucidated. Methods: Next generation sequencing-based methodswere performed to identify genes and pathways regulated by TWEAK in VSMCs. Flow-citometry, wound-healing scratch experiments and transwellmigration assays were used to analyze VSMCs proliferation and migration. Mouse wire injury model was done to evaluate the role of TWEAK/Fn14 during neointimal hyperplasia. Findings: TWEAK up-regulated 1611 and down-regulated 1091 genes in VSMCs. Using a gene-set enrichment method,we found a functionalmodule involved in cell proliferation defined as the minimal network connecting top TWEAK up-regulated genes. In vitro experiments in wild-type or Tnfrsf12a deficient VSMCs demonstrated that TWEAK increased cell proliferation, VSMCs motility and migration. Mechanistically, TWEAK increased cyclins (cyclinD1), cyclin-dependent kinases (CDK4, CDK6) and decreased cyclin-dependent kinase inhibitors (p15lNK4B) mRNA and protein expression. Downregulation of p15INK4B induced by TWEAK was mediated by mitogen-activated protein kinase ERK and Akt activation. Tnfrsf12a or Tnfsf12 genetic depletion and pharmacological intervention with TWEAK blocking antibody reduced neointimal formation, decreasing cell proliferation, cyclin D1 and CDK4/6 expression, and increasing p15INK4B expression compared with wild type or IgG-treated mice in wire-injured femoral arteries. Finally, immunohistochemistry in human coronary arteries with stenosis or in-stent restenosis revealed high levels of Fn14, TWEAK and PCNA in VSMCs enriched areas of the neointima as compared with healthy coronary arteries. Interpretation: Our data define a major role of TWEAK/Fn14 in the control of VSMCs proliferation and migration during neointimal hyperplasia after wire injury in mice, and identify TWEAK/Fn14 as a potential target for treating in-stent restenosis.This work was supported by Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias ISCiii/FEDER PI13/00395; PI16/01419; PI17/ 01495) and Spanish Biomedical Research Centre in Cardiovascular Disease (CIBERCV) and Metabolic Diseases and Diabetes (CIBERDEM). PM was supported by ISCIII Miguel Servet Program (CP16/00116). CGM was supported by Fundación Conchita Rábago. NMB and VE were supported by the Spanish Ministry of Economy and Competitiveness (Juan de la Cierva IJCI-2016-29630 and Ramón y Ramón Cajal Program RyC-2013-12880, respectively). JMM has been supported a postdoctoral fellowship fromthe American Diabetes Association (Grant 1-15-MI-03) and a postdoctoral fellowship fromthe American Heart Association

Nonuniversality due to inhomogeneous stress in semiconductor surface nanopatterning by low-energy ion-beam irradiation

A lack of universality with respect to ion species has been recently established in nanostructuring of semiconductor surfaces by low-energy ion-beam bombardment. This variability affects basic properties of the pattern formation process, like the critical incidence angle for pattern formation, and has remained unaccounted for. Here, we show that nonuniform generation of stress across the damaged amorphous layer induced by the irradiation is a key factor behind the range of experimental observations, as the form of the stress field is controlled by the ion/target combination. This effect acts in synergy with the nontrivial evolution of the amorphous-crystalline interface. We reach these conclusions by contrasting a multiscale theoretical approach, which combines molecular dynamics and a continuum viscous flow model, with experiments using Xe+ and Ar+ ions on a Si(100) target. Our general approach can apply to a variety of semiconductor systems and conditions.This work has been partially supported by MICINN (Spain) Grant MAT2011-13333-E, and MINECO (Spain) Grants FIS2012-38866-C05-01, FIS2012-38866-C05-05, FIS2013-47949-C2-2-P and FIS2012-32349. TEM work has been conducted at LABMET laboratory, associated with Red de Laboratorios of CAM, Spain. A.M.-B. acknowledges support from MINECO, through FPI scolarship BES-2010-036179. A.R.C. acknowledges funding from Juan de la Cierva program (Spain) under Contract No. JCI-2012-14509.Publicad