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

Rhamnogalacturonase lyase gene downregulation in strawberry and its potential on mechanical fruit properties

Strawberry softening is one of the main factors that reduces fruit quality and leads to economically important losses. Textural changes during fruit ripening are mainly due to the dissolution of middle lamellae, a reduction in cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Functional studies of genes encoding pectinase enzymes (polygalacturonase, pectate lyase and -galactosidase) support a key role of pectin disassembly in strawberry softening. Evidence that RG-I may play an important role in strawberry texture has been obtained from the transient silencing of a RG-lyase gene. Pectins are major components of fruit cell walls and highly dynamic polysaccharides, but due to their heterogeneity the precise relation between the structures and functions is incomplete. In this work, stable transgenic strawberry lines with a rhamnogalacturonate lyase gene (FaRGLyase1) down-regulated have been analyzed. Several transgenic lines showing more than 95% silencing of FaRGLyase1 displayed fruit firmness values higher than control. Cell walls from these lines were extracted and analyzed by ELISA and Epitope Detection Chromatography (EDC). This last technique is based on the detection of specific cell wall oligosaccharide epitopes and provides information on sub-populations of pectins containing homogalacturonan and RG-I domains, but also reveals potential links with other cell wall polysaccharides such as xyloglucan. The results obtained indicate that the silencing of FaRGLyase1 reduces degradation of RG-I backbones, but also homogalacturonan, in cell walls, especially in pectin fractions covalently bound to the cell wall. These changes contribute to the increased firmness of transgenic fruits.This research was supported by FEDER EU Funds and the Ministerio de Economía y Competitividad of Spain (grant reference AGL2014-55784-C2), a Marie Curie IEF within the 7th European Community Framework Programme (reference: PIEF-2013-625270) for SP and a FPI fellowship (BES-2015-073616) to support PR-V. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Isolation and transfection of strawverry protoplasts for gene editing

Strawberry is the most economically important soft fruit. The improvement of the organoleptic qualities of ripe fruit and the postharvest shelf life are main objectives of strawberry breeding programs. Fruit softening is mainly due to the disassembly of cell walls and the dissolution of middle lamella. In strawberry, functional analyses of genes encoding polygalacturonases (PGs) indicate that these enzymes play a key role in fruit softening, i.e. the antisense downregulation of PG genes FaPG1 or FaPG2 increased fruit firmness and postharvest shelf life (Paniagua et al., 2020). These results suggest that PG encoding genes are excellent targets for gene editing to improve strawberry fruit quality. Transfection of protoplasts with CRISPR/Cas9 ribonucleoprotein complexes is currently being explored in many species to produce DNA-free edited plants. In this research, a protocol for strawberry protoplasts transfection has been optimized with the final goal of producing non-transgenic strawberry plants with the FaPG1 gene edited. Protoplasts were isolated from 9 weeks old in vitro grown plants of Fragaria x ananassa, cv. ‘Chandler’, micropropagated in Murashige and Skoog (MS) medium supplemented with 2 mg/L of BA. Protoplast extraction and purification was performed as described by Barceló et al. (2019). Using this protocol, a yield of 1 x 105 protoplast/g fresh tissue was obtained and nearly 50-70% of them were viable. Protoplasts were transfected with the plasmid pHBT-sGFP(S65T)-NOS using a PEG-mediated transformation system, as reported by Yoo et al. (2007). To improve the efficiency of protoplast transfection, different variables were evaluated: PEG concentration, time of incubation on PEG and DNA concentration. At 48 h after transfection, the highest percentage of protoplasts showing GFP expression, 18%, was obtained with 15 minutes incubation in 20% of PEG and 5 µg of DNA

Caracterización de indicadores de la calidad del fruto en líneas de fresa transgénicas con genes silenciados que codifican para enzimas pectinolíticas

Se han evaluado algunos indicadores de calidad del fruto en líneas transgénicas de fresa con los genes de poligalacturonasa FaPG1 (líneas PG) o pectato liasa FaplC (líneas APEL) silenciados. Se analizaron dos líneas independientes por genotipo transgénico. No se observaron diferencias en el contenido de sólidos solubles entre las líneas transgénicas y el control. De igual forma, la acidez total y el pH fueron similares en las líneas PG29, APEL21 y el control; sin embargo, la acidez de los frutos de las líneas PG62 y APEL39 fue superior al control. Los parámetros de color L*, a* y b* fueron similares en todos los genotipos; sin embargo, el contenido en antocianos fue menor en la línea APEL21. Los valores más altos de firmeza de fruto, estimada mediante un ensayo de extrusión, se observaron en las dos líneas transgénicas PG y en la línea APEL39. En cuanto a las pérdidas por goteo (drip loss), la línea APEL39 presentó un valor mayor que el control, pero la línea APEL21 registró valores menores. El contenido de compuestos fenólicos se analizó en la línea PG29, no encontrándose diferencias estadísticas con respecto al control. Finalmente, la capacidad del fruto para captar radicales libres fue ligeramente menor en la línea PG29 que en el control. Los resultados indican que el silenciamiento de los genes de pectinasas incrementa significativamente la firmeza de la fresa sin modificar sustancialmente parámetros de calidad del fruto maduro como color, acidez, sólidos solubles o contenido en antocianos.Some quality traits of transgenic strawberry fruits with low levels of expression of the pectinase genes FaPG1 (PG lines) or FaplC gene (APEL lines) were evaluated. Two independent lines per transgenic genotype were analyzed. Soluble solids were similar in control and transgenic lines. Similarly, pH and titratable acidity was similar in lines PG29, APEL21 and control; however, lines PG62 and APEL39 showed acidity values higher than the control. The color parameters L*, a* and b* were similar in control and transgenic fruits; however, line APEL21 displayed a lower value of anthocyanin content. The highest values of fruit firmness, measured with an extrusion test, were observed in both PG transgenic lines and in the APEL39 line. Regarding the drip loss, APEL39 line showed a higher value than the control, but the APEL21 line displayed lower values. The content of phenolic compounds was analyzed in line PG29, not observing significant differences with the control. Finally, the antiradical activity of the fruit was slightly lower in the line PG29 than in the control. The results obtained indicate that the silencing of the pectinase genes increases the firmness of the fruit without substantially modifying other quality parameters such as color, acidity, soluble solids or anthocyanin content

Obtención de poliploides en Fragaria para su uso en mejora

La fresa cultivada, Fragaria × ananassa Duch., es una especie octoploide procedente de un híbrido interespecífico entre las especies silvestres F. virginiana Duch. y F. chiloensis L. Los cruces intraespecíficos de F × ananassa se utilizan extensivamente para la obtención de nuevos cultivares con características agronómicas mejoradas. A pesar de esto, esta especie muestra una alta susceptibilidad a patógenos fúngicos y bacterianos y una baja tolerancia a estreses abióticos. Especies silvestres de menor ploidía, e.g. F. vesca (2x), pueden ser una fuente genética de gran valor para la mejora de esos caracteres en la fresa cultivada, pero las diferencias en ploidía representan una barrera reproductiva para los cruzamientos interespecíficos. La duplicación del número de cromosomas mediante tratamientos con colchicina de meristemos procedentes de estolones se ha utilizado para facilitar la hibridación interespecífica entre fresas diploides y comerciales. En este trabajo, se ha puesto a punto la metodología para la obtención de poliploides en fresa mediante la aplicación de colchicina durante la regeneración de brotes in vitro.Esta investigación ha sido financiada por los fondos FEDER EU y el Ministerio de Economía y Competitividad de España (AGL2017-86531-C2-1-R

Dimorphic Fungal Coinfection as a Cause of Chronic Diarrhea and Pancolitis

Histoplasma capsulatum and Paracoccidioides brasiliensis are dimorphic fungi that cause systemic mycosis mostly in tropical South America and some areas of North America. Gastrointestinal involvement is not uncommon among these fungal diseases, but coinfection has not previously been reported. We report a patient with chronic diarrhea and pancolitis caused by paracoccidioidomycosis and histoplasmosis

Chronic Diarrhea and Pancolitis Caused by Paracoccidioidomycosis: A Case Report

South American blastomycosis is a systemic micosis caused by infection with Paracoccidioides brasiliensis. The most frequently affected sites are the lower lip buccal mucous membrane, palate, tongue, sublingual region, lymph glands, and lungs. However, colonic involvement is not a common expression of Paracoccidioidomycosis. We report a case of chronic diarrhea and pancolitis caused by Paracoccidioidomycosis with fatal outcome

Antioxidant activity and prevention of pork meat lipid oxidation using traditional Mexican condiments (pasilla dry pepper, achiote, and mole sauce)

Antioxidant activity and prevention of pork meat lipid oxidation using traditional Mexican condiments (pasilla dry pepper, achiote, and mole sauce

An Exploratory Study of Healing Circles as a Strategy to Facilitate Resilience in an Undocumented Community

Within the United States, the COVID-19 pandemic highlighted critical inequalities affecting undocumented communities and resulting in particularly heightened stress for members of these communities. In addition to the stress associated with COVID-19, immigrants in the United States were more than ever subjected to a hostile antiimmigrant climate under Trump’s administration. Given this compounded stress, the impact of the pandemic on mental health is likely to be disproportionately experienced by undocumented immigrants. In response, a group of psychologists partnered with a leading immigrant rights advocacy organization and formed a reciprocal collaboration to support undocumented communities. A major focus of the collaboration is to foster learning, supporting members of the immigrant community to contribute to their own well-being and others in the community. Accordingly, the collaborative developed and delivered a web-based mental health education session to the immigrant community and to practitioners serving this population. The session presented the use of healing circles as a strength-based approach to building resilience and also sought feedback regarding specific features of healing circles that can enhance their effectiveness in managing distress. Survey data and qualitative findings from this study show that those who participated in the web-based program perceived the session as validating and informative. Findings also underscored the need for creating safe spaces for community members to be vulnerable about their lived experiences while promoting ownership of their narratives. We discuss practical implications pertaining to the development and facilitation of social support groups for immigrants led by nonspecialist community members trained for this role. Impact Statement We describe a reciprocal collaboration between psychologists and an immigrant-led advocacy organization for the purposes of supporting undocumented immigrants in tailoring culturally congruent therapeutic approaches for fostering resilience as they face multiple stressors due to interlocking crises, such as the COVID-19 pandemic and antiimmigrant policies. The collaboration led to the development and delivery of a web-based session that provided immigrant community members and practitioners with recommendations for facilitating healing circles as a strength-based and culturally responsive approach to fostering peer-led social support during stressful times. Findings highlight the need for creating such safe spaces for community members to be vulnerable about their lived experiences and feel validated

Implementación de un sistema piloto de telemedicina en Mendoza

La telemedicina se caracteriza por utilizar las nuevas tecnologías informáticas y de comunicaciones para ofrecer acceso a la salud, independientemente del lugar donde se encuentre quien lo necesite. Este informe hace referencia a la utilización de dichas tecnologías al plantear el desarrollo e implementación de un sistema de telemedicina, para la transmisión y monitoreo de señales cardíacas, con la finalidad de suplir atención sanitaria en comunidades rurales, donde la distancia constituye un factor crítico. Los actores principales que conforman la arquitectura tecnológica y la red son: 1) Sistemas Embebidos, 2) Adquisidores de Señales Cardíacas Inalámbricos, 3) Computadoras Personales PC, 4) Asistentes Digitales Personales PDA 5) Software y 6) Infraestructura de Comunicación. Según se agrupen estos elementos es posible distinguir tres alternativas o unidades, que de acuerdo con sus ventajas y desventajas serían aplicables a diferentes usos. En todos los casos la transmisión es realizada hacia un servidor, donde los datos correspondientes a las variables biológicas son almacenados en bases de datos, pudiendo ser luego objeto de análisis y diagnóstico. Hasta el momento los resultados obtenidos en ambas alternativas han sido satisfactorios, tanto en la fabricación del adquisidor de señales y como en la transmisión de los datos.Telemedicine is characterized for offering access to healthcare by using the new computer and communication technologies, independently of where the individual in need is. This report refers to the use of these technologies while outlining the development and implementation of a system for the monitoring and transmission cardiac signals with the purpose of replacing sanitary services in rural communities, where distance constitutes a critical factor. The main elements that make up the technological architecture and the network are: 1) the embedded systems, 2) Wireless Cardiac signal data loggers 3) Personal Computers PCs, 4) Personal Digital Assistants PDA, 5) Software and 6) Communication Infrastructure. Depending on the way we group these elements it is possible to distinguish three different alternatives, each one with its pros and cons for different applications. In all these alternatives, the data is sent to a server where the data of the biological variables are stored in a database, subject to later analysis and diagnosis. So far, the results obtained have been satisfactory both in the manufacturing of the data logger as well as the data transmissionWorkshop de Arquitecturas, Redes y Sistemas Operativos (WARSO)Red de Universidades con Carreras en Informática (RedUNCI