Esta producción y el trabajo experimental presentado en este documento refleja la ayuda generosa y entusiasta de Reynaldo Pareja amablemente con el apoyo de Lina Aguirre, Gilberto Córdoba, William Mera, Ximena Bonilla, y Viveros Darío. También agradecemos a John Miles y tres revisores anónimos por los comentarios que nos ayudó a mejorar el vídeo y el manuscrito.

1. Plantas <ol><li> Esquejes solo tallo de las plantas maduras se usan como unidades experimentales. Los esquejes se cortan a 10 cm para asegurar la uniformidad del material de siembra. </li><li> Para prevenir la contaminación por patógenos de las plantas, los cortes se lavan en una solución de hipoclorito de sodio al 3% durante 2 minutos y luego enjuagar con agua del grifo thoughroughly. </li><li> Cada corte se siembra en ca. 36 g de suelo estéril en un tubo cilíndrico de PVC (5,3 cm de diámetro exterior de 6,5 cm de altura) sellado en el extremo inferior con un vaso de plástico y cubierto con un manguito de PVC. El corte se mantiene en su lugar por un anillo de espuma colocada en la abertura central del buje. </li><li> Las plantas son fertilizados y regados según sea necesario para mantener la humedad adecuada del suelo. </li><li> Las plantas crecen durante un mes antes de ser cuestionado por los insectos, lo que permite el desarrollo de raíces superficiales, que sirven como lugares de alimentación de las ninfas del salivazo raíz de alimentación del xilema. </li><li> Tubos de PVC con estacas enraizadas se invierten durante 8 días antes de la infestación para estimular aún más el crecimiento de las raíces superficiales, parte de ajustar las diferencias inherentes a la arquitectura de las raíces. Este paso requiere de una fuente artificial de luz, que nos ofrecen para el día 24 ha. Para evitar escapes de confusión de los herbívoros con resistencia a los herbívoros, de cada planta se reproduce con raíces superficiales pocas o ninguna inversión después de este tratamiento son eliminados del experimento. Esta eliminación debe ser poco frecuente, no conduce a la eliminación del genotipo que representan. </li></ol> 2. Los insectos <ol><li> Salivazo adultos son recogidos en el campo, identificaron a las especies, e introdujeron a las jaulas de oviposición en el que se proporcionan follaje de brachiariagrasses susceptibles a alimentarse. Las hembras ponen sus huevos en el suelo contenido en una bandeja de poca profundidad extraíble colocada en el fondo de las jaulas. </li><li> Después de varios días, la tierra está suspendida en el agua y pasa a través de una serie de tamices (42, 60 y malla 150) para recoger los huevos. La suspensión del material tamizado en una solución salina al 30% hace que los óvulos maduros para flotar, dejando tras de óvulos inmaduros y cualquier materia orgánica residual. </li><li> Para prevenir la contaminación por agentes patógenos de insectos, los huevos son desinfectados en una solución de hipoclorito de sodio al 0,5% durante 5 minutos y luego enjuagar con agua destilada. </li><li> Cada planta (unidad experimental) está infestada en la superficie del suelo con seis huevos maduros. Sólo los óvulos maduros cerca de la eclosión se seleccionan para las infestaciones. Se los puede identificar por la presencia de dos puntos rojos en su parte anterior, que corresponde a los ojos de los insectos, marcas de color rosa en su parte posterior, correspondiente al abdomen del insecto, y un opérculo totalmente expandido. </li><li> El éxito de eclosión se confirma cuatro días más tarde, momento en el que cualquier huevo sin eclosionar se sustituye por un salivazo recién nacido de nuestra colonia. </li><li> Las plantas están dispuestos en un diseño de bloques completos al azar con seis repeticiones por genotipo. </li></ol> 3. Evaluaciones <ol><li> El experimento se evalúa ca. 30 días después de la infestación (tiempos exactos varían dependiendo de las especies de insectos estudiados). </li><li> Los daños se califica en una escala de 1 a 5 visual, donde 1 corresponde a ningún daño visible y 5 corresponde a una planta muerta (Figura 1). Daños a las plantas de puntuación proporciona una estimación de la tolerancia. </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="Figura 1" /> Figura 1. Daños escala utilizada para evaluar la tolerancia al salivazo herbivoría en Brachiaria spp. 1 = sin daño detectable, 2 = 0-25% hojas necróticas, 3 = 25-50% de hojas necróticas, 4 = 50-75% de hojas necróticas y 5 = 75-100% de hojas necróticas. <ol start="3"><li> La supervivencia del insecto se evaluó contando los insectos o bien llegar a su estadio final o el estado adulto (Figura 2). La puntuación para la supervivencia del insecto se ofrece una estimación de la antibiosis. </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="Figura 2" /> Figura 2. Salivazo desarrollo de primer estadio (izquierda) a la edad adulta (derecha). Insectos sólo llega por lo menos en su estadio final (flechas) en la fecha de evaluación se consideran como &quot;sobrevivientes&quot;. 4. Los resultados representativos Los resultados de un ensayo de híbridos de Brachiaria representante de la detección de resistencia a la reducta salivazo Aeneolamia se presentan en la Figura 3. <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="Figura 3" /> Figura 3. Los resultados de detección híbridos de Brachiaria resistentes a salivazo Aeneolamia reducta. Consideramos que las plantas resistentes, si su respuesta a la herbivoría cae en la mitad inferior de esta trama. Aumenta antibiosis de derecha a izquierda y aumenta la tolerancia de arriba a abajo. Las flechas señalan nuestros cheques resistentes.

<ol>
	<li>Painter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect resistance in crop plants. , (1951).</li><li>Kogan, M., Ortman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antixenosis-a+new+term+proposed+to+define+Painter's+'Nonpreference'+modality+of+resistance.">Antixenosis-a new term proposed to define Painter's 'Nonpreference' modality of resistance.</a> Bull. Entomol. Soc. Am. 24, 175-176 (1978).</li><li>Kennedy, G., Gould, F., Deponti, O., Stinner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+agricultural,+genetic,+and+commercial+considerations+in+the+deployment+of+insect-resistant+germplasm.">Ecological, agricultural, genetic, and commercial considerations in the deployment of insect-resistant germplasm.</a> Environ. Entomol. 16, 327-338 (1987).</li><li>Holmann, F., Peck, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+damage+of+grassland+spittlebugs+(Homoptera+Cercopidae)+in+Colombia:+a+first+approximation+of+impact+on+animal+production+in+Brachiaria+decumbens.">Economic damage of grassland spittlebugs (Homoptera Cercopidae) in Colombia: a first approximation of impact on animal production in Brachiaria decumbens.</a> Neotrop. Entomol. 31, 275-284 (2002).</li><li>Miles, J. W., Cardona, C., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+selection+in+a+synthetic+brachiariagrass+population+improves+resistance+to+three+spittlebug+species.">Recurrent selection in a synthetic brachiariagrass population improves resistance to three spittlebug species.</a> Crop Sci. 46, 1088-1093 (2006).</li><li>Ferrufino, A., Lapointe, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+resistance+in+Brachiaria+grasses+to+the+spittlebug+Zulia+colombiana.">Host plant resistance in Brachiaria grasses to the spittlebug Zulia colombiana.</a> Entomol. Exp. Appl. 51, 155-162 (1989).</li><li>Lapointe, S. L., Serrano, M. S., Arango, G. L., Sotelo, G., Cordoba, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+to+spittlebugs+(Homoptera:+Cercopidae)+in+accessions+of+Brachiaria+spp.">Antibiosis to spittlebugs (Homoptera: Cercopidae) in accessions of Brachiaria spp.</a> J. Econ. Entomol. 82, 1764-1766 (1992).</li><li>Cardona, C., Miles, J. W., Sotelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+methodology+for+massive+screening+of+Brachiaria+spp.+genotypes+for+resistance+to+Aeneolamia+varia+(Homoptera:+Cercopidae).">An improved methodology for massive screening of Brachiaria spp. genotypes for resistance to Aeneolamia varia (Homoptera: Cercopidae).</a> J. Econ. Entomol. 92, 490-496 (1999).</li><li>Cardona, C., Fory, P., Sotelo, G., Pabon, A., Diaz, G., Miles, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiosis+and+tolerance+to+five+species+of+spittlebug+(Homoptera:+Cercopidae)+in+Brachiaria+spp.:+implications+for+breeding+for+resistance.">Antibiosis and tolerance to five species of spittlebug (Homoptera: Cercopidae) in Brachiaria spp.: implications for breeding for resistance.</a> J. Econ. Entomol. 97, 635-645 (2004).</li><li>Smith, C., Khan, Z., Pathak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques for evaluating insect resistance in crop plants. , (1994).</li><li>Lapointe, S., Sotelo, G., Arango, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+technique+for+rearing+spittlebugs+(Homoptera:+Cercopidae).">Improved technique for rearing spittlebugs (Homoptera: Cercopidae).</a> J. Econ. Entomol. 82, 1764-1766 (1989).</li><li>Lopez, F., Cardona, C., Miles, J. W., Sotelo, G., Montoya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+resistance+to+adult+spittlebugs+(Hemiptera:+Cercopidae)+in+Brachiaria+spp.:+methods+and+categories+of+resistance.">Screening for resistance to adult spittlebugs (Hemiptera: Cercopidae) in Brachiaria spp.: methods and categories of resistance.</a> J. Econ. Entomol. 102, 1309-1316 (2009).</li></ol>

Discriminar entre los mecanismos de resistencia a los herbívoros pueden iluminar los esfuerzos de mejoramiento de cultivos 3. Resistencia basada en antibiosis fuerte podría, en determinadas circunstancias, crean presiones de selección de biotipos de plagas más agresivas. Por otro lado, la resistencia basada en la tolerancia podría permitir a las poblaciones de plagas para incrementar la tolerancia hasta que finalmente se siente abrumado. Mejoramiento de la resistencia durable, por lo tanto, requiere una cuidadosa consideración de lo que los mecanismos de resistencia que confiere más estabilidad para un determinado cultivo / sistema de plagas [ver Kennedy et al. (1987) para una discusión amplia sobre el tema] 3. Este video de demostración se basa en varios estudios previos diseñados para estimar las contribuciones relativas de antibiosis y tolerancia a la resistencia al salivazo en brachiariagrasses 6-9. En contraste con las pruebas de selección o los experimentos de campo, no hay pruebas de selección asegurar que todas las plantas reciben la presión de los herbívoros mismo, proporcionando así el control de las diferencias que surjan a partir del comportamiento de herbívoros (por ejemplo, la preferencia de acogida, la agregación). Por esta razón, sin embargo, no escogidos no son adecuados para evaluar la resistencia de antixenosis. Crianza artificial de las colonias de insectos es el método preferido para obtener insectos de prueba para los ensayos de detección de resistencia 10. Las colonias son una fuente confiable y uniforme de salivazo de una época conocida por los experimentos que se llevó a cabo en cualquier época del año 11. Puede, sin embargo, será necesario inyectar periódicamente individuos silvestres en la colonia para asegurarse de que la colonia no se desvía genéticamente de las poblaciones de campo pertinente 10. Evaluar la idoneidad de estos herbívoros artificialmente criados, y monitorear posibles cambios en su agresividad a través del tiempo, es importante que todas las proyecciones son los adecuados controles de los niveles de resistencia conocida. Varios parámetros que debe definirse claramente el diseño de un ensayo de resistencia a un examen adecuado. A unos pocos parámetros clave que hemos considerado para diseñar nuestro examen son, entre la edad de las plantas huésped, la especie y etapa de desarrollo del salivazo, el nivel de infestación, y la duración del contacto planta-insecto 8,9,12. Varias rondas de la experimentación y el perfeccionamiento del protocolo puede ser necesario para llegar a una rápida y rentable, ensayo fiable, que predice adecuadamente la resistencia a los herbívoros en el campo.

characterizing herbivore resistance mechanisms: spittlebugs on brachiaria spp. as an example

Las plantas pueden resistir el daño de herbívoros a través de tres mecanismos principales: antixenosis, antibiosis y tolerancia 1. Antixenosis es el grado en que se evita la planta cuando el herbívoro es capaz de seleccionar las plantas de otras dos. Antibiosis es el grado en que la planta afecta al buen estado de los herbívoros se alimentan de ella 1. La tolerancia es el grado en que la planta puede soportar o reparar el daño causado por los herbívoros, sin comprometer el crecimiento del herbívoro y la reproducción de uno. La durabilidad de la resistencia a los herbívoros en un entorno agrícola depende en gran medida en el mecanismo de resistencia favorecidos durante los esfuerzos de mejoramiento de los cultivos 3. Se demuestra un experimento sin elección diseñado para estimar la contribución relativa de antibiosis y tolerancia a la resistencia al salivazo en Brachiaria spp. Varias especies de pastos africanos del género Brachiaria son forrajes valiosos y plantas forrajeras en el Neotrópico, pero pueden ser severamente cuestionado por varias especies nativas de salivazo (Hemiptera: Cercopidae). 4 Para evaluar la resistencia al salivazo, las plantas propagadas vegetativamente son por estacas y se dejan crecer durante aproximadamente un mes, lo que permite el crecimiento de las raíces superficiales en los que puede alimentar a salivazo. En ese momento, cada planta es de forma individual desafiados con seis huevos de salivazo cerca de la eclosión. Las infestaciones se les permite el progreso de un mes antes de la evaluación de daños a las plantas y la supervivencia del insecto. Daños a las plantas de puntuación proporciona una estimación de la tolerancia y anotó la supervivencia del insecto se ofrece una estimación de la antibiosis. Este protocolo ha facilitado nuestro objetivo cultivo de plantas para mejorar la resistencia al salivazo en brachiariagrases comerciales 5.

Watch this Scientific Journal Video about Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example at JoVE.com

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Este vídeo explica los mecanismos de resistencia en las plantas a la herbivoría y demuestra una prueba de no-elección que estima la contribución relativa de antibiosis y tolerancia al salivazo de resistencia en<em&gt; Brachiaria</em&gt; Spp.

Caracterización de los mecanismos de resistencia Herbívoro: salivazo en Brachiaria Spp. como ejemplo

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

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Research

JoVE Journal

Biology

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

13.7K vistas.  CIAT. Este vídeo explica los mecanismos de resistencia en las plantas a la herbivoría y demuestra una prueba de no-elección que estima la contribución relativa de antibiosis y tolerancia al salivazo de resistencia en Brachiaria Spp.

Video: Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Investigación

Biología

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Inducida por herbívoros-Volátiles Blueberry y señalización dentro de la planta

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Medición de la osmótica de agua Coeficiente de Permeabilidad (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos de plantas aisladas como ejemplo

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La técnica de infiltración-centrifugación para extracción de apoplástico fluido procedente de hojas de la planta Uso<em&gt; Phaseolus vulgaris</em&gt; Como ejemplo

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

Proyección de imagen de FITC-dextrano como reportero para exocitosis regulada

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

Explorando los epitrancrismos de m6A y m5C sobre la infección viral: un ejemplo con el VIH

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Caracterización de las propiedades mecánicas de la pared celular primaria en órganos vegetales vivos mediante microscopía de fuerza atómica

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

Un método de electroporación para transformar Rickettsia spp. con un vector lanzadera fluorescente que expresa proteínas en líneas celulares de garrapatas

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Investigación del mecanismo de transporte de zinc en células de mamíferos con el ensayo FluoZin-3

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Autor destacado: Explorando la regulación celular del zinc a través de la funcionalidad de ZnT1 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Caracterización de transportadores de zinc en mamíferos mediante un ensayo de transporte de zinc in vitro

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...