Esta produção e do trabalho experimental aqui apresentadas refletem a ajuda dedicada e entusiasta de Reynaldo Pareja gentilmente apoiado por Lina Aguirre, Córdoba Gilberto, Mera William, Bonilla Ximena, e Darío Viveros. Agradecemos também a John Miles e três revisores anônimos pelos comentários que nos ajudou a melhorar o vídeo e manuscrito.

1. Plantas <ol><li> Estacas única a partir de plantas adultas são utilizadas como unidades experimentais. As estacas são cortadas a 10 cm para garantir a uniformidade do material de plantio. </li><li> Para evitar a contaminação por patógenos de plantas, os cortes são lavados em uma solução de hipoclorito de sódio a 3% para 2 min e depois enxaguados com água da torneira thoughroughly. </li><li> Cada corte é plantada em ca. 36 g de solo estéril em um tubo de PVC cilíndricos (5,3 centímetros de diâmetro externo de 6,5 cm de altura) selado na extremidade inferior com uma taça de isopor e revestido por uma bucha de PVC. O corte é mantida no lugar por um anel de espuma colocada na abertura central da bucha. </li><li> Plantas são fertilizadas e regado conforme necessário para manter a umidade do solo adequada. </li><li> Plantas vão crescer durante um mês antes de ser desafiado por insetos, permitindo o desenvolvimento de raízes superficiais, que servem como locais para alimentação das ninfas-raiz xilema alimentação cigarrinha. </li><li> Tubos de PVC com estacas enraizadas são invertidos para 8 d antes de infestação para estimular ainda mais o crescimento de raízes superficiais, parcialmente o ajuste para diferenças inerentes na arquitetura de raiz. Esta etapa requer uma fonte de luz artificial, que prevê 24 dias ha. Para evitar confusão escapa da herbivoria com resistência herbívoro, planta individual repetições, com pouca ou nenhuma raiz superficial após este tratamento inversão são eliminados do experimento. Esta eliminação deve ser freqüente, nunca levando à eliminação do genótipo que eles representam. </li></ol> 2. Insetos <ol><li> Cigarrinhas adultas são coletados a partir do campo, identificou a espécie, e introduziu a gaiolas de oviposição onde são prestados folhagem de brachiariagrasses suscetíveis para se alimentar. As fêmeas colocam seus ovos no solo contidos em uma bandeja rasa removíveis colocados no fundo das gaiolas. </li><li> Depois de vários dias, o solo está em suspensão na água e passou por uma série de peneiras (42, 60 e 150 mesh) para coletar os ovos. Suspendendo o material peneirado em uma solução salina 30% faz com que os ovos maduros para flutuar, deixando para trás os ovos imaturos e qualquer matéria orgânica residual. </li><li> Para evitar a contaminação por patógenos de insetos, ovos são desinfetados com uma solução de hipoclorito de sódio 0,5% por 5 min e depois enxaguados com água destilada. </li><li> Cada planta-teste (unidade experimental) está infestada na superfície do solo com seis óvulos maduros. Apenas óvulos maduros perto de incubação são selecionados para infestações. Eles podem ser identificados pela presença de dois pontos vermelhos em sua parte anterior, correspondente aos olhos do inseto; marcas-de-rosa em sua parte posterior, correspondente ao abdômen do inseto, e um opérculo totalmente expandida. </li><li> Incubação de sucesso é confirmado 4 d tarde, altura em que qualquer ovo unhatched é substituído por um recém-nascido cigarrinha da nossa colônia. </li><li> Plantas de teste são dispostos em um delineamento em blocos casualizados com seis repetições por genótipo. </li></ol> 3. Avaliações <ol><li> O experimento é avaliada ca. 30 d após a infestação (horários exatos variam dependendo da espécie de inseto estudada). </li><li> Dano é pontuados em uma escala 1-5 visual, onde 1 corresponde a nenhum dano visível e 5 corresponde a uma planta morta (Figura 1). Pontuação danos às plantas fornece uma estimativa da tolerância. </li></ol><img src="/files/ftp_upload/3047/3047fig1.jpg" alt="Figura 1" /> Figura escala Danos 1. Utilizado para avaliar a tolerância a cigarrinha herbivoria em Brachiaria spp. 1 = nenhum dano detectável; 2 = folhagem 0-25% de necrose; 3 = folhas 25-50% de necrose; 4 = folhas 50-75% de necrose; 5 = folhagem 75-100% de necrose. <ol start="3"><li> Sobrevivência do inseto é avaliada através da contagem de insetos atingindo tanto instar seu final ou fase adulta (Figura 2). De pontuação para a sobrevivência do inseto fornece uma estimativa da antibiose. </li></ol><img src="/files/ftp_upload/3047/3047fig2.jpg" alt="Figura 2" /> Figura 2. Cigarrinha desenvolvimento de primeiro ínstar (esquerda) para adultos (direita). Insetos só chegar, pelo menos, instar os seus finais (setas) na data da avaliação são contadas como &quot;sobreviventes&quot;. 4. Resultados representativos Resultados de um ensaio de híbridos representante Brachiaria triagem para resistência à cigarrinha Aeneolamia reducta são apresentados na Figura 3. <img src="/files/ftp_upload/3047/3047fig3.jpg" alt="Figura 3" /> Figura 3. Resultados da triagem híbridos de Brachiaria para resistência à cigarrinha Aeneolamia reducta. Consideramos plantas resistentes, se sua resposta à herbivoria cai na metade inferior desta trama. Antibiose aumenta da direita para a esquerda e para a tolerância aumenta de cima para baixo. As setas apontam no nosso cheques resistente.

<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>

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Discriminar entre os mecanismos de resistência herbívoro pode iluminar os esforços de melhoramento das culturas 3. Resistência antibiose baseado em forte poderia, em algumas circunstâncias, criar pressões de seleção para os biotipos de pragas mais agressivas. Por outro lado, a resistência baseada na tolerância poderia permitir que as populações de pragas a aumentar até que, eventualmente, a tolerância é sobrecarregado. Melhoramento para resistência durável, portanto, requer uma análise cuidadosa do que mecanismos de resistência que conferem maior estabilidade para uma determinada cultura / sistema de pragas [ver Kennedy et al. (1987) para uma discussão abrangente sobre o tema] 3. Esta demonstração de vídeo baseia-se em vários estudos anteriores para estimar as contribuições relativas de antibiose e tolerância à resistência à cigarrinha em brachiariagrasses 6-9. Em contraste com testes de escolha ou experiências de campo, nenhuma escolha-testes garantir que todas as plantas recebem a pressão herbívoro mesma, proporcionando assim controlar as diferenças resultantes do comportamento de herbívoros (por exemplo, a preferência host, agregação). Por esta razão, no entanto, sem chance de escolha testes são inadequados para avaliar a resistência por antixenose. Artificialmente criação de colônias de insetos é o método preferido para obter insetos de teste para testes de resistência de triagem 10. Colônias fornecer uma fonte confiável e uniforme das cigarrinhas de uma era conhecido por experimentos a ser realizado em qualquer época do ano 11. Pode, no entanto, ser necessário infundir periodicamente indivíduos selvagens para a colônia para garantir que a colônia não se desvia de populações geneticamente campo relevante 10. Para avaliar a aptidão destes herbívoros artificialmente criados, e monitorar possíveis alterações na sua agressividade ao longo do tempo, é importante que todas as sessões incluem controlos adequados dos níveis de resistência conhecida. Vários parâmetros devem ser cuidadosamente definidos para projetar um ensaio de rastreio apropriado resistência. Alguns parâmetros-chave que têm considerado a projetar nosso teste incluem a idade das plantas hospedeiras, as espécies e estágio de desenvolvimento da cigarrinha, o nível de infestação, ea duração do contato planta-inseto 8,9,12. Várias rodadas de experimentação e refinamento do protocolo pode ser necessário para chegar a uma resposta rápida, de custo-benefício, ensaio, confiável, que prevê de forma adequada resistência herbívoro no campo.

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

As plantas podem resistir a danos de herbívoros através de três mecanismos principais: antibiose antixenose e tolerância 1. Antixenose é o grau em que a planta é evitado quando o herbívoro é capaz de selecionar outras plantas 2. Antibiose é o grau em que a planta afeta a aptidão do herbívoro alimentando-se de que 1. Tolerância é o grau em que a planta pode suportar ou reparar os danos causados ​​pelo herbívoro, sem comprometer o crescimento dos herbívoros e reprodução 1. A durabilidade da resistência de herbívoros em ambientes agrícolas depende, em grande medida, o mecanismo de resistência favorecidos durante os esforços de melhoramento das culturas 3. Demonstramos um experimento sem chance de escolha para estimar as contribuições relativas de antibiose e tolerância à resistência cigarrinha em Brachiaria spp. Várias espécies de gramíneas Africano do gênero Brachiaria são forrageiras valiosas e plantas de pastagem na região Neotropical, mas eles podem ser severamente desafiada por várias espécies nativas de cigarrinhas (Hemiptera: Cercopidae). 4 Para avaliar a sua resistência às cigarrinhas, as plantas são propagadas vegetativamente- por estaquia e permitiu a crescer por aproximadamente um mês, permitindo o crescimento de raízes superficiais em que cigarrinhas pode alimentar. Nesse ponto, cada planta teste é individualmente desafiados com seis ovos próximos cigarrinha incubação. Infestações são permitidos para o progresso de um mês antes de avaliar danos às plantas e sobrevivência do inseto. Pontuação danos às plantas fornece uma estimativa da tolerância ao marcar sobrevivência do inseto fornece uma estimativa da antibiose. Este protocolo tem facilitado o nosso objetivo melhoramento de plantas para aumentar a resistência cigarrinha em brachiariagrases comercial 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 os mecanismos de resistência da planta hospedeira à herbivoria e demonstra um teste sem chance de escolha que as estimativas da contribuição relativa de antibiose e tolerância a cigarrinha resistência em<em&gt; Brachiaria</em&gt; Spp.

Mecanismos Herbívoro caracterizando Resistance: Spittlebugs em Brachiaria Spp. como Exemplo

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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Biology

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

13.7K visualizações.  CIAT. Este vídeo explica os mecanismos de resistência da planta hospedeira à herbivoria e demonstra um teste sem chance de escolha que as estimativas da contribuição relativa de antibiose e tolerância a cigarrinha resistência em Brachiaria Spp.

Vídeo: 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.

Estudos genéticos de proteínas de reparo do DNA Humano Utilizando Levedura como um sistema modelo

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

Biologia

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.

Herbívoro induzida Voláteis Blueberry e planta-Intra Sinalização

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.

Medindo o Coeficiente de Permeabilidade osmótica de água (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos planta isolada como um exemplo

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.

A técnica de infiltração-centrifugação para Extração de apoplásticas Fluid das folhas da planta Usando<em&gt; Phaseolus vulgaris</em&gt; Como exemplo

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.

Imagem latente FITC-dextrano como repórter por exocitose 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.

Calibração e uso de desempenho da MRM Microcoil demonstrados em raízes de truncatula medicago a 22 T

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 epitransomos m6A e m5C sobre infecção viral: um exemplo com 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 ...

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.

Caracterizando as Propriedades Mecânicas da Parede Celular Primária em Órgãos de Plantas Vivas Usando Microscopia de Força Atômica

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.

Um método de eletroporação para transformar Rickettsia spp. com um vetor shuttle fluorescente de expressão de proteínas em linhagens celulares de carrapatos

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

Investigando o mecanismo de transporte de zinco em células de mamíferos com o ensaio 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 em destaque: Explorando a regulação celular do zinco por meio da funcionalidade 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.

Caracterização de Transportadores de Zinco em Mamíferos Utilizando um Ensaio de Transporte de Zinco 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 ...