Pesquisa no laboratório Ghanim foi apoiado por bolsa de pesquisa no. 908-42.12/2006 da Fundação Alemã-Israelense (GIF), conceder nenhuma. IS-4062-07 dos Estados Unidos-Israel Binacional Fundo das Nações Agrícola Pesquisa e Desenvolvimento (BARD) e bolsa de pesquisa no. 884/07 da Fundação para a Ciência Israel (ISF) para MG

1. Preparativos Geral Whitefly, Flora, e vírus para análise FISH <ol><li> B traseiro e Q whiteflies biótipo de plântulas de algodoeiro (Gossypium hirsutum L. cv. Acala) e manter dentro de gaiolas à prova de insetos e salas de crescimento sob condições padrão de 25 ± 2 ° C, 60% de umidade relativa, e uma luz de 14 horas / fotoperíodo escuro de 10 horas. </li><li> Executar PCR para testar a infecção com endosimbiontes utilizando iniciadores específicos de endossimbiontes como anteriormente descrito 15-19. </li><li> Compre mudas de tomate (Solanum esculentum cv. Beefsteak) a partir de um viveiro comercial ou de plantas de tomate sementes. </li><li> Manter sob as mesmas condições de criação descritos acima até que as plantas atingem o estágio de quatro folhas verdadeiras, a idade mais adequada para a inoculação mediada por mosca branca de TYLCV 15 (ver secção 4.1 abaixo). </li></ol> 2. Manipulação de insetos, Fixação, Probe projeto, hibridização e visualização para Endosimbionte FISH <ol><li> Colete 10 whiteflies adultos por aspiração de plantas de algodão, ou pegar 10 3 ª ou 4 ª ninfas de folhas de algodão. </li><li> Mergulhar imediatamente em fixador de Carnoy (ácido clorofórmio-etanol-ácido acético glacial, 06:03:01, v / v) em tubos Eppendorf. Correção para 2 horas durante a noite (de preferência durante a noite). </li><li> Remover completamente o fixador e descolorir em 6% de H 2 O 2 em etanol durante 2 horas. </li><li> Preservar as amostras em etanol absoluto por vários dias a várias semanas à temperatura ambiente, ou prosseguir para a próxima etapa. </li><li> Conceber sondas de DNA primers reversos como complementares com base nos genes de ARN ribossomal 16s de cada bactéria. Para projetar os testes, levar em conta as mesmas considerações para a concepção de iniciadores de PCR. Utilizar as sondas oligonucleotídicas BTP1 5&#39;-Cy5-TGTCAGTGTCAGCCCAGAAG-3 &#39;, 5&#39;-Rb1 Cy3 TCCACGTCGCCGTCTTGC-3&#39;, 5&#39;-Cy3 BTH-CCAGATTCCCAGACTTTACTCA-3 &#39;e 5&#39;-Ars2 Cy3TCATGACCACAACCTCCAAA-3 &#39;para o direcionamento específico de Portiera, Rickettsia, Hamilonella e Arsenophonus, respectivamente. </li><li> Hibridizam amostras durante a noite, no escuro, em tampão de hibridação (20 mM Tris-HCl, pH 8,0, 0,9 M de NaCl, 0,01% [peso / vol] de sulfato de dodecilo de sódio a 30% [vol / vol] formamida) contendo 10 pmol de sonda fluorescente por ml . Combinar mais do que uma sonda com diferentes corantes fluorescentes, se necessário. Use whiteflies não infectadas com a bactéria alvo e amostras não-sonda como controles negativos. </li><li> Monte as amostras de todo, numa lâmina de microscópio, em tampão de hibridação contido com um bloqueador de líquido, cubra com uma lamela, selar com unha polonês, e vista sob um microscópio confocal de fluorescência ou. </li></ol> 3. Mosca branca Órgão Dissection, Fixação, hibridização e visualização para TYLCV FISH <ol><li> Colete whiteflies adultos permanentemente criados em plantas de tomate TYLCV infectados por aspiração e anesthetize por exposição a toalha de papel impregnado com acetona. Expor whiteflies apenas para 1-2 min como demasiado longa exposição à acetona pode consertar tecidos moscas brancas. Use whiteflies nonviruliferous criados em plantas de tomate não infectadas e nenhuma amostra de sonda para controles para confirmar a hibridação específica. </li><li> Mount whiteflies sobre depressão lâminas de microscópio para dissecção de órgãos usando um microscópio estéreo. </li><li> Puxe a cabeça inseto do protórax e dissecar as glândulas salivares em 1x PBS suplementado com 1% de azul de toluidina mancha para melhor visualização, devido ao seu pequeno tamanho. Permitir que a absorção da mancha para 2-3 min. </li><li> Puxe o inseto além na junção entre o tórax eo abdômen para observar e expulsar o intestino médio. Expulse o conteúdo do abdômen, empurrando com uma pinça na ponta do abdome. </li><li> Remova cuidadosamente o PBS e azul de toluidina a partir de dissecações de sucesso e adicione delicadamente 300 ml de fixador de Carnoy para corrigir os órgãos para 5 min. </li><li> Add fixador de um dos lados do poço deprimido e não a partir do topo para evitar o movimento não controlado dos órgãos dissecados. Uma vez que tocar no fixador, os órgãos vai aderir ao vidro, e ao longo do processo que não se move. </li><li> Remover fixador e adicionar 500 ul de tampão de hibridação suplementadas com 10 pmoles da sonda de DNA fluorescente Cy5 5&#39;-GGAACATCAGGGCTTCGATA-3 &#39;, que é complementar a uma sequência no gene de proteína de revestimento de TYLCV. </li><li> Hibridizar durante a noite à temperatura ambiente no escuro por incubação da lâmina com a amostra no interior de uma pequena câmara húmida, composta de pequena caixa de plástico com toalha de papel húmido no fundo. </li><li> Após a hibridização, pegar os órgãos com uma ferramenta de dissecção fina e rapidamente movê-los para uma lâmina de microscópio fresco contendo 30 mL de novo tampão de hibridação suplementadas com DAPI (0,1 mg / ml em PBS 1x) e contidos com bloqueador de líquido. </li><li> Cubra as amostras com uma lamela, selar sagacidadeh unha polonês e vista sob um microscópio confocal de fluorescência ou. </li></ol> 4. Manipulação de Plantas, Mão-corte, fixação e hibridização para TYLCV FISH <ol><li> Inocular mudas de tomate com TYLCV usando inoculação mediada por mosca branca viróticas. O vírus pode ser detectado utilizando este método de FISH em plantas sem sintomas de uma semana após a inoculação. O sintoma típico são visíveis três semanas após a inoculação. Use plantas de tomate não infectadas e nenhuma amostra de sonda para controles para confirmar a hibridação específica. </li><li> Use uma lâmina de barbear histológica afiada para preparar 2-4 longos cortes longitudinais ou transversais cm cortados à mão de tomate caules e folhas. </li><li> Em tubos Eppendorf, fixar as secções em fixador de Carnoy, durante 2 horas a durante a noite à temperatura ambiente. </li><li> Remover fixador e preservar as seções em etanol absoluto por vários dias a várias semanas à temperatura ambiente, ou prosseguir para a próxima etapa. </li><li> Lavar as secçõestrês vezes, 1 min cada, em tampão de hibridação. </li><li> Hibridar as secções com 500 ul de tampão de hibridação suplementadas com 10 pmol do oligonucleótido sonda fluorescente Cy3-5&#39;-GGAACATCAGGGCTTCGATA-3 &#39;, que é complementar a uma sequência no gene de proteína de revestimento de TYLCV. </li><li> Lavar as secções três vezes, 1 min cada, em tampão de hibridação. </li><li> Mount seções inteiras em tampão de hibridação suplementadas com DAPI (0,1 mg / ml em PBS 1x) e contidos com bloqueador de líquido, cubra com uma lamela, selar com unha polonês e vista sob um microscópio confocal de fluorescência ou. </li></ol>

<ol>
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O protocolo aqui descrito para a localização de um vírus de plantas, na sua planta hospedeira e vector de insecto, bactérias e endosymbiotic no seu hospedeiro mosca branca específico, pode ser adaptado para a localização de outros vírus em plantas e até mesmo em tecidos animais. Além disso, o protocolo pode ser utilizado para localizar bactérias endosymbiotic e patogénicos e outros micro-organismos em sistemas de plantas e animais. Os métodos descritos dependem de conceito simples de hibridação entre uma ...

Ao estudar as interacções entre os vírus de plantas e outros organismos patogénicos com as suas plantas hospedeiras infectadas, é importante para visualizar os patógenos e seus respectivos ácidos nucleicos in situ, independentemente de causar efeitos negativos sobre os seus hospedeiros. Isto é muito importante quando se estuda o movimento patógeno dentro e entre as células de plantas. Localização in situ de produtos de genes do agente patogénico é um passo essencial que complementa outras abordagens para estudar o processo de patogenicidade. Muitos patógenos de plantas, principalmente os vírus, são transmitidas por insetos, com interações complexas e íntimas com seus vetores. A localização destes vírus em seus vectores é importante para estudar o caminho para a transmissão, e os possíveis locais de interacção no interior do vector. A transmissão de alguns vírus de plantas vetorizado por insetos é auxiliado por bactérias endosymbiotic que residem dentro desses insetos. Para melhor estudar a transmissão desses vírus de plantas by seus vectores, é também essencial para visualizar as bactérias endosymbiotic em geral, e aqueles que estão envolvidos na transmissão do vírus, em particular. Co-localização das bactérias e vírus endosymbiotic é assim desejado para investigar as possíveis relações entre esses organismos dentro de seu exército de insetos. Além da transmissão de vírus, bactérias endosymbiotic influenciar vários aspectos da biologia dos vectores de insecto, assim, a localização espacial destas bactérias dentro de insectos é de grande interesse e importância. Tomato virus (TYLCV) (Begomovirus, Geminiviridae) é o complexo de doença viral mais importante do tomateiro cultivado em todo o mundo 1-4. TYLCV é um vírus limita-floema e é exclusivamente vetorizado pela mosca branca Bemisia tabaci 5,6. Um modelo que descreve a translocação de begomovirus nos seus vectores de mosca branca tem sido proposto 6-9. Duas barreiras específicas estão ativamente cruzou durção esta transmissão circulativa: o intestino médio / hemolinfa e as barreiras da glândula hemolinfa / salivares. Este processo de transmissão é a hipótese de ser mediada pelos receptores desconhecido, que reconhecem a cápside do vírus. TYLCV é pensado para atravessar B. tabaci intestino 6,10, e é absorvida através da glândula salivar primário 6 antes de ser injectado para dentro da fábrica. Na hemolinfa mosca branca, TYLCV interage com uma proteína GroEL produzido pela bactéria endossimbiótica secundária inseto Hamiltonella. Essa interação garante o transporte seguro de TYLCV na hemolinfa, e protege-lo de um ataque pelo sistema imunológico do inseto 11. Hamiltonella e Portiera, o endosymbiont primário de moscas brancas, estão alojados em bacteriocytes, células de insetos encontrados na hemolinfa e casa endosymbiotic bactérias 11. B. tabaci abriga bactérias endosymbiotic adicionais, incluindo Rickettsia, Arsenophonus, Wolbachia e FritschEA, que pode ser localizada no interior ou no exterior das bacteriocytes, e têm efeitos diversos sobre a biologia do insecto 12. Vários relatórios têm tentado estudar a localização de TYLCV em plantas e moscas brancas, usando protocolos demorados e dispendiosos, como a microscopia eletrônica de transmissão (TEM), anticorpos e RNA in situ na seção de espessura microscópica 10,13, um estudo recente descreveu a localização de vírus de plantas dentro de suas plantas hospedeiras e de vetor usando um protocolo simples 14. Aqui nós descrevemos um protocolo simples para a localização de TYLCV em B. tabaci dissecado intestino médio e glândulas salivares, e nas seções preparados a partir de plantas TYLCV infectados. Descrevemos ainda mais a localização de Portiera, o endosymbiont primária de B. tabaci, e seus endossimbiontes secundárias Hamitonella, Rickettsia e Arsenophonus. Este protocolo é baseado no uso de sondas de DNA curtas, quesão marcadas por fluorescência em sua extremidade 5 &#39;, e hibridam especificamente com sequências complementares em sequências de genes virais ou bacterianas. O processamento das amostras é relativamente fácil e que o sinal obtido é altamente específico. O protocolo descrito pode ser utilizado para localizar os vírus, bactérias e outros organismos patogénicos nas plantas, animais e insectos hospedeiros, e ainda pode ser utilizado para localizar o ARNm em qualquer dado tecido.

<table border="0" cellpadding="0" cellspacing="0" width="653">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fluorescently labeled&#160; Probes</td>
			<td style="width:95px;">Metabion</td>
			<td style="width:129px;">20 bp HPLC purified</td>
			<td style="width:212px;">Sequence designed by customer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Toluidine blue</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">89640</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">sodium dodecyl sulfate</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">L3771</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Formamide</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">F9037</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Tris-HCl</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">T5941</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Glacial Acetic Acid</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">320099</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Blocker</td>
			<td>Ted Pella Inc.</td>
			<td>22309</td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues

Hibridização fluorescente in situ (FISH) é um nome dado a uma variedade de técnicas vulgarmente utilizadas para a visualização de transcritos de genes em células eucarióticas e pode ser ainda modificado para visualizar os outros componentes da célula, tais como a infecção com os vírus e as bactérias. Localização espacial e visualização de vírus e bactérias, durante o processo de infecção é um passo essencial que complementa a expressão profiling experiências como microarrays e RNAseq em resposta a diferentes estímulos. Compreender as infecções espaço-temporais com esses agentes complementa experiências biológicas destinadas a compreender a sua interação com componentes celulares. Várias técnicas para visualizar os vírus e as bactérias, tais como sistemas de gene repórter ou métodos imuno-histoquímicos são demoradas, e alguns estão limitados a trabalhar com organismos modelo e as metodologias complexas. PEIXES que tem como alvo de RNA ou DNA de espécies na célula é um me relativamente fácil e rápidoCTO para o estudo de localização espaço-temporal de genes e para fins de diagnóstico. Este método pode ser robusto e relativamente simples de implementar quando os protocolos empregar curto hibridação, as sondas comercialmente adquiridos, que não são caros. Isto é particularmente robusto, quando a preparação da amostra, a fixação, a hibridação, e a visualização microscópica não envolvem etapas complexas. Aqui nós descrevemos um protocolo para a localização de bactérias e vírus em tecidos de insetos e plantas. O método baseia-se na preparação simples, a fixação, e hibridação de insectos montagens inteiras e órgãos ou partes de plantas dissecadas feitos à mão, com 20 pares de bases de DNA sondas curtas conjugados com corantes fluorescentes na sua 5 &#39;ou 3&#39;. Este protocolo foi aplicado com sucesso a um número de tecidos de insecto e vegetais, e pode ser usada para analisar a expressão de ARNm ou de outras espécies de RNA ou de DNA na célula.

O sistema estudado neste manuscrito é mostrado na Figura 1 e inclui uma planta infectada com TYLCV, um adulto e uma ninfa da mosca branca B. tabaci, ea anatomia interna da mosca-branca, mostrando o caminho para a translocação TYLCV no inseto. Figura 2 mostra dupla FISH em uma mosca adulta para o simbionte primário Portiera eo simbionte secundário Arsenophonus. Figura 3 mostra dupla FISH para Portiera e Hamiltonella e <st...

Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues | Immunology and Infection | Video

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Descrevemos aqui um simples fluorescência de hibridação in situ (FISH), o método para a localização de vírus e bactérias, em tecidos de plantas e de insectos. Este protocolo pode ser estendido para a visualização de mRNA em toda montagem e cortes microscópicos.

Fluorescência<em&gt; In situ</em&gt; Hibridizações (FISH) para a localização de vírus e bactérias Endosymbiotic em Plant e Insect Tecidos

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

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Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

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23.2K visualizações.  Volcani Center.  Descrevemos aqui um simples fluorescência de hibridação in situ (FISH), o método para a localização de vírus e bactérias, em tecidos de plantas e de insectos. Este protocolo pode ser estendido para a visualização de mRNA em toda montagem e cortes microscópicos. 

Vídeo: Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluxo de ácido nucléico bloqueado Citometria de fluorescência In situ Hibridação (LNA fluxo FISH): um método para detecção de RNA bacteriano Pequenas

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Imunologia e Infecção

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescência métodos de microscopia para determinar a viabilidade de bactérias em associação com células de mamíferos

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

A detecção in situ de bactérias dentro dos tecidos de parafina-encaixados Usando um marcado com Digoxina DNA Probe Segmentação 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificação de sequências de localização Plasmodesmal em proteínas em Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protocolos para investigar a distribuição no tecido hospedeiro, o modo de transmissão e o efeito na aptidão do hospedeiro de um Densovírus na lagarta do algodoeiro

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

-Fluorescência baseado Neuraminidase Ensaio de Inibição Para avaliar a susceptibilidade do vírus influenza A de neuraminidase Inibidor de Classe Antivirais

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Hibridação in situ da fluorescência quantitativa (peixes) e immunofluorescence (se) de produtos específicos do gene em pilhas KSHV-infectadas

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Microscopia de Fluorescência de células vivas para investigar a localização de proteínas subcelulares e alterações da morfologia celular nas bactérias

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

Localização e Quantificação de Begomovírus em Tecidos Whitefly por Imunofluorescência e PCR Quantitativo

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...