This work was funded by the Creative Inquiry Program for Undergraduate Research and the Calhoun Honors College at Clemson University.

1.-Geral Trypanosoma Pecuária <ol><li> Pesar sólidos para a preparação dos meios SDM79 (Tabela 1). </li><li> Armazenar a 4 ° C em 50 mL cónico ou um saco plástico. NOTA: Os reagentes são estáveis ​​durante pelo menos 6 meses. </li><li> Descongelar soro fetal bovino (FBS) em um banho de água a 37 ° C, e misturar periodicamente por inversão. NOTA: FBS é recebida do fornecedor, como uma solução estéril. Filtro de esterilização FBS reduz a sua capacidade para suportar o crescimento do parasita. </li><li> Uma vez que toda a garrafa é descongelada, calor inactivar soro durante 30 minutos num banho de água a 56 ° C, a mistura periodicamente para minimizar a precipitação de componentes do soro. </li><li> Descongelar a penicilina / estreptomicina (pen / strep), hemina (2 mg / ml em NaOH a 50 mM), e solução basal águia vitamina meio à temperatura ambiente. </li><li> Para uma proveta graduada de 1000 ml, adicionar 20,2 g de sólidos SDM79 (Tabela 1) para os componentes líquidos (Tabela 2) e misture bem com uma placa de agitação. </li><li> Ajustar o pH a 7,35, e perfazer o volume de 850 ml com água. </li><li> Filtrar esterilizar mídia ligando uma mangueira de vácuo para o fundo de uma garrafa de filtro. Aplicar vácuo até que a solução é filtrada. </li><li> Remover top filtro no gabinete de biossegurança e adicione 150 ml de inactivado pelo calor FBS. Remover cobertura plástica de tampa estéril e garrafa mídia selo. </li><li> Prepare mídia SDM80 da mesma maneira como SDM79 com as seguintes excepções; não adicione açúcar ou glucosamina e usar MEM sem glutamina. Em lugar de 150 ml de FBS inactivado pelo calor, adicionar 135 ml de dialisado, FBS inactivado pelo calor, e 15 mL de FBS inactivado pelo calor. </li><li> Cultura parasitas PCF em cada 25 cm2 frasco com 10 ml de meio apropriado a 27 ° C e 5% de CO 2. NOTA: As células devem ser contadas diariamente utilizando um hemocitômetro ou um citômetro de fluxo (ver secção 6) e culturas devem ser mantidas em densidades entre 1 x 10 5 e 5 x 10 6 células / ml. </li></ol> 2 Transfection de PCF Parasitas com a construção repórter fluorescente NOTA: Para seguir glicossomo dinâmica, uma proteína repórter, contendo a sequência de direccionamento de peroxissomas (PTS2) de aldolase fundido reforçado a proteína fluorescente amarela é expresso nos parasitas. A sequência que codifica a proteína de fusão foi clonado no pXS2bla 12, que contém o promotor procyclin e as regiões intergénicas de tubulina, que a recombinação homóloga no genoma directa e o gene de resistência à blasticidina para selecção. Linhas celulares de pró-cíclicos, contendo os genes que codificam a polimerase de T7 e tetraciclina repressor (PF29-13) são transformadas com a construção de direccionamento, pXS2: PTS2eYFP. <ol><li> Prepare cytomix misturando os componentes listados na Tabela 3. Filtre esterilizar e armazenar a RT. </li><li> DNA de plasmídeo linearizado com Mlu I, pipetando 10 mL de ADN (1 mg / mL), tampão de enzima de restrição de 5 ul, 33 ul de água, e 2 μ; L de enzima Mlu I para um tubo de microcentrífuga. Incubar a 37 ° C durante 1 hora. </li><li> Purifica-se a digestão de restrição por adição de um volume de tampão de ligação com a enzima de restrição digest. Misturar tampão de ligação e DNA digerido com vórtex. Resumidamente, centrifugar para recolher a amostra no fundo do tubo. </li><li> Inserir uma coluna de ADN de ligação para um tubo de recolha de 2 ml, e a transferência de ADN digerido / ligante solução tampão à coluna. </li><li> Centrifugar por 10.000 xg por 1 min. Após a centrifugação, descartar filtrado do tubo de coleta e retornar a coluna para o tubo de coleta. </li><li> Adicionar 700 ul de tampão de lavagem SPW e centrifugar a 10000 xg durante 1 min. </li><li> Filtrado Descarte de tubo de coleta, retornar a coluna de tubo de coleta e repetir SPW etapa de lavagem e centrifugação. </li><li> Descartar filtrado, coluna de retorno ao tubo de recolha, e centrifugar coluna vazio durante 3 minutos a 10000 xg para remover o etanol residual. </li><li> Em uma cabine de segurança biológica, jogue fora collection tubo e lugar da coluna em um tubo de microcentrífuga estéril. </li><li> Para eluir o ADN, adicionar 25 ul de água estéril para coluna e incuba-se à temperatura ambiente durante 2 min. Centrifuga-se a 10000 xg durante 1 min. </li><li> Adicionar mais 25 ul de água estéril na capa de biossegurança coluna e incuba-se à temperatura ambiente durante 2 min. </li><li> Centrifugar a 10.000 velocidade xg por 1 min. </li><li> Numa câmara de biossegurança transferir todo o volume de ADN para um novo tubo de microcentrifuga estéril. Nota: Esta etapa evita a contaminação da tampa aberta durante a centrifugação. </li><li> Adicionar o ADN purificada estéril,, linearizado (10 ng) a 400 ul de cytomix esterilizada por filtração. </li><li> Contagem de células, como descrito na seção 6 e colher 5 x10 7 a 10 8 células por centrifugação a 800 xg por 15 minutos em temperatura ambiente. </li><li> Decantar o sobrenadante e ressuspender o sedimento de células em 450 ul de ADN + cytomix usando uma pipeta de 1 ml serológico. </li><li> Solução de transferência contendo ce<html> lls, ADN, e cytomix para uma cuvete estéril 4 milímetros lacuna e lugar da cuvete na câmara de electroporação. </li><li> Selecione &quot;decaimento exponencial&quot; e digitar manualmente as seguintes configurações: Tensão: 1,5 kV, capacitância: 25 mF, Resistance: Ω, e Cuvette: 4 mm. Pressione pulso. Uma vez que o impulso está completa, remover cuvete da câmara de electroporação e retornar para o armário de biossegurança. </li><li> Para um novo frasco estéril, pipeta 10 ml de SDM79. Transferência das células transformadas para o balão com 10 ml de SDM79. </li><li> Incubar SEM selecção de drogas durante 24 horas a 27 ° C, 5% de CO 2. Após 24 horas, adicionar G418 (15 mg / mL), higromicina (50 ug / ml), e blasticidina (10 ug / ml). Passar 1 ml de células transformadas com droga em 9 ml de SDM79 com droga. </li><li> Analise as células diariamente conforme descrito nas seções 6 e 7 Quando as células são fluorescentes, e chegaram a uma densidade celular de 1 x 10 7 / ml, fazer stabilates para armazenamento de longo prazo (3,1-3,3). </li></ol>ve_title &quot;&gt; 3. Fazer Stabilates <ol><li> Para fazer com que meios de congelação, adicionar um volume igual de glicerol a 100% e para cytomix filtro de esterilização. </li><li> Adicionar 200 ul de meio de congelação a 800 ul de células (1 x 10 7) em um frasco de congelação, misturar suavemente por inversão e lugar entre duas prateleiras de esferovite e congelar a -80 ° C. </li><li> Uma vez congelado (~ 24 horas), as células de transferência para nitrogênio líquido. NOTA: É importante mover em células LN 2, após 24 h, como um armazenamento mais longo à temperatura de -80 ° C, leva a uma diminuição da viabilidade celular. </li></ol> 4. Stocks descongelamento congelados <ol><li> Remover o frasco congelado de -80 ° C e descongelamento à temperatura ambiente durante cerca de 10 min. </li><li> Pipetar 9 ml de SDM79 com droga para um novo frasco estéril. Adicionar células descongeladas para este balão. Passa células 1:10, adicionando 1 ml desta cultura a 9 ml de SDM79 em um novo frasco (concentração final de 1 x 10 5 / ml). </li><li> Antes de as células atingirem uma densidade de 10 7 / ml, começam citómetro definire ensaios de diluição. </li></ol> 5. Setup Cytometer <ol><li> Ligue o citômetro de fluxo e abrir o programa cflow Plus. NOTA: Antes de executar quaisquer amostras, os fluidos devem ser backflushed e enxaguado acordo com os passos 5,2-5,7. </li><li> Substitua o tubo de água nanopura em que o gole é armazenado com um tubo vazio. </li><li> Selecione o botão &quot;fluxo reverso&quot;. </li><li> Depois de retrolavagem é completa, remova o tubo de microcentrífuga de gole e descarte. </li><li> Colocar um novo tubo de microcentrifuga contendo 1 ml de água nova nanopura na sorvo. </li><li> Selecione um novo poço no programa cflow. Em &quot;Limites Run&quot; definir o tempo de 2 min. Em &quot;Fluidics&quot;, selecione &quot;rápido&quot; e &quot;Run&quot;. </li><li> Uma vez que o limite de tempo de 2 min é completa, clique no botão &quot;Apagar dados de exemplo&quot;. </li></ol> 6. contagem celular usando citômetro de fluxo <ol><li> Substituir a água com a amostra a ser contadas. Defina &quot;Limites Executar &quot;para 30 segundos,&quot; Fluidics &quot;para&quot; rápida &quot;e selecione&quot; Executar &quot;. </li><li> Após o prazo de 30 seg é completa, cflow mais irá fornecer os eventos / l em &quot;Last Run&quot;. Contagem Repita para cada cultura. NOTA: Antes de células chegar a 1 x 10 7 células / ml, prossiga com ensaio de diluição. As células devem ser interrompido após um ensaio de diluição for concluída. Células cultivadas por períodos mais longos perdem a capacidade de responder às condições ambientais. </li></ol> 7 Fluorescência de medição usando citômetro de fluxo <ol><li> Para avaliar a fluorescência, definir &quot;limites Run&quot; para 10 mil eventos e &quot;Fluidos&quot; para &quot;lento&quot;. Selecione &quot;Definir limite&quot;. Em &quot;Threshold Primária&quot;, selecione &quot;permanentemente eliminar eventos do&quot;, &quot;FSC-H&quot; (na caixa drop-down) e entre menos de 30.000. Clique em &quot;Aplicar&quot; e depois em &quot;fechar&quot;. </li><li> Selecione o220; &quot;botão em uma das novas janelas de plotagem e selecione&quot; Histograma FSC-A &quot;A partir da lista drop-down, selecione&quot; FL1-A &quot;. NOTA: medidas FL1-A fluorescência no comprimento de onda de 530 nm. </li><li> Selecione &quot;Run&quot; e repita para todas as culturas. </li><li> Executar a solução de limpeza através da linha de fluidos durante 2 min e água durante 2 min. </li></ol> 8. Diluição Ensaios <ol><li> Passar as células a uma densidade de 1 x 10 5 células / ml em 3 ml de SDM79 num balão de cultura de 2 25 cm e medir a fluorescência imediatamente após a passagem e, em seguida, em 3 horas e 24 horas. </li></ol> Análise dos dados do 9 <ol><li> Selecione &quot;Analisar&quot; guia sobre cflow Plus. </li><li> Clique no botão &quot;Histograma&quot; em &quot;Fazer uma nova trama.&quot; Selecionar &quot;FSC-A&quot; e uma lista drop-down aparecerá. Selecione o canal &quot;FL1-A.&quot; </li><li> Realce um bem para analisar. Selecione o botão &quot;Gate&quot;, e desenhar manualmente uma portapara a população de interesse. </li><li> Fluxo Plus fornecerá celular &quot;Contagem&quot; dentro desse portão e &quot;% dessa trama&quot;. </li></ol>

<ol>
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The authors have nothing to disclose.

Glicossomos são, dinâmico, organelas essenciais específicos do parasita. Os processos que regulam a biogênese, manutenção, proliferação e remodelação dessas organelas provavelmente incluem alvos de medicamentos que poderiam ser exploradas para fins terapêuticos. Apesar do elevado potencial de abundância de tais alvos de drogas, o campo de glicossomo biogênese ficou para trás o estudo de processos similares em outros organismos, predominantemente, devido à falta de um sistema tratável, de alto rendimento ...

Trypanosoma brucei causa a doença do sono Africano em seres humanos e uma doença debilitante, nagana, em bovinos. As drogas utilizadas no tratamento das referidas doenças são antiquado e extremamente tóxico, as vacinas não estão disponíveis, e o potencial para o desenvolvimento de resistência às drogas necessita da busca de novos alvos de medicamentos 1. Durante seu ciclo de vida, T. brucei, alterna entre um inseto vetor e hospedeiro mamífero; dois hosts que se apresentam muito diferentes ambientes em que o parasita deve sobreviver. Uma série de alterações metabólicas e morfológicas ocorrer como o parasita está exposta a diferentes condições ambientais. Algumas das mudanças mais dramáticas são observadas em microcorpos específicas do parasita único delimitado por membrana, denominado glicossomos 13. Os níveis de glicose são relativamente elevados (~ 5 mm) na corrente sanguínea e parasitas na corrente sanguínea (BSF) gerar ATP exclusivamente através wh glicólisemetabolismo mitocondrial ile é reprimida 14. Ao contrário de outros eucariotas, em que a glicólise ocorre no citoplasma, T. brucei compartimenta a maioria das enzimas glicolíticas em glicossomos 14,15. Os parasitas são absorvidos pela mosca tsé-tsé durante a farinha de sangue e experimentar uma queda em glicose, que cai para níveis indetectáveis ​​dentro de 15 min de ser ingerido pela mosca. O metabolismo de inseto, forma procíclicos (PCF), parasitas é mais flexível e glicose, bem como aminoácidos, tais como prolina, pode ser usado na síntese de ATP 16-18. Estudos proteômicos comparativos revelam mudanças de ciclo de vida dependentes em glicosomal e proteínas mitocondriais com proteínas glicolíticas aumentou em parasitas na corrente sanguínea e proteínas mitocondriais envolvidos no ciclo TCA e cadeia respiratória 13,19. Enquanto muitos estudos têm-se centrado sobre as diferenças entre BSF e glicossomos PCF, pouco se sabe sobre as mudanças no glicossomos PCF que ocorrem em resposta aos envmudanças ironmental. No intestino posterior da mosca, os níveis de glicose estão baixos com aumentos transitórios durante uma alimentação 20. Na maioria dos estudos in vitro, parasitas PCF são cultivadas em meios contendo glicose. No entanto, estudos recentes têm demonstrado que mudanças no metabolismo PCF significativamente em resposta à glicose disponibilidade 17. Na ausência da glucose, a absorção de prolina e prolina aumento da actividade de desidrogenase 18. Esta mudança no metabolismo mitocondrial é provavelmente acompanhada por uma alteração na morfologia e composição glicossomo, no entanto, isto não foi avaliado directamente. Electron e microscopia de fluorescência são técnicas comuns usadas para estudar a dinâmica glicossomo em T. brucei 2,21-24. Estes protocolos são o tempo e trabalho intensivo, caro e difícil de adaptar-se a estudos em tempo real e os protocolos de alto rendimento. Para superar esta limitação, um sistema repórter fluorescente organelo-used para estudar organelos em sistemas de mamífero e de levedura foi modificada para utilização em T. brucei 12. Sistemas repórter fluorescente-organelas têm sido amplamente utilizados em eucariotos superiores, tais como fungos, plantas e células de mamíferos 25-27. Em tais sistemas, uma proteína fluorescente é fundido com uma sequência de aminoácidos que tem como alvo a proteína de organelos específicos. A degradação ou a síntese das proteínas-alvo é medida por meio de fluorescência e as mudanças na composição do organelo são reflectidas pelas alterações na fluorescência celular. Quando a grelha de leitura aberta de maior proteína fluorescente amarela (EYFP) é fundido a uma sequência de direccionamento peroxissomal tipo II (PTS2) 12, a proteína é importado para PTS2eYFP, glicossomos maduros importação-competente e fluorescência podem ser monitorizadas por meio de citometria de fluxo. As variações na composição glicossomo são refletidas por mudanças na fluorescência celular. Este sistema pode auxiliar no resolVing os mecanismos que regulam as mudanças induzidas pelo ambiente em glicossomo composição. Este manuscrito descreve a geração de um sistema repórter em glicossomo PCF parasitas em conjunção com citometria de fluxo para controlar a dinâmica glicossomo em tempo real em parasitas vivos e fornece um exemplo de como ele foi usado para seguir alterações na composição glicossomo em resposta a diferentes ambientes. Em resumo, glicossomo composição é influenciada pela concentração de glucose extracelular e a passagem de culturas em fase logarítmica em meio fresco provoca mudanças na composição glicossomo. Este sistema pode ser modificado para estudar o comportamento dinâmico de outros organelos nos tripanossomas e outros parasitas.

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using fluorescent proteins to monitor glycosome dynamics in the african trypanosome

Trypanosoma brucei é um parasita que causa cinetoplastida tripanossomíase humana Africano (HAT), ou doença do sono, e uma doença debilitante, nagana, em bovinos 1. Os suplentes do parasita entre na corrente sanguínea do hospedeiro mamífero eo vetor mosca tsé-tsé. A composição de muitas organelas celulares muda em resposta a essas condições extracelulares diferentes 2-5. Glicossomos peroxissomas são altamente especializados, em que muitas das enzimas envolvidas na glicólise estão compartimentados. Mudanças na composição glicossomo de uma forma de desenvolvimento e regulamentada ambientalmente 4-11. Atualmente, as técnicas mais comuns usadas para estudar glicossomo dinâmicas são microscopia eletrônica e de fluorescência; técnicas que são caros, tempo e mão de obra intensiva, e não é facilmente adaptada para análises de elevado rendimento. Para superar essas limitações, um sistema repórter fluorescente glicossomo emo que aumentou a proteína fluorescente amarela (EYFP) é fundido a uma sequência de direccionamento de peroxissoma (PTS2), que dirige a proteína de fusão para glicossomos 12, foi estabelecido. Após a importação da proteína de fusão PTS2eYFP, glicossomos se tornar fluorescente. Degradação organelo e reciclagem resulta na perda de fluorescência que pode ser medido por citometria de fluxo. Um grande número de células (5000 células / seg) podem ser analisados ​​em tempo real, sem grande preparação de amostras, tais como a fixação e montagem. Este método oferece uma maneira rápida de detectar mudanças na composição de organelas em resposta às variações das condições ambientais.

Neste sistema, uma alteração dependente de glucose em glicossomo composição foi observado. Quando as células são cultivadas em meios contendo glucose, duas populações são observados; um brilhante e uma fraca (Figura 2A). Células Dim abrigar glicossomos imaturas, que não tenham importado a PTS2eYFP enquanto as células vivas abrigam uma mistura de maduros e imaturos glicossomos 12. Quando a glicose está presente na mídia, mislocalization de glicossomo proteínas é letal 15,2...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizando Proteínas Fluorescentes para monitorar glicossomo Dynamics do Trypanosoma Africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

using-fluorescent-proteins-to-monitor-glycosome-dynamics-african

Research

JoVE Journal

Immunology and Infection

8.9K visualizações.  Clemson University Eukaryotic Pathogens Innovation Center. Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Vídeo: Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

O Uso de diacetato carboxifluoresceína Succinimidyl Ester (CFSE) para monitorar a proliferação de linfócitos

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Imunologia e Infecção

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Um Ensaio visual para o Monitor T6SS mediada Competição bacteriana

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Utilização de<em&gt; In vivo</em&gt; Imagem para monitorar a progressão da infecção pelo citomegalovírus Experimental Rato em recém-nascidos

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Usando proteínas fluorescentes para visualizar e Quantificar<em&gt; Chlamydia</em&gt; Vacúolo Crescimento Dynamics em células vivas

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

A linfa mesentérica Duct canulada Rat Modelo: Aplicação para a Avaliação de Intestinal Linfática transporte da droga

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Bioluminescência de imagem para detectar a infecção fase tardia Africano Tripanossomíase

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Fagossomo Encerramento Ensaio&quot; para Visualize Formação fagossomo em Três Dimensões Usando Total Internal Reflection Microscopia fluorescente (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Desenvolvimento de um Sistema Reporter vírus da hepatite B de acompanhar as fases iniciais do ciclo de replicação

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpressão e purificação do ser humano<em&gt; Cis</em&gt; -preniltransferase em<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualização de candida albicans no trato gastrointestinal murina usando fluorescente in situ hibridização

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...