Trabalho em laboratório Duennwald é suportado por concessões da Federação Americana para Pesquisas sobre o Envelhecimento (AFAR), a Fundação doença hereditária (HDF) e da Fundação William Wood.

1. Expressão de Tóxicos poliQ expansão de Proteínas em Leishmania Uma análise sistemática estabeleceu a sequência de ácido preciso amino de uma proteína poliQ-expansão que é necessário para produzir a toxicidade em levedura 7. Esta proteína poliQ expansão-tóxico contém um amino-terminal FLAG-tag seguido por 17 aminoácidos a partir da sequência original da proteína huntingtina, uma região poliQ, e uma fusão carboxi-terminal para uma proteína fluorescente (GFP quer ou PCP, ver Figura 1 um). A expressão de proteínas com expansões poliQ de 46 glutaminas ou mais (por exemplo 72 e 103 glutamines) produz toxicidade em levedura quando expresso sob o controlo do induzível e relativo promotor GAL1 forte 7, 9. Como descrito anteriormente, poliQ toxicidade em levedura é apenas aparente em células que transportam o Rnq1p proteína na sua conformação prião, [RNQ +], por exemplo, a estirpe de levedura W303 9. A r proteína tóxica poliQ-expansãoecapitulates aspectos centrais de poliQ biologia-em leveduras, tais como toxicidade poliQ comprimento-dependente (ver abaixo) e agregação (Figura 1 b). Notavelmente, tóxicos poliQ expansão de proteínas não matar células de levedura imediatamente, pois eles sim prejudicar ou prender o ciclo celular e devision célula, assim, retardar ou inibir o crescimento de colônias de leveduras em placas ou culturas líquidos (os nossos dados não publicados). 2. Possíveis problemas com células de levedura Expressando tóxicos poliQ expansão de Proteínas As células de levedura que expressam as proteínas de expansão de outra forma tóxicos poliQ pode não apresentar qualquer defeito de crescimento 9. A natureza genética destes supressores de poliQ-toxicidade não parece ser com base em simples mutações Mendelian e, portanto, pode ocorrer com frequência relativamente elevada (nossos resultados não publicados). Especula-se que estes supressores espontâneas são causados ​​pela cura de priões não identificados que funcionam de forma semelhante ao [RNQ +] na determinação toxicity.Thes poliQde e supressores espontâneos de toxicidade poliQ pode comprometer o êxito de qualquer experimento que tem por objetivo caracterizar a toxicidade poliQ ou para identificar e caracterizar modificadores de toxicidade poliQ. A fim de evitar a ocorrência frequente destes supressores espontâneos, siga as medidas de precaução descritas abaixo, que provaram ser muito eficazes: <ol><li> Use células de levedura fresca para experimentos de toxicidade. Não armazenar levedura, por períodos de tempo prolongados. Freqüentemente recuperar colônias de leveduras frescas provenientes das existências de congelados. </li><li> Frequentemente monitorizar a expressão e agregação de tóxicos poliQ-expansão proteínas por microscopia de fluorescência. </li><li> Manter as células de levedura em meios que reprimem a expressão da toxicidade poliQ tóxico em todos os momentos (isto é, em meios selectivos, contendo glucose como única fonte de carbono) antes de iniciar quaisquer medições de toxicidade. </li><li> Use pelo menos três transformantes independentes para cada experimento poliQ toxicidade. &lt;/ Li&gt; </ol> 3. Ensaios de crescimento <ol><li> Para cada condição experimental, inocular um colónia de cada a partir de três transformantes independentes de células de levedura ancorando o proteína poliQ expansão-tóxico em 3 ml de meio de levedura selectivos com glucose como única fonte de carbono. </li><li> Incubar estas culturas durante a noite a 30 ° C. Sob estas condições, as células não estão expressando a proteína poliQ-expansão, porque a glucose no meio reprime a sua expressão. Não deixe que estes overgrow culturas; manter o OD 600 (absorção de luz de comprimento de onda de 600 nm) das culturas noturnas abaixo de 1. </li><li> Rotineiramente usar três ensaios diferentes para monitorar os defeitos de crescimento de células de levedura que expressam a proteína poliQ expansão-tóxico: </li></ol> 3,1 Crescimento em placas <ol><li> Diluir as culturas de leveduras durante a noite (cultivadas com 220 agitação rpm) para uma DO600 de 0,0005 (isto é, uma diluição de 1:1000 de um OD600 = 0,5 cultura) em selectiva media 7 contendo glucose. </li><li> Uniformemente distribuído 50 uL (resultando em CA. 700 colónias por placa) de cada cultura diluídos em um prato (10 cm de diâmetro) com meio selectivo contendo glucose como única fonte de carbono e uma placa com meio selectivo contendo galactose como única fonte de carbono. </li><li> Incubar as placas durante três a quatro dias a 30 ° C. Após a incubação, tirar fotografias de cada placa e contar o número de colónias no glicose e do número de colónias nas placas de galactose. Sob condições ideais, não deve haver nenhum ou apenas muito poucas colónias sobre a placa de galactose ao utilizar células de levedura que expressa uma proteína altamente tóxico poliQ expansão-(103.oQ, a Figura 2a). </li></ol> 3.2 ensaios Spotting Este ensaio é mais quantitativa do que o ensaio de plaqueamento descrito acima e pode, assim, revelam mesmo diferenças subtis na toxicidade poliQ com a mesma experiência na mesma placa. <ol><li> Dilui-se a overnculturas ireito crescidas em meio com glicose a uma OD 600 de 0,1. </li><li> Pipetar 200 destas culturas diluídas em estéreis placas de 96 poços e preparar cinco diluições em série de cinco vezes em água estéril, utilizando uma pipeta multi-canal. </li><li> Usando um frogger, (também chamado um removedor, com 8 x 6 pinos para a transferência de suspensões de células) transferir as suspensões de células em placas contendo meio selectivo com glucose como única fonte de carbono e placas contendo meio selectivo com galactose como única fonte de carbono. </li><li> Permitir que as placas a secar antes de incubando-as a 30 ° C durante três a quatro dias. </li><li> Após a incubação, tirar fotografias de cada placa (Figura 2b). </li></ol> 3,3. Monitorização poliQ toxicidade em levedura por crescimento de culturas líquidas Este protocolo é o mais quantitativa (OD600 números) dos três ensaios descritos aqui e pode até mesmo detectar diferenças muito subtis na toxicidade poliQ. O referido ocrência de supressores espontâneos, no entanto, pode potencialmente produzir resultados enganosos. Por conseguinte, combinando recomendam neste ensaio, com pelo menos um dos dois ensaios de revestimento descritas acima. Propomos a utilização do instrumento BioscreenC para estas experiências. O BioscreenC é um instrumento que mede automaticamente a densidade óptica de culturas de leveduras em 100 poços enquanto incubadas a temperaturas definidos, com agitação definido. Outros métodos para o crescimento de células de levedura e medindo a sua densidade óptica pode também aplicar. <ol><li> Lave as células de levedura a partir de meio mínimo contendo glucose como única fonte de carbono três vezes em 3 ml de água estéril. </li><li> Diluir as culturas durante a noite cultivadas em meio com galactose a uma OD 600 de 0,1. </li><li> Encher cada poço da placa 100-BioscreenC bem com 300 uL das culturas de leveduras. </li><li> Abra o programa Experimento Fácil Bioscreen. Determinar o número de amostras que você deseja monitorar (incluindo espaços em branco e meio-somentecontroles), definir a temperatura a 30 ° C, definir a duração dos experimentos 3 dias, definir os intervalos de medição de 15 minutos, definir o filtro para 600 nm / Brown, e definir o modo de agitação para 15 segundos antes de cada medição de força média. </li><li> O instrumento BioscreenC eo software anexo irá produzir planilhas do Excel de cada ponto de dados obtidos durante o experimento. </li><li> Preparar curvas de crescimento com o Excel para cada amostra e comparar o crescimento de amostras diferentes (Figura 2c). Uma descrição detalhada da análise dos dados produzidos por experiências BioscreenC foi fornecida antes de 10. </li></ol> 4. Os resultados representativos <img alt="A Figura 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> Figura 1. A levedura modelo poliQ. a) representação esquemática do poliQ expansão-b proteína tóxica.) A microscopia de fluorescência mostrando células de levedura que expressam um curto, não-tóxico poliQcélulas de proteína de expansão (25Q, painel da esquerda) e levedura que expressa um longo, a proteína poliQ expansão tóxico (painel, 103.oQ direita). <img alt="A Figura 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> Figura 2. Os resultados representativos de ensaios de crescimento de células de levedura que expressam poliQ de expansão-proteínas. a) ensaio de plaqueamento. Aproximadamente 700 células de levedura foram espalhadas em placas e incubadas durante três dias a 30 ° C. O painel superior mostra uma placa de meio contendo glucose, ou seja, a expressão da proteína poliQ expansão tóxico (103.oQ) não é induzida. O painel inferior mostra uma placa de levedura contendo o meio de galactose, ou seja, a expressão da proteína poliQ expansão-tóxico é induzida. Note-se que na experiência mostrada aqui, nenhum supressor espontânea ocorreu. B) Spotting ensaio. Cinco série de cinco diluições de células de levedura ou abrigando um não-tóxico proteína poliQ (25Q) ou um tóxico poliQ-expansão protein (103.oQ) foram manchados em placas de glicose que reprimem a expressão das proteínas (painel da esquerda) ou em placas de galactose, que induzem a sua expressão. As placas foram então incubadas durante três dias a 30 ° C. c) experimento BioscreenC. O crescimento de culturas de células de levedura que expressam, quer uma proteína não-tóxico poliQ (25Q) ou uma proteína poliQ expansão tóxico (103.oQ) foram monitorizados pela instrumento BioscreenC. As condições experimentais e da análise dos experimentos BioscreenC são descritos no texto principal.

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Este manuscrito descreve três abordagens complementares experimentais para medir poliQ toxicidade no organismo levedura modelo baseado na redução do crescimento de células de levedura que expressam tóxicos poliQ expansão de proteínas. Trabalho em levedura ofereceu profundos insights sobre os mecanismos básicos celulares e moleculares da misfolding proteína e sua toxicidade que se seguiu, incluindo o misfolding e toxicidade de poliQ expansão de proteínas 9,11,12. Experimentos baseados em protocolos aqui apresentados já ajudou a identificar e caracterizar potenciadores genéticos ou supressores de toxicidade poliQ, modificadores de moléculas pequenas de toxicidade poliQ e mecanismos celulares subjacentes poliQ toxicidade 5-8,13,14. Os protocolos apresentados podem ser facilmente adaptados para explorar outros modificadores de poliQ toxicidade, tais como diferentes condições de crescimento ou mutações genéticas que reduzir ou exacerbar toxicidade poliQ. Além disso, o protocolo experimental apresentoupode ser facilmente ajustado para testar a toxicidade de outras proteínas deformadas tóxicos em levedura.

<table border="1"><tbody><tr><td> Nome de instrumento </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Frogger (6x8 pinos) </td><td> V &amp; P Scientific, San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves EUA </td><td> 5101370 </td></tr><tr><td> 100 poços de favo de mel </td><td> Growthcurves EUA </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Misfolding proteína está associada com várias doenças humanas, doenças neurodegenerativas particularmente, tais como a doença de Alzheimer, doença de Parkinson e doença de Huntington 1. Doença de Huntington (DH) é causada pela expansão anormal de uma região poliglutaminas (poliQ) dentro do huntingtina proteína. O poliQ-expandida proteína huntingtina atinge uma conformação aberrante (ie misfolds) e provoca toxicidade celular 2. Pelo menos oito outras doenças neurodegenerativas são causadas por poliQ de expansões, incluindo o AEC e doença de Kennedy 3. A levedura organismo modelo facilitou insights significativos para a base celular e molecular de poliQ toxicidade, incluindo o impacto de fatores intra e inter-molecular de poliQ-toxicidade, bem como a identificação de caminhos celulares que são deficientes em células expressando poliQ expansão proteínas 3-8. Importantely, muitos aspectos poliQ-toxicidade que foram encontrados em levedura foram reproduzidas em outros sistemas experimentais e em certa medida em amostras de pacientes de HD, demonstrando assim a significância do modelo de levedura para a descoberta de mecanismos básicos subjacentes poliQ-toxicidade. Uma forma direta e relativamente simples para determinar poliQ toxicidade em levedura é medir defeitos de crescimento de células de levedura que expressam poliQ expansão de proteínas. Este manuscrito descreve três complementares abordagens experimentais para determinar a toxicidade poliQ-em levedura através da medição do crescimento de células de levedura que expressam poliQ-expansão proteínas. As duas primeiras abordagens experimentais controlar o crescimento de leveduras em placas, a terceira abordagem monitora o crescimento de culturas de leveduras líquidos utilizando o instrumento BioscreenC. Além disso, este manuscrito descreve dificuldades experimentais que podem ocorrer ao manusear modelos poliQ levedura e traça estratégias que ajudarão a evitar ouminimizar estas dificuldades. Os protocolos aqui descritos podem ser utilizados para identificar e caracterizar as vias genéticos e pequenas moléculas que modulam poliQ-toxicidade. Além disso, os ensaios descritos podem servir como modelos para análises precisas da toxicidade causada por outras doenças associadas a proteínas deformadas em modelos de levedura.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Este manuscrito descreve três protocolos complementares para avaliar a toxicidade de poliglutaminas (poliQ)-expansão proteínas na levedura<em&gt; Saccharomyces cerevisiae</em&gt;. Estes protocolos podem ser facilmente modificados para monitorar a toxicidade de outras proteínas deformadas em levedura.

Ensaios de crescimento para avaliar a toxicidade poliglutamina em Levedura

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

growth-assays-to-assess-polyglutamine-toxicity-in-yeast

Nanyang Technological University

Research

JoVE Journal

Biology

13.5K visualizações.  Boston Biomedical Research Institute. Este manuscrito descreve três protocolos complementares para avaliar a toxicidade de poliglutaminas (poliQ)-expansão proteínas na levedura Saccharomyces cerevisiae. Estes protocolos podem ser facilmente modificados para monitorar a toxicidade de outras proteínas deformadas em levedura.

Vídeo: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Medição Span vida replicativa na levedura de brotamento

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Biologia

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identificação de fenótipos inibição do crescimento induzido pela expressão de Effectors Tipo III bacteriana em Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado Sistema Yeast-Two-Hybrid para identificar proteínas Interagindo com o Fator de Crescimento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Métodos para avaliar Subcelulares Compartimentos de músculo em<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Análise comparativa da hormona de crescimento humano no soro utilizando SPRI, Nano-SPRI e ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: um método em larga escala para a quantificação multiparamétrica do crescimento de fermento

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Efeito de proteínas fluorescentes na fusão parceiros usando Polyglutamine ensaios de toxicidade em levedura

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ensaios de triagem para avaliação dos efeitos de Ca2 +médio-produção-sinalização e reacção acrossómica no esperma humano

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Ensaios de crescimento para avaliar a toxicidade poliglutamina em Levedura

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