Name: a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators
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Description: Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

Os autores gostaria de agradecer Eric Fonteyn e Geert Schoofs excelente assistência técnica. Este trabalho foi apoiado pelo KU Leuven (conceder n. PF/10/018), o Fonds voor Wetenschappelijk Onderzoek (FWO, conceder n. G.485.08) e a Fondation Dormeur Vaduz.

Nota: Todos os passos descritos nas seções 1 e 2 são realizados em condições estéreis em um fluxo laminar.
1. manutenção do u-87. Células CD4.hCXCR4
<ol><li>Crescem as células em frascos de cultura T75 em 37 ° C e 5% CO2 em uma incubadora umidificada. Nota: A linha celular em vitro utilizada neste protocolo é uma linhagem de células de glioblastoma u-87 estàvel expressando cluster de diferenciação 4 (CD4) e humanos CXCR4 e tem sido descrito anteriorment...

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O Ca2 +-ensaio de mobilização aqui descrito anteriormente foi mostrado para ser uma ferramenta valiosa para identificar e caracterizar os antagonistas dos receptores CXCR417de direcionamento. No entanto, prevê-se que este método pode ser mais geralmente aplicado a um grupo grande de outros GPCRs que desencadeiam um citosólico Ca2 + lançamento após a sua ativação, conforme ilustrado pelo receptor relacionados chemokine CCR5. Considerando que, no caso de CCR5 poderiam ...

GPCRs são uma importante superfamília de proteínas de superfície celular que ativam cascatas de transdução de sinal com ligação ligante extracelular. Eles podem ser ativados por uma grande variedade de estímulos, incluindo hormônios proteicos, peptídeos, aminas biogênicas e lipídios, o que resulta no início de diversas vias de sinalização intracelulares e respostas biológicas eventualmente1,2 . Além disso, GPCRs estão envolvidos em muitos, se não todos, processos fisiológicos e de desenvolvimento e muitas doenças humanas são associadas com superexpressão de sinalização ou receptor GPCR disfuncional. GPCRs são, portanto, entre os alvos farmacológicos mais validados em medicina1,3.
Normalmente, um fluxo de trabalho de descoberta de drogas GPCR começa com ensaios de rastreio celular permitindo a identificação de compostos como pequenas moléculas, anticorpos monoclonais e peptídeos que podem modular a atividade de um determinado GPCR. Na descoberta da droga GPCR muitos tipos diferentes de ensaios existem para pesquisar por tais compostos, a maioria dos quais são compatíveis com campanhas de meados - a seleção da elevado-produção. Os ensaios mais utilizados incluem experiências de ligação do receptor, fluorescência ou luminescência com base em ensaios de detecção de flutuações no nível dos chamados mensageiros secundários (por exemplo, Ca2 +, adenosina monofosfato cíclica (AMP)), fenotípicos ensaios de triagem e recrutamento de β-arrestin ensaios4. A escolha de um determinado tipo de ensaio pode depender de vários fatores, mas também é determinada pelo conhecimento prévio das propriedades de sinalização de um determinado GPCR. Agonista obrigatório para um GPCR induz uma mudança conformacional catalisar a troca de difosfato de guanidina (PIB) para trifosfato de guanidina (GTP) sobre a subunidade α-de proteínas via G. Posteriormente a subunidade de -GTP Gαdissocia-se da subunidadeβγ de G... e as duas subunidades iniciará novas vias de sinalização. Hidrólise da molécula de GTP e subsequente re-associação da Gα- PIB e Gβγ subunidades irão restaurar a proteína G em sua conformação inativa5,6. Com base na similaridade da sequência de subunidadeα G são definidos diferentes tipos de proteínas G (G,s, G,eu, Gq, G12/13)7. Sinalização através da Gα subunidade dá origem a várias respostas típicas, tais como o aumento (via Gs) ou diminuir (via Geu) de produção de AMP cíclico e Ca2 + mobilização intracelular (via Gq)5 ,7. Gβγ subunidades são também capazes de induzir vias efetoras intracelulares. Por exemplo, após a ativação de Geu-GPCRs acoplados, Gβγ diretamente podem estimular a fosfolipase C (PLC-β) para produzir o inositol trifosfato (IP3) que provoca a liberação de Ca2 + do intracelulares lojas7 . Após a ativação do receptor, GPCRs são fosforiladas por quinases GPCR (GRKs), que promove a interação com β-arrestins. Este processo termina a sinalização de proteína G e leva a internalização e, eventualmente, a dessensibilização do receptor. Β-arrestins também são capazes de formar complexos multi moleculares que podem desencadear outras vias de sinalização independentes da proteína G sinalização8.
Dentro da subfamília de chemokine receptores, o Geu-CXCR4 acoplado é um GPCR que suscitou muito interesse como um alvo promissor para a descoberta de medicamentos. Dado seu papel estabelecido como um grande receptor de co para vírus de imunodeficiência humana 1 (HIV-1) entrada viral e infecção, compostos visando CXCR4 foram inicialmente desenvolvidos como anti-VIH droga candidatos9. Mais recentemente, um conjunto crescente de evidências tem apontado um papel importante para CXCR4 em metástase tumorigênese e câncer, tornando-se um alvo terapêutico bem validado em Oncologia também10. CXCR4 é altamente expressa em mais de vinte tipos de câncer humano e sobrevivência de células de tumor de controles, proliferação e migração, bem como relacionados ao tumor angiogênese10. Antagonistas de CXCR4 de diferentes classes químicas anteriormente foram descritos11,12, mas só a pequena molécula que AMD3100 atualmente é aprovado para uso na clínica como um agente de mobilização de células-tronco utilizado durante o tratamento de linfoma e pacientes de mieloma13,14. Ensaios clínicos estão em andamento para avaliar a segurança e a eficácia dos vários outras antagonistas de CXCR4 em diferentes doenças humanas, mas com um forte foco na oncologia12. Tendo em conta as muitas aplicações potenciais para CXCR4 antagonistas, a busca de novos compostos com propriedades farmacocinéticas melhoradas, melhor biodisponibilidade ou potencialmente menos efeitos colaterais é garantida.
Neste documento, um ensaio de celular baseado em fluorescência cinético usada principalmente para tela para compostos capazes de inibir o CXCR4 é descrita. A medição fluorescente desse método baseia-se o aumento transiente de Ca2 + concentração intracelular evocado após a ativação de CXCR4 pelo seu agonista endógena, o ligante de chemokine CXCL12 (anteriormente conhecido como células do estroma derivadas fator 1 α ( SDF1-α)) e a inibição de potencial desta resposta2 + Ca CXCL12-induzida por compostos particulares. Neste ensaio, células de glioblastoma humano u-87 estàvel expressando o receptor CXCR4 humano são utilizadas. Ao mesmo tempo, estas células faltam expressão endógena de CXCR7, um receptor do chemokine relacionados que também vincula CXCL1215,16,17. CXCR7 anteriormente também foi mostrado para ser capaz de formar heterodímeros com CXCR4, desse modo, modulando as propriedades sinalização deste último receptor18. Flutuações no nível de intracelular Ca2 + mediada por CXCR4 são monitoradas por carregar o CXCR4+ células com éster acetoximetil fluo-2 (AM), uma célula-permeável de alta afinidade fluorescente Ca2 +-vinculação a tintura. Fluo-2 AM é uma molécula fluorescente único comprimento de onda que pode ser excitada em 490 nm, enquanto sua emissões a fluorescência é medida em 520 nm. Esta fluorescência de emissão aumenta em cima de Ca2 + ligação, com um grande alcance dinâmico entre a Ca2 +-vinculado e não vinculado a estado. O aumento do sinal fluorescente é transitório, ocorrendo dentro de um intervalo de tempo de alguns minutos e irá decair depois. A altura da emissão fluorescente mais correlaciona-se com o nível de ativação do receptor. O ensaio em si é executado usando um leitor de microplacas de fluorescência equipado com uma câmera CCD intensificou-se (ICCD) que possui um sistema de pipetagem integrado que permite a padronização das etapas pipetagem no ensaio (ver Tabela de materiais) . Além disso, a medição simultânea do sinal fluorescente em todos os poços de uma microplaca é outra grande vantagem do leitor de fluorescência que é usado. Durante a primeira parte do ensaio os compostos sob investigação (por exemplo, um painel de pequenas moléculas com uma concentração fixa ou em uma série de diluição) são adicionados à AM fluo-2 carregado CXCR4+ u-87 células seguiram por um ~ período de incubação de 10 min durante o qual o potencial efeito agonístico dos compostos é continuamente medido em tempo real. Em seguida, o agonista endógeno (i.e., CXCL12) é adicionado às células para evocar um aumento transiente CXCR4-mediada do nível intracelular Ca2 +. Durante esta parte do ensaio pode ser avaliada a potencial atividade antagonista dos compostos testados. Uma visão esquemática do fluxo de trabalho geral do ensaio é apresentada na Figura 1.
Embora este ensaio de Ca2 + mobilização tem sido usado principalmente para identificar e determinar a potência inibitória dos antagonistas competitivas de CXCR4 (ou seja, compostos que impedem que o agonista endógeno para vincular e estimulam o receptor), também pode identificar agonistas dos receptores e, além disso, os compostos que exercem sua função por ligação alostéricos locais (ou seja, sites que topograficamente diferem o sítio de ligação do orthosteric ocupado pelo agonista endógeno). Exemplos desta última categoria de compostos agonistas alostéricos e PAMs e NAMs19,20. Considerando que os antagonistas dos receptores específicos, bem como NAMs iria inibir a resposta2 + Ca induzida por CXCL12, PAMs aumentaria essa resposta (Veja também discussão seção). Embora o ensaio aqui descritos especificamente metas CXCR4, prevê-se que este método pode ser aplicado a outros GPCRs com esforço de otimização mínima, pelo menos se eles sinal através do lançamento do intracelular Ca2 +.

<table>
	<tbody>
		<tr>
			<td>Fluo-2 AM</td>
			<td>Abcam</td>
			<td>ab142775</td>
			<td>fluorescent Ca2+ sensitive dye</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td>pluronic acid</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>AMD3100</td>
			<td>Sigma</td>
			<td>A5602-5 mg</td>
			<td>specific CXCR4 antagonist</td>
		</tr>
		<tr>
			<td>Maraviroc</td>
			<td>kind gift of AnorMed</td>
			<td></td>
			<td>antiretroviral drug, CCR5 antagonist</td>
		</tr>
		<tr>
			<td>Chemokine ligand CXCL12</td>
			<td>PeproTech</td>
			<td>300-28A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemokine ligand CCL5</td>
			<td>PeproTech</td>
			<td>300-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco (Life Technologies)</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1933-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco (Life Technologies)</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10 x), calcium, magnesium, no phenol red</td>
			<td>Gibco (Life Technologies)</td>
			<td>14065-049</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco (Life Technologies)</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0,25 %), phenol red</td>
			<td>Gibco (Life Technologies)</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>Gibco (Life Technologies)</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>227 261</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flask (T75)</td>
			<td>Corning</td>
			<td>353024</td>
			<td></td>
		</tr>
		<tr>
			<td>Black plate, 96-well, clear bottom, with lid</td>
			<td>Costar/Fisher Scientific</td>
			<td>10530753</td>
			<td>assay plate (96-well), for cell seeding</td>
		</tr>
		<tr>
			<td>Polypropylene (PP) plates</td>
			<td>Thermo Scientific (VWR)</td>
			<td>732-2661</td>
			<td>plates used to prepare the compound plates and chemokine plates, round bottom</td>
		</tr>
		<tr>
			<td>FLIPR Tetra high throughput cellular screening system</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Fluorescent plate reader with integrated pipettor head and ICCD camera</td>
		</tr>
		<tr>
			<td>FLIPR Tetra LED Module 470 - 495 nm</td>
			<td>Molecular Devices</td>
			<td>0200-6128</td>
			<td>Light emitting diodes for excitation of the fluorescent Ca2+ sensitive dye</td>
		</tr>
		<tr>
			<td>FLIPR Tetra Emission Filter 515 - 575 nm</td>
			<td>Molecular Devices</td>
			<td>0200-6203</td>
			<td>emission filter compatible with the fluorescent dye</td>
		</tr>
		<tr>
			<td>FLIPR Tetra 96 Head</td>
			<td>Molecular Devices</td>
			<td>0310-4536</td>
			<td>96-well pipettor head, integrated within the fluorescent plate reader</td>
		</tr>
		<tr>
			<td>ScreenWorks</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>software package used for data analysis and visualization on the FLIPR Tetra</td>
		</tr>
		<tr>
			<td>Vi-CELL</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>cell viability analyzer</td>
		</tr>
		<tr>
			<td>Corning CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates, with Lid, Sterile</td>
			<td>Corning</td>
			<td>3340</td>
			<td>Pre-coated 96-well assay plates that may represent an alternative for manual coating of the assay plate.</td>
		</tr>
	</tbody>
</table>

a kinetic fluorescence-based ca2+ mobilization assay to identify g protein-coupled receptor agonists, antagonists, and allosteric modulators

G receptores (GPCRs) são de grande importância para a indústria farmacêutica como eles estão envolvidos em muitas doenças humanas e incluem metas bem validadas para a intervenção terapêutica. Descoberta de compostos de chumbo, incluindo pequenas moléculas sintéticas, que inibem especificamente a função do receptor, é um passo inicial importante no desenvolvimento de drogas e se baseia em ensaios de cell-based sensíveis, específicos e robustos. Aqui, descrevemos um ensaio cinético de celular com um visor fluorescente projetado principalmente para identificar os antagonistas de receptores específicos que inibem o intracelular Ca2 + lançamento evocado mediante a ativação do receptor chemokine CXC 4 (CXCR4) por seu ligante endógeno, o ligante de chemokine CXC 12 (CXCL12). A principal vantagem deste método é que ele também permite o rastreio dos compostos dotados de propriedades intrínsecas de agonísticos (ou seja, compostos, provocando um aumento de Ca2 + concentração intracelular na ausência de CXCL12) ou compostos função do receptor através da interação com sítios de ligação alostéricos de modulação (ou seja, moduladores alostéricos positivos e negativos (PAMs e NAMs, respectivamente)). No lado de baixo, autofluorescent compostos podem interferir com a leitura do ensaio, prejudicando, assim, interpretação de dados fiáveis. Muito provavelmente este ensaio pode ser implementado, com mínimas adaptações, como um ensaio de descoberta de medicamentos genéricos para muitos outros GPCRs cuja a ativação leva à liberação de Ca intracelular2 +.

O efeito da estimulação CXCL12 na mobilização2 + Ca intracelular no u-87. CD4.CXCR4+ e u-87. Células CD4 foi avaliada com o ensaio de Ca2 + mobilização. Em vez de 20 µ l de composto de teste que normalmente seria adicionado durante a primeira etapa de pipetagem do protocolo (Figura 1), buffer de ensaio foi adicionado para o fluo-2AM carregado u-87. CD4.CXCR4+ células numa placa de medição. Durante a segun...

Watch this Scientific Journal Video about A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators at JoVE.com

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

O ensaio de celular descrito destina-se a identificação do receptor do chemokine do CXC 4 (CXCR4)-interação agentes que inibem ou estimulam, competitivos ou qual alostericamente, o intracelular Ca2 + lançamento iniciado pela ativação de CXCR4.

Um cinética baseada em fluorescência Ca2 + mobilização ensaio para identificar agonistas do Receptor acoplado à proteína G, antagonistas e moduladores alostéricos

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

The overall goal of this cell-based assay is to identify and characterize molecules capable of stimulating or inhibiting G protein-coupled receptor-mediated intracellular calcium mobilization. This method can be a valuable assay for G protein-coupled receptor drug discovery because it can be used to screen for molecules that modify receptor-mediated intracellular calcium bodies. The main advantage of this technique is that it allows molecules with agonistic or antagonistic properties to be identified within the same assay.This assay may help in the discovery of novel drug candidates that act on G protein-coupled receptors promising therapeutic targets that play a role in many human diseases. On the day of cell passaging, coat each well of a black walled polystyrene 96-well plate with clear bottoms with 100 microliters of 0.1%gelatin solution and incubate the plates for two hours at room temperature. While the gelatin is solidifying, use a trypsin EDTA solution to lift the cells of interest from the bottom of their culture flask, resuspending the cells in fresh growth medium in a 50 milliliter conical tube once they have detached.After counting, collect the cells by centrifugation and resuspend the pellet in fresh growth medium to a one times 10 to the fifth viable cells per milliliter concentration. When the plates are ready, discard any excess gelatin solution and dry the plates on a lab tissue. Then wash the wells with 200 microliters of PBS and replace the wash with 200 microliters of cells per well for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide.The next morning, tap the plate to discard the growth medium from the wells and dry the plate upside on a paper towel. Then label the cells in each well with 100 microliters of loading dye solution per well and incubate the plate for 45 minutes in the dark at room temperature. While the cells are incubating, add 75 microliters of CXCL12 per well to the chemokine plate and 50 microliters of the compound of interest to each well of the compound plate.Next, turn on the cooler unit of the fluorescence plate reader followed by the plate reader itself. After allowing the system a few minutes to initiate, open the plate reader software. To set up the assay protocol, under the settings tab select read mode and the appropriate excitation and emission wavelengths for the loading dye.Select the mix with transfer fluid tab for automatic mixing of the compounds in the compound plate. In the transfer fluid tab, set 20 microliters of each well from the compound plate to be transferred into the measurement plate and position the pipette tips at 20 microliters below the liquid surface. Set the speed of aspiration to 50 microliters per second and the speed of dispensing to 25 microliters per second and click read with transfer fluid.During the first interval, select 60 reads with a read interval time of one second with 10 reads to be recorded before the compounds are dispensed into the measurement plate and 50 reads to be completed after. Set the second interval with a read interval time of 30 seconds and 18 reads. Select three tip washes for the protocol, setting each wash step to include the appropriate wash fluid, one wash cycle, a fast pump speed, and five strokes per wash.Use the pause pipettor function to include the pipettor pause of 300 seconds and use the mix with transfer fluid setting to allow automatic mixing of the CXCL12 solution within the chemokine plate. In the subsequent transfer fluid tab, set 25 microliters from each well of the chemokine plate to be transferred to the measurement plate and position the tips 20 microliters below the liquid surface. Set the aspiration speed to 50 microliters per second and the dispensing speed to 25 microliters per second.Use the read with transfer fluid button to set the first interval to include 145 reads with a read interval time of one second. Set the second interval to a read interval time of six seconds and 20 reads. Select three more wash tip cycles.Then click set stage temperature and set the device temperature to 37 degrees Celsius. At the end of the loading dye incubation, discard the buffer from the measurement plate and dry the plate on a lab tissue. Wash the cells with 150 microliters per well of assay buffer replacing the buffer with 80 microliters of assay buffer after two minutes.Place the measurement plate in the read position, the compound plate at the source two position, the chemokine plate at the source three position, and a box of black tips at the source one position. Close the device and click protocol signal test to determine the background relative light units and fluorescence variance over the plate. Select test signal in the popup window.If background values of 8, 000 to 10, 000 relative light units are obtained, select update and click save to save the main protocol. Then push run to start the assay. In this graph, a dose-dependent increase in fluorescence correlating with the dose-dependent increase in the release of calcium can be observed.Increasing levels of CXCL12-induced fluorescence allow the generation of a dose response curve based on the difference between the minimum and maximum response over baseline between the first measurement after addition addition of the chemokine and the final measurement in the protocol. No fluorescent calcium-related response was evoked by CXCL12 even at a high concentration when cells lacking the corresponding functional chemokine receptor CXCR4 were used demonstrating the receptor specificity of the CXCL12-evoked measurement. A key application for this cell-based assay is to identify small molecules that, following pre-incubation, dose dependently inhibit CXCL12-induced calcium mobilization in the cells as compared to control non-active compounds that do not inhibit the response.To determine the inhibitory potency of small molecules, a dose response curve can be generated for the inhibitory molecules using nonlinear regression. This assay can be used to study other G protein-coupled receptors with no or only minimal further optimization as well as in this experiment in which a dose-dependent increase in fluorescence was observed when another chemokine receptor, CCR5, was stimulated with its natural chemokine agonist CCL5. Once mastered, this technique can be used to study many other G-protein coupled receptors that signal via the mobilization of intracellular calcium ions upon receptor activation.After watching this video, you should have a good understanding of how this cellular assay can be used to identify agonists and antagonists of the CXCL4 chemokine receptor.

a-kinetic-fluorescence-based-ca2-mobilization-assay-to-identify-g

Research

JoVE Journal

Medicine

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

A estratégia para identificar mutações de novo em Distúrbios comuns, tais como autismo e esquizofrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Medicina

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure&gt;150 mmHg), proteinuria (&gt; 50 mg/24 hr) and mild uremia (&gt;10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (&lt;10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.

5/6th Nephrectomy em combinação com uma dieta alta de sal e Óxido Nítrico Sintase inibição para induzir o Doença Renal Crônica no Rato Lewis

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria de Fluxo Ensaio Celular para detectar anticorpos em N-metil-D-aspartato ou dopamina-2 Receptor em Soro Humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

Testes dinâmicos visuais para identificar e quantificar danos Visual e Reparação Após desmielinização na neurite óptica Pacientes

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilização do ensaio de formação de colónia agar mole para identificar inibidores de tumorigenicidade em células de cancro da mama

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

A Rat carótida Balão Injury modelo para testar Therapeutics remodela Anti-vasculares

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e cultura de células endoteliais primárias de Artérias e Veias canina

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

Um ensaio clínico avaliando a segurança, eficácia e entrega de sacos de três câmaras à base de azeite para nutrição parenteral

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Um ensaio à base de fluorescência para caracterização e quantificação da formação de gotas lipídicas em organóides intestinais humanos

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Um protocolo simples de ensaio de poços para visualizar e quantificar a resorção osteoclástica in vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...