O estudo atual foi concedido pelo "Estudo sobre as principais questões do efeito curativo de Koumiss nas doenças regionais da medicina mongol" em 2018 Projeto Apoiado do programa de ciência e tecnologia do Departamento de Ciência e Tecnologia da Região Autônoma da Mongólia Interior.

NOTA: Consulte a Tabela de Materiais para obter detalhes relacionados a todos os materiais, instrumentos e reagentes usados neste protocolo.
1. Cultura de células
<ol><li>Cultura de células HepG2 em frascos de cultura contendo Meio de Eagle Modificado (DMEM) de Dulbecco (contendo 10% de soro fetal bovino [FBS], 100 unidades/mL de penicilina e 100 μg/mL de estreptomicina). Manter os frascos de cultura a 37 °C numa incubadora de CO2 a 5%.</li>
</ol>2. Efeito do ácido oleico na viabilidade celular medida pelo kit de contagem de células-8
<ol><li>Dissolver um volume específico de OA em dimetilsulfóxido (DMSO) até atingir uma concentração de 200 mM. Conservar a solução a -20 °C para utilização futura.</li>
	<li>Semeie células HepG2 em uma placa de 96 poços com uma densidade celular de 6 × 103 células por poço. Adicione 100 μL de DMEM a cada poço. Incubar e cultivar as células a 37 °C em uma incubadora de CO5% 2 por 24 h. Divida as células HepG2 em dois grupos:<ol><li>Grupo controle: adicionar meio de cultura celular.</li>
		<li>Grupo OA: adicione OA ao meio de cultura celular para atingir concentrações finais de 0,125 mM, 0,25 mM, 0,5 mM e 1 mM.</li>
	</ol></li>
	<li>Após 24 h de incubação inicial, descartar o sobrenadante de cada alvéolo e adicionar OA de acordo com o agrupamento especificado, adicionando 100 μL por alvéolo. Para o grupo de controle, adicione 100 μL de meio de cultura de células a cada poço. Continue a incubar as células por mais 24 horas. NOTA: Certifique-se de que cada grupo tenha seis poços replicados. Para evitar a evaporação, adicione 100 μL de solução salina tamponada com fosfato (PBS) ao anel externo de poços na placa de 96 poços.</li>
	<li>Após 24 h de incubação, adicione 10 μL de kit de contagem de células-8 (CCK-8) a cada poço, misture delicadamente e incube por 2 h no escuro. Retire a placa de 96 poços da incubadora, coloque-a no leitor de microplacas e meça o valor de absorbância a 450 nm (A450). Os valores do IG50 foram contados de acordo com a DO.</li>
</ol>3. Coloração Oil Red O para observar a formação de gotículas lipídicas intracelulares
<ol><li>Semeie células HepG2 em placas de cultura de células de 6 poços a uma densidade de 5 × 105 células por poço e cultive as placas em uma incubadora de temperatura constante por 24 h. Consulte a Figura 1 para obter uma representação visual das etapas descritas.</li>
	<li>Após 24 h de cultura celular, adicione 2 mL de meio de cultura celular contendo OA a cada poço, atingindo concentrações finais de 0,125 mM, 0,25 mM, 0,5 mM e 1 mM. Após mais 24 horas, remover o meio de cultura celular de cada alvéolo e lavar duas vezes com PBS. Adicione 1 mL de fixador ORO a cada poço e incube por 30 min.</li>
	<li>Prepare a solução de coloração ORO misturando a solução de coloração A com a solução de coloração B na proporção de 3:2. Deixe a mistura repousar em temperatura ambiente por 10 min e, em seguida, filtre-a uma vez com um filtro de 0,45 μm. Conservar a solução filtrada num tubo de centrifugação protegido da luz até à utilização.</li>
	<li>Descarte o fixador e lave duas vezes com água destilada. Adicione 1 mL de isopropanol a 60% em cada poço e incube por 30 s. Descarte 60% da solução de isopropanol e adicione 1 mL de solução de coloração ORO recém-preparada em cada poço antes de incubar por 20 min. Descarte a solução de coloração ORO, adicione 1 mL de isopropanol a 60% em cada poço e incube por 30 s. Lave 5x com água para remover o excesso de corante.</li>
	<li>Cubra as células com água destilada e observe ao microscópio. Depois que as imagens forem coletadas, descarte o líquido na placa e deixe secar. Em seguida, adicione 2 mL de isopropanol a cada poço e agite a placa em um agitador orbital por 10 min. Transfira o líquido para uma nova placa de 96 poços, com 16 poços em cada grupo, adicionando 100 μL por poço. Calcular o teor de lípidos medindo a densidade ótica (DO) de cada alvéolo utilizando um leitor de microplacas a 510 nm (A510).</li>
</ol>4. Efeitos de diferentes concentrações de ácido oleico sobre triglicerídeos totais em sobrenadantes de células HepG2
<ol><li>Equilibre o kit em temperatura ambiente por 20 min e prepare as placas necessárias para o experimento.</li>
	<li>Recolher o sobrenadante da célula e centrifugar a 1.570 × g durante 10 min. Defina poços padrão e teste de poços de amostra. Adicione 50 μL de concentração padrão ([S0 → S5] seguido por: 0, 0,5, 1, 2, 4, 8 mmol / L) aos poços padrão. Além dos poços em branco e padrão, adicione 10 μL de amostras diferentes aos poços de amostra, seguido pela adição de 40 μL de diluente de amostra a cada poço. Adicionar 100 μL de peroxidase de anticorpo-rábano de detecção a cada alvéolo, selar com uma membrana de placa e incubar a 37 °C durante 1 h numa estufa de temperatura constante.</li>
	<li>Descarte o sobrenadante, seque em papel livre de poeira e lave cada poço com 1x solução de lavagem. Deixe repousar em temperatura ambiente por 1 min. Repita o processo de lavagem 5x.</li>
	<li>Adicione 50 μL de substrato A e 50 μL de substrato B a cada poço. Misture delicadamente e incube por 15 min a 37 °C. Adicione 50 μL de solução de terminação a cada poço e meça o valor OD de cada poço a 450 nm (A450) em 15 min.</li>
	<li>Plote a concentração do padrão ao longo do eixo x e o valor de absorbância (OD) correspondente ao longo do eixo y para realizar a regressão linear e derive a equação da curva para calcular o valor da concentração de cada amostra.</li>
</ol>5. Análise estatística
<ol><li>Determine diferenças significativas nos dados quantitativos.</li>
	<li>Calcule a média ± desvio padrão (DP) e represente graficamente os dados. Considere P < 0,05 estatisticamente significativo.</li>
</ol>

Células HepG2 induzidas por ácido oleico para análise de doenças metabólicas 

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Os autores declaram não haver conflitos de interesse.

A MASLD é uma síndrome clínico-patológica caracterizada pela deposição excessiva de gordura intracelular nos hepatócitos devido a fatores além do álcool e outros agentes prejudiciais ao fígado estabelecidos18. A MASLD está intrinsecamente ligada à lesão hepática por estresse metabólico adquirido, notadamente associada à resistência à insulina e à suscetibilidade genética. Para estudar e rastrear efetivamente os medicamentos para MASLD, é crucial selecionar um modelo experiment...

A doença hepática esteatótica associada à disfunção metabólica (MASLD) engloba uma variedade de condições, incluindo esteatose simples, esteato-hepatite não alcoólica (EHNA), cirrose e carcinoma hepatocelular 1,2,3,4,5,6, todas atribuídas a outros fatores além do consumo de álcool 7. A MASLD é a doença hepática mais prevalente causada por lesão hepática metabólica, afetando quase um quarto da população mundial 8,9,10,11,12. Embora a patogênese precisa do MASLD ainda não tenha sido elucidada, várias teorias tentam explicar seu desenvolvimento. Uma noção predominante sugere um afastamento da teoria clássica de "dois golpes" em direção a um modelo de "múltiplos golpes"1. Central para essas hipóteses é o papel da resistência à insulina, que se acredita ser fundamental na patogênese da MASLD13. Pesquisas indicam que a resistência à insulina nos hepatócitos leva ao aumento dos níveis de ácidos graxos livres, formando posteriormente triglicerídeos armazenados no fígado14,15.
Os pesquisadores usaram modelos in vivo e in vitro para simular a deposição de gordura em MASLD; No entanto, replicar totalmente seu mecanismo patológico continua sendo um desafio. Apesar dessa limitação, esses modelos têm sido fundamentais no estudo de potenciais alvos terapêuticos para MASLD. No entanto, o desenvolvimento de um modelo estável de MASLD é crucial. Embora os modelos animais sejam eficazes, eles são demorados e caros, destacando assim o crescente interesse em modelos celulares in vitro . Esses modelos geralmente usam ácidos graxos livres únicos ou múltiplos, como ácido oleico (OA) e ácido palmítico, para recriar MASLD induzido por dieta. Dentre estes, a linhagem celular de hepatoblastoma humano HepG2 é frequentemente utilizada para estabelecer modelos celulares in vitro de MASLD.
A indução da OA estimula as células HepG2 a replicar a deposição de gordura semelhante ao MASLD, um método com uma história bem estabelecida. O objetivo deste estudo foi demonstrar a viabilidade, a coloração Oil Red O (ORO), o conteúdo lipídico e o nível de triglicerídeos (TG) de células HepG2 tratadas com OA 0,25 mM. O objetivo deste experimento foi fornecer mais evidências para o desenvolvimento de estudos de modelagem MAFLD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1x) Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filter</td>
			<td>Millex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Crygenic Vials</td>
			<td>CORNING</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 Cell Culture Flask</td>
			<td>CORNING</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>CORNING</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well cell culture plate</td>
			<td>CORNING</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Count Plate</td>
			<td>Shanghai Jing Jing Biochemical Reagent &#38; Instrument Co.</td>
			<td>02270113</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8 assays</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>CA1210-1000T</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>NUAIRE</td>
			<td>NU-5710E</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;DMSO Dimethyl sulfoxide&#160;</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>D8371</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>8122691</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme Labeling Equipment</td>
			<td>Tecan</td>
			<td>Spark</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>HepG2 cells line</td>
			<td>Beijing North China Chuanglian Biotechnology Research Institute (BNCC)</td>
			<td>221031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Triglyceride (TG) ELISA instruction</td>
			<td>Nanjing Jiacheng Bioengineering Institute</td>
			<td>20170301</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope for Cell Culture</td>
			<td>Leica</td>
			<td>DMi1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Tianjin Zhiyuan Chemical Reagent Co.</td>
			<td>2021030141</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red Stain Kit, For Cultured Cells</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>G1262</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid&#160;</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td>A502071</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 24.0</td>
			<td></td>
			<td>Statistics software</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro modeling of fat deposition in metabolic dysfunction-associated steatotic liver disease

A prevalência de doença hepática esteatótica associada à disfunção metabólica (MASLD) aumentou devido a mudanças nos padrões econômicos e de estilo de vida, levando a desafios de saúde significativos. Relatórios anteriores estudaram o estabelecimento de modelos animais e celulares para MASLD, destacando diferenças entre eles. Neste estudo, foi criado um modelo celular induzindo o acúmulo de gordura em MASLD. As células HepG2 foram estimuladas com o ácido graxo insaturado ácido oleico em várias concentrações (0,125 mM, 0,25 mM, 0,5 mM, 1 mM) para emular MASLD. A eficácia do modelo foi avaliada usando ensaios de contagem de células-8, coloração Oil Red O e análise do conteúdo lipídico. Este estudo teve como objetivo criar um modelo celular simples de operar para células MASSLD. Os resultados dos ensaios do kit de contagem de células-8 mostraram que a sobrevivência das células HepG2 foi dependente da concentração de ácido oleico, com um GI50 de 1,875 mM. A viabilidade celular nos grupos 0,5 mM e 1 mM foi significativamente menor do que no grupo controle (P < 0,05). Além disso, a coloração Oil Red O e a análise do conteúdo lipídico examinaram a deposição de gordura em concentrações variadas de ácido oleico (0,125 mM, 0,25 mM, 0,5 mM, 1 mM) em células HepG2. O conteúdo lipídico dos grupos 0,25 mM, 0,5 mM e 1 mM foi significativamente maior do que o do grupo controle (P < 0,05). Além disso, os níveis de triglicerídeos nos grupos OA foram significativamente maiores do que os do grupo controle (P < 0,05).

Efeito do ácido oleico na viabilidade celular As células HepG2 foram expostas a concentrações variadas de OA (0 mM, 0,125 mM, 0,25 mM, 0,5 mM, 1 mM), resultando em uma diminuição nas taxas de sobrevivência celular em 0,125 mM, 0,25 mM, 0,5 mM e 1 mM em comparação com 0 mM. Observou-se significância estatística em 0,5 mM (P < 0,05) e 1 mM (P < 0,05) quando comparados a 0 mM. Os resultados do impacto da OA na viabilidade celular, conforme avaliado pelo kit CCK-8, são mostra...

Autor em destaque: Estabelecendo modelos de células MASLD para investigar mecanismos de doenças e os efeitos hipolipemiantes de Koumiss

Este artigo descreve o uso de células HepG2 induzidas por ácido oleico como modelo para doença hepática esteatótica associada à disfunção metabólica.

Modelagem in vitro da deposição de gordura na doença hepática esteatótica associada à disfunção metabólica

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has surged due to changes in economic and lifestyle patterns, leading ...

in-vitro-modeling-fat-deposition-metabolic-dysfunction-associated

Research

JoVE Journal

Biology

In Vitro Modeling of Fat Deposition in Metabolic Dysfunction-Associated Steatotic Liver Disease

412 visualizações.  Inner Mongolia Medical University. Nossa direção de pesquisa é o estudo da medicina tradicional da Mongólia, estabelecendo um modelo de célula de doença hepática esteatótica associada à disfunção metabólica. O papel e o mecanismo da terapia koumiss são estudados. Os resultados do estudo de presença serão vantajosos no futuro, permitindo o estabelecimento de modelos celulares de doença hepática esteatótica associada à disfunção metabólica e a investigação dos mecanismos ...

Vídeo: Autor em destaque: Estabelecendo modelos de células MASLD para investigar mecanismos de doenças e os efeitos hipolipemiantes de Koumiss

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Biologia

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modelagem do Morbid Genoma Humano utilizando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Metabólica Rotulagem de Leucina rico Repita Kinases 1 e 2 com Radioactive Fosfato

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

Detecção de proteínas de ligação a ARN pela<em&gt; In Vitro</em&gt; RNA Pull-down em Cultura adipócito

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análise da Função Ligase E3 Ubiquitin

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Isolamento de Células Valvares Primárias Humanas para Modelagem de Doenças In Vitro

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

In vitro Ensaio Tridimensional de Angiogênese Utilizando Células-Tronco Embrionárias de Camundongos para Modelagem de Doenças Vasculares e Testes de Fármacos

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is often not detected until drugs have reached clinical stages, risking patients' safety and leading to substantial losses for the pharma industry. Taking into account that standard 2D models have limitations in detecting DILI it is essential to develop in vitro models that are more predictive to improve data translatability. To understand causality and mechanistic aspects of DILI in detail, a human liver MPS consisting of human primary liver parenchymal and non-parenchymal cells (NPCs) and cultured in 3D microtissues on an engineered scaffold under perfusion has been developed. Cryopreserved primary human hepatocytes (PHHs) and Kupffer cells (HKCs) were cocultured as microtissues in the MPS platform for up to two weeks, and each compound of interest was repeatably dosed onto liver microtissues at seven test concentrations for up to four days. Functional liver-specific endpoints were analyzed (including clinical biomarkers such as alanine aminotransferase, ALT) to evaluate liver function. Acute and chronic exposure to compounds of various DILI severities can be assessed by comparing responses to single and multi-dosed microtissues. The methodology has been validated with a broad set of severe and mildly hepatotoxic compounds. Here we show the data for pioglitazone and troglitazone, well-known hepatotoxic compounds withdrawn from the market for causing liver failures. Overall, it has been shown that the liver MPS model can be a useful tool to assess DILI and its association with changes in hepatic function. The model can additionally be used to assess how novel compounds behave in distinct patient subsets and how toxicity profiles may be affected by liver disease states (e.g., viral hepatitis, fatty liver disease).

Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro

Drug-induced liver injury (DILI) is a major cause of drug failure. A protocol has been developed to accurately predict the DILI liability of a compound using a liver microphysiological system (MPS). The liver model uses the coculture of primary hepatic cells and translationally relevant endpoints to assess cellular responses to treatment.

Sistema Microfisiológico do Fígado Humano para Avaliação da Toxicidade Hepática Induzida por Drogas In Vitro

DILI is a major cause of attrition in drug development with over 1000 FDA-approved drugs known to potentially cause DILI in humans. Unfortunately, DILI is ...