Este estudo foi suportado pela bolsa de investigação intramural Instituto de Coreia da ciência e tecnologia (2E27513) e o elevado valor acrescentado alimentos tecnologia desenvolvimento programa (IPET) financiado pelo Ministério de agricultura, alimentação e Assuntos rurais (315067-03).

Nota: todo o experimento deve ser realizado em um laboratório isolado limpo a 20 ° C, com a poeira baixa e com a minimização da contaminação durante o worm e manipulação bacteriana. Para essa finalidade, devem ser realizados experimentos sob a chama de uma lâmpada de álcool ou usando uma bancada limpa.
 1. manutenção de c. elegans e ovo de preparação para o teste químico <ol><li>manutenção c. elegans N2 (var. Bristol) sobre uma placa de ágar NGM alimentada com live Escherichia coli OP50 a 20 ° C 12 , 13.</li>
	<li>Preparar ovos de idade-sincronizado usando um alvejante solução tratamento 5 ou o tempo de ovo postura método 6 , 14 , 15. 
	Nota: antes de preparação do ovo, casaco placa contendo etoposido NGM com calor-inativado Escherichia coli OP50 conforme descrito abaixo. Inactivados por calor E coli OP50 é usado para minimizar a atividade metabólica de Escherichia coli 6 , 16.</li>
</ol> 2. Preparação de calor-inativado Escherichia coli OP50 feed C. elegans durante o ensaio de toxicidade <ol><li>preparar 500 mL de meio de duplo levedura triptona (DYT) conforme descrito na tabela 1 e inocular uma única colônia de Escherichia coli OP50 cultivadas na um ágar Luria-Bertani (LB) placa 5.</li>
	<li>Incubar a Escherichia coli OP50 numa incubadora tremendo para 14 h a 37 ° C e 150 rpm.</li>
	<li>Após a centrifugação por 30 min no 3.220 x g e 20 ° C, retire todo o sobrenadante médio DYT e adicionar uma pré-mistura de 25 mL de tampão-S e 250 µ l de 1m MgSO 4 25 µ l de colesterol de 5 mg/mL (dissolvido em etanol). Todas essas soluções são estéreis.</li>
	<li>Ressuspender as bactérias e transfira para um tubo de plástico descartável de 50 mL.</li>
	<li>Para a calor-inactivação, incubar a ressuspensão OP50 de Escherichia coli em banho-maria por 30 min no 65 ° C.</li>
	<li>Esfriar até a temperatura ambiente e loja a 4 ° C até o uso. Isso pode ser armazenado por até um mês.</li>
</ol> 3. Preparação de NGM placas contendo ou 1% DMSO ou 750 µM Etoposide <ol><li>Prepare duas garrafas de vidro de 200ml limpa. Adicione 0,6 g de NaCl, 0,5 g de peptona, 3,4 g de ágar-ágar, 195 mL de água destilada e um agitador magnético para uma garrafa. Adicionar um agitador magnético para a outra garrafa vazia.</li>
	<li>Garrafas de autoclave estes dois por 15 min a 121 ° C e em seguida esfriar os frascos em banho-maria por 30 min a 55 ° C.</li>
	<li>Adicionar 0,2 mL de 1 M CaCl 2, 0,2 mL de colesterol de 5 mg/mL, 0,2 mL de 1 M MgSO 4 e 5 mL de 1 M KPO 4 (todas essas soluções são estéreis) e em seguida, misturá-los por agitação magnética, um prato quente em 55 ° C.</li>
	<li>Dividir o meio NGM misto em duas garrafas com o mesmo volume (100 mL cada uma). Em seguida, adicionar 1 mL de DMSO para uma garrafa, misture-o novamente usando a agitação magnética. Armazene o outro frasco num banho de água a 55 ° C até o próximo passo. Alíquota 3 mL do meio a cada 35 x 10 mm 2 placa de Petri contendo DMSO. Aproximadamente 33 placas contendo DMSO NGM podem ser feitas desta etapa.</li>
	<li>Misturar o outro frasco usando a agitação magnética, adicionar 1 mL de 75mm etoposide (estoque dissolvido em DMSO), misturar novamente e repita o procedimento de alíquota (passo 3.4). Aproximadamente 33 etoposide (750 µM)-com NGM chapas pode ser feita desta etapa. 
	Nota: No correlata anterior papel 6, nós testamos 0, 250, 500 e 1.000 µM de etoposide. Com base nos dados do, escolhemos a 750 µM de etoposide dose neste trabalho.</li>
	<li>Cool para baixo as placas NGM que contenham produtos químicos à temperatura ambiente por deixá-los em temperatura ambiente por cerca de 3 h e armazená-los em 4 ° C até o uso. 
	Nota: No caso de produtos químicos sensíveis à luz, bloqueie a luz para minimizar a decomposição induzida por luz. 
	Nota: Opcionalmente, fazer uma placa NGM normal sem produtos químicos para o ensaio, que pode ser realizado usando duas condições diferentes de tratamento químico, conforme descrito abaixo de postura de ovos.</li>
	<li>Adicione 100 µ l de calor-inativada Escherichia coli OP50 (como descrito na seção 2) e espalhe sobre cada placa NGM. Deixe secar durante a noite em uma incubadora de c. elegans, a 20 ° C para o teste de química</li>
</ol> 4. Medição do crescimento de c. elegans <ol><li>transferência a idade-sincronizado c. elegans ovos à placa NGM suplementada com 1% DMSO ou 750 µM etoposide.</li>
	<li>Incubar c. elegans em uma incubadora a 20 ° C por 4 dias. Observem os vermes, usando um microscópio estéreo e tomar imagens microscópicas todos os dias. Geralmente, ampliação de 20 X (1 X objectiva, ampliação de zoom X 2 e 10 X ocular lente) é apropriado para a observação do crescimento de c. elegans. Também levar uma imagem microscópica de micrômetro de palco o microscópio para a calibração de tamanho worm. 
	Nota: A fome afeta o ensaio de toxicidade. Portanto, alimentar com bactérias suficientes ou ajustar o transferidos c. elegans números no início de ovo. Observar até 3 dias para excluir a possibilidade de fome.</li>
	<li>Medir o comprimento de corpo de c. elegans, tratados com DMSO ou etoposide em cada ponto de tempo usando software ImageJ. 
	Nota: Para cada medição de amostra, 50 vermes são suficientes para a análise estatística.
	<ol><li>No ImageJ, abra a imagem de micrômetro de palco de microscópio (&#39; arquivo &#39; &#8594; &#39; abrir &#39;). 
		Nota: A imagem de micrômetro e imagens de worm (etapa 4.2) devem ser a mesma ampliação.</li>
		<li>Arrastar uma linha de 1.000 µm na imagem de calibração de micrômetro usando &#39; linha reta &#39; ferramenta.</li>
		<li>Clique &#39; Analyze &#39; &#8594; &#39; definir escala de &#39; e entrada de 1.000 e µm para a &#39; conhecida a distância &#39; caixa e &#39; unidade de comprimento &#39; caixa, respectivamente. Verificar &#39; Global &#39; e clique em &#39; Okey &#39;.</li>
		<li>Abrir as imagens de worm (&#39; arquivo &#39; &#8594; &#39; Open &#39;). Desenhar uma linha ao longo da característica de cada worm usando &#39; Freehand linha &#39; ferramenta. Obter os dados de comprimento de corpo clicando &#39; Analyze &#39; &#8594; &#39; medida &#39;.</li>
		<li>Repita essas etapas para 50 vermes.</li>
	</ol></li></ol> 5. C. elegans ovo deitado ensaio <ol><li>incubar a idade-sincronizado ovos sobre a NGM placa contendo DMSO ou etoposide para 64 h (antes do nascimento de primeiro) até a fase adulta jovem em 20 ° C. 
	Nota: É opcional usar os vermes das experiências de medição de crescimento. É conveniente fazer a medição de crescimento e postura ensaio de ovo juntos.</li>
	Placas de <li>transferir 5 vermes adultos para a nova NGM sem produtos químicos (condição 1, tratamento químico dentro de um determinado período de tempo, de ovos para fase adulta jovem) ou nova placa NGM, contendo o mesmo pré-tratamento químico (condição 2, a exposição contínua de produtos químicos durante o período experimental em geral) e então eles incubar por 24 h. 
	Nota: É aconselhável fazer réplicas usando placas NGM de 3-5 para cada tratamento; Estas repetições podem ser usadas para análise estatística.</li>
	<li>Transferir para a nova chapa NGM como descrito acima e contar o número de ovos todos os dias. Contar os vermes que rastejou fora, morreram e foram internamente escotilhaEd.</li>
	<li>Repita estes passos até os vermes ovos sem mais, geralmente em 5 dias.</li>
	<li>Calcular o número de ovos postos por worm de cada prato e somar esses valores todos os dias durante 5 dias determinar o número total de ovos postos. 
	Nota: Excluir o número de minhocas que rastejou fora e internamente eclodiram no cálculo de.</li>
</ol>

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Neste artigo, descrevemos a avaliação da toxicidade de produtos químicos em c. elegans, um nematódeo de solo, usando etoposide como um exemplo tóxica. Para este fim, usamos duas condições experimentais. No primeiro set, c. elegans foram cultivadas na etoposide contendo placas de ovos para a fase de adulta jovem, e então os vermes foram autorizados a colocar ovos em placas NGM normais sem produtos químicos. No segundo conjunto experimental, c. elegans continuamente foram tratados com et...

Avaliação toxicológica é essencial para o desenvolvimento de produtos farmacêuticos, nutracêuticos e cosmecêuticos, bem como a avaliação de risco de várias toxinas ambientais. O modelo de roedor é um dos mais populares na vivo sistemas experimentais para o estudo de toxicologia; Alternativamente, organismos não-mamíferos, tais como c. elegans são também amplamente utilizados. Modelos de avaliação toxicológica mamiferas são benéficos devido não só animais questões éticas, mas também sua conveniência e utilidade, considerando a relação custo-eficácia, facilidade de manutenção, velocidade e reprodutibilidade1,2 ,3,4.
C. elegans, um solo redonda worm, tem sido explorada como um animal modelo em várias pesquisas básicas e aplicadas biologia e química. É um 1 mm de comprimento, o nematódeo transparente, que é simplesmente mantido em sólido ou líquido nematódeo crescimento Media (NGM) alimentados com a estirpe bacteriana Escherichia coli OP50. C. elegans tem um ciclo de vida curto, e o selvagem-tipo N2 c. elegans põe aproximadamente 300 ovos. Portanto, ela é facilmente propagada para serem utilizados como materiais experimentais3,4,5. C. elegans também tem sido amplamente utilizada nos estudos toxicológicos de muitas drogas e poluentes ambientais6,7,8,9.
Porque muitas drogas anticâncer alvo rapidamente dividindo as células cancerosas, podem também danificar dividindo rapidamente as células normais como as células do folículo piloso, epitélio intestinal e medula óssea. Por exemplo, drogas anticâncer inibidores de topoisomerase alvo o processo de replicação do DNA de células cancerosas; Portanto, eles também inibem dividindo rapidamente as células normais. Todos os organismos vivos tem topoisomerases e estes topoisomerase inibidores mais provável impacto ambiental de ecossistemas6,10,11. Assim, uma plataforma de avaliação toxicológica de drogas usando um animal modelo é valiosa para tanto no desenvolvimento de fármacos e avaliação dos riscos ambientais.
Neste artigo, descrevemos os protocolos detalhados para testar a toxicidade de etoposide, que é um agente anticâncer clínico que alvos topoisomerase II, como um produto químico tóxico de modelo em c. elegans. Para este fim, descreveremos o método de medição do tamanho do corpo e o número total de ovos postos em c. elegans , tratados com etoposide.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Affymetrix, USA</td>
			<td>10906</td>
			<td></td>
		</tr>
		<tr>
			<td>Caenorhabditis elegans N2</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Wild type</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma, USA</td>
			<td>C3045</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma, USA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli OP50</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide</td>
			<td>Sigma, USA</td>
			<td>E1383</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software (ver 1.4)</td>
			<td>Natinoal Institute of Health, USA</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Jenopitk, Progress Gryphax, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Merck, USA</td>
			<td>107213</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#215; 10 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10035</td>
			<td></td>
		</tr>
		<tr>
			<td>90 &#215; 15 mm Petri dish</td>
			<td>SPL Life Sciences, South Korea</td>
			<td>10090</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Nikon, Japan</td>
			<td>SMZ800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Becton Dickinson, USA</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effect of chemicals on the growth and reproduction of caenorhabditis elegans

Avaliação toxicológica é crucial para entender os efeitos das substâncias químicas sobre organismos vivos nos campos da ciência biológica básica e aplicada. Um solo não-mamíferos redondo verme Caenorhabditis elegans, é um organismo modelo valioso para estudos de toxicologia, devido à sua conveniência e a falta de questões de ética animal, em comparação com sistemas de animais mamíferos. Neste protocolo, é descrito um procedimento detalhado de avaliação toxicológica de substâncias químicas em c. elegans . Uma droga anticancerígena clínica, etoposide, quais destinos humano topoisomerase II e inibe a replicação do DNA de células cancerosas humanas, foi selecionada como um modelo de testes químicos. Idade-sincronizado c. elegans ovos foram expostos a dimetilsulfóxido (DMSO) ou etoposide, e então o crescimento de c. elegans foi monitorado diariamente por 4 dias pela observação de microscópio estéreo. O número total de ovos assente de c. elegans , tratados com DMSO ou etoposide foi também contada usando o microscópio estéreo. Tratamento de Etoposide afetado significativamente o crescimento e a reprodução de c. elegans. Por comparação do número total de ovos de vermes com períodos de tratamento diferentes de produtos químicos, pode decidir-se que a toxicidade reprodutiva de produtos químicos na reprodução de c. elegans é reversível ou irreversível. Estes protocolos podem ser úteis para tanto no desenvolvimento de várias drogas e avaliação de riscos de tóxicos ambientais.

O tratamento de etoposide (24-96 h) retardado significativamente o crescimento de c. elegans. Após 96 horas de incubação, vermes etoposide-tratados cresceram 0,86 mm de comprimento, enquanto os vermes de veículo-tratados cresceram a 1,04 mm (Figura 1). Retardo de crescimento também aparentemente foi observado sob observação microscópio estéreo (Figura 2). Começamos a ver os ovos do veículo-tratados vermes a 72...

Watch this Scientific Journal Video about Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans at JoVE.com

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Um protocolo básico para avaliar a toxicidade de produtos químicos em um animal modelo, Caenorhabditis elegans, é descrito. O método é conveniente e útil para o desenvolvimento de produtos farmacêuticos, bem como para a avaliação de risco de diversos poluentes ambientais.

Medir o efeito de produtos químicos sobre o crescimento e a reprodução de Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

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Medicine

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

16.8K visualizações.  Korea Institute of Science and Technology. Um protocolo básico para avaliar a toxicidade de produtos químicos em um animal modelo, Caenorhabditis elegans, é descrito. O método é conveniente e útil para o desenvolvimento de produtos farmacêuticos, bem como para a avaliação de risco de diversos poluentes ambientais.

Vídeo: Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Measuring the Strength of Mice

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score maximum on this task; it is a quick but insensitive gross screen, and the weights test described in this article will provide a finer measure of muscular strength.
There are also several strain gauge-based pieces of apparatus available commercially that will provide more graded data than the inverted screen test, but their cost may put them beyond the reach of many laboratories which do not specialize in strength testing. Hence in 2000 a cheap and simple apparatus was devised by the author. It consists of a series of chain links of increasing length, attached to a "fur collector" a ball of fine wire mesh sold for preventing limescale build up in hard water areas. An accidental observation revealed that mice could grip these very tightly, so they proved ideal as a grip point for a weight-lifting apparatus. A common fault with commercial strength meters is that the bar or other grip feature is not thin enough for mice to exert a maximum grip. As a general rule, the thinner the wire or bar, the better a mouse can grip with its small claws.
This is a pure test of strength, although as for any test motivational factors could potentially play a role. The use of scale collectors, however, seems to minimize motivational problems as the motivation appears to be very high for most normal young adult mice.

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

Medir a força dos ratos

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score ...

Medicina

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

A coloração imuno-histoquímica de B7-H1 (PD-L1) na histológicos em parafina lâminas de tecido adenocarcinoma do pâncreas

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

Medindo fragilidade em indivíduos infectados pelo HIV. Identificação de pacientes frágeis é o primeiro passo para a Melhoria e Reversão de fragilidade

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

The Influence of Liver Resection on Intrahepatic Tumor Growth

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not detectable by routine clinical imaging, may be stimulated by hepatic regeneration factors after liver resection. It is not entirely clear, however, which factors are crucial for tumor recurrence.
The presented mouse model may be useful to explore the mechanisms that play a role in the development of recurrent malignant lesions after liver resection. The model combines the easy-to-perform and reproducible techniques of defined amounts of liver tissue removal and tumor induction (by injection) in mice. The animals were treated with either a single laparotomy, a 30% liver resection, or a 70% liver resection. All animals subsequently received a tumor cell injection into the remaining liver tissue. After two weeks of observation, the livers and tumors were evaluated for size and weight and examined by immunohistochemistry.
After a 70% liver resection, the tumor volume and weight were significantly increased compared to a laparotomy alone (p &#60;0.05). In addition, immunohistochemistry (Ki67) showed an increased tumor proliferation rate in the resection group (p &#60;0.05).
These findings demonstrate the influence of hepatic regeneration mechanisms on intrahepatic tumor growth. Combined with methods like histological workup or RNA analysis, the described mouse model could serve as foundation for a close examination of different factors involved in tumor growth and metastatic disease recurrence within the liver. A considerable number of variables like the length of postoperative observation, the cell line used for injection or the timing of injection and liver resection offer multiple angles when exploring a specific question in the context of post-hepatectomy metastases. The limitations of this procedure are the authorization to perform the procedure on animals, access to an appropriate animal testing facility and acquisition of certain equipment.

A high incidence of tumor recurrence after resection of liver metastases remains an unsolved problem. The illustrated mouse model may be useful to investigate the reasons for such recurrences. It combines a liver resection model with intrahepatic tumor cell injection for the first time.

A Influência do fígado ressecção no crescimento do tumor intra-hepática

The high incidence of tumor recurrence after resection of metastatic liver lesions remains an unsolved problem. Small tumor cell deposits, which are not ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Fósforo-31 Magnetic Resonance Spectroscopy: uma ferramenta para medir<em&gt; In Vivo</em&gt; Capacidade mitocondrial fosforilação oxidativo no músculo esquelético humano

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetria em plaquetas humanas lavadas: O efeito do inibidor Pannexin1-Brilliant Blue FCF na agregação induzida por colagénio

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Um modelo de trofoblasto humano primário para estudar o efeito da inflamação associada com obesidade materna no Regulamento de autofagia na Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

Medindo os efeitos de bactérias e produtos químicos sobre a permeabilidade intestinal de Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Efeito das formulações de lágrimas artificiais na atividade metabólica das células epiteliais da córnea humana após exposição à dessecação

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic nerve cross-sectional assessment and quantification using 3 T MRI for image acquisition and ImageJ's Fiji software for image processing quantification. Image acquisition was performed using 3 T MRI, with proper instructions for the patient to ensure straight fixation during imaging. A T2-weighted fat suppressed sequence was used. A coronal cut taken 3 mm behind the globe and perpendicular to the optic nerve axis should be uploaded to the software. Using the threshold function, the white matter area of the optic nerve is selected and quantified, thus, eliminating inter-individual measurement bias. We also described the normal limits for the optic nerve cross-sectional area according to age, based on previously published literature. We used the described protocol to assess optic nerve of a suspected glaucoma patient. The optic nerve cross-sectional area was found to be within the normal limits, a finding further confirmed via optical coherence tomography of the optic nerve.

We provided a detailed protocol for a standardized method of optic nerve assessment and quantification using MRI, utilizing a widely available imaging sequence, and open access software for image analysis. Following this standardized protocol would provide meaningful data for comparison between different patients and different studies.

Quantificação da Área Transversal do Nervo Óptico na Ressonância Magnética: Um novo protocolo usando o software Fiji

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic ...