Este estudo foi parte da Agriculture and Agri-Food Canada A-base do projeto &quot;Desenvolvimento de técnicas de alto rendimento para amostras de carne para reduzir o gap phenomic para características de qualidade multivariadas na seleção assistida&quot;. A assistência especializada da Unidade de Processamento de Carne de pessoal Meat no Centro de Pesquisa Lacombe estão sinceramente apreciado. Os autores também gostaria de agradecer a assistência técnica dedicada de Christine Burbidge-Boyd, Fran Costello, Glynnis Croken e Rhona Thacker.

1. Coleção Steak <ol><li> Após a graduação 24 horas - 48 horas pós abate, no músculo Longissimus dorsi (ou o fim torácica e / ou lombar) é removido a partir de carcaças de carne bovina e coletadas para análise de qualidade. </li><li> O músculo é cortado de gordura subcutânea e off quadrado. O músculo é então cortada em 2,5 cm (1 in) bifes seguindo protocolos de manipulação de alimentos e de segurança. Após o corte das carnes individuais, as etiquetas apropriadas (o número de animais, o lado, a localização do músculo, ou tempo) são colocados nas respectivas carnes. </li><li> Bifes são então preparadas para armazenamento. Caso devam permanecer fresca, eles são colocados num tabuleiro e levado para a grelha. Se os bifes frescos não podem ser preparados imediatamente, eles precisam de ser cobertas com plástico, até se pode cozinhar, para evitar a desidratação. Se bifes devem ser envelhecido em um refrigerador, ou congelado, o peso é registrado com duas casas decimais antes de serem colocados em um oxigênio impermeável vácuo pacote bag e selados usando uma máquina de embalagem a vácuo. Bifes são então colocadas nos seus respectivos locais de armazenamento de longo prazo. </li></ol> 2. Cooking Procedimento <ol><li> Se a amostra foi congelada, retire do freezer e coloque em um 4 ° C mais frio para descongelar durante a noite. </li><li> No dia da cozinha, pré-aqueça uma grelha open-forno a 210 ° C para a amostra de cozinhar, e preparar o sistema logger de temperatura e computador, inserindo os termopares para as saídas do dispositivo de digitalização. Unte levemente a grelha com gordura vegetal cerca de 10 min antes de cozinhar começa. </li><li> Antes de começar a cozinhar, retire bifes do refrigerador. Retirar a amostra da embalagem e secar o excesso de umidade do bife com toalha de papel. Anote o peso bruto do bife com duas casas decimais. </li><li> Inserir um termopar no ponto médio da carne ao longo do seu eixo longitudinal. Inicie a digitalização termopares para gravar dados de cozinha. </li><li> Coloque abifes na grelha, mantendo o paralelo termopares à superfície cozedura da carne e da grade. Se a sonda não é mantido paralelo, que pode resultar em leituras de temperatura impróprias. </li><li> Cozinhar carnes a uma temperatura interna de 35,5 ° C, e depois virar a recolha de amostras durante e continue a cozinhar até uma temperatura final interno de 71 ° C. Uma vez que a temperatura interna é de 71 ° C, remover a amostra da grelha e puxar para fora o termopar da carne. </li><li> Se um peso de cozimento é desejado, deixe o bife para esfriar um pouco (1-2 min), em seguida, apagar a amostra com um papel toalha para remover o excesso de umidade da amostra e pesar até duas casas decimais. </li><li> Se forem necessários dados de culinária, salvar os dados de verificação de termopares. </li></ol> 3. Ajustar o ângulo e Preparação de Amostras <ol><li> Embora a amostra ainda está quente da grelha, faça um corte de 1 - 2 cm da extremidade lateral do bife para criar uma ponta quadrada. </li><li> A seção 5 centímetros em todo o widpo de o bife é preparado usando uma &quot;caixa de dimensionamento amostra&quot; com um entalhe de faca, a 5 cm, em paralelo com o corte inicial. </li><li> O ângulo de orientação da fibra muscular pode ser medida colocando um prolongador na superfície de lâmina de corte da carne. </li><li> A caixa de corte de ângulo variável é, então, definida como o ângulo correspondente da amostra, deslizando a placa de metal menor personalizado com os furos inferiores para guiar a faca de lâmina dupla. Apertar os botões para fixar a placa de metal inferior ao ângulo correspondente. </li><li> Coloque a secção de 5 cm de carnes na área de corte da caixa de corte ângulo variável, de modo que a direcção das fibras musculares é paralelo ao ângulo da faca de lâmina dupla. </li><li> A faca de lâmina dupla é utilizado para cortar uma fatia de 1 cm através da previamente cortado 5 centímetros peça, desse modo, assegurar que a faca irá passar através de toda a amostra, com o ângulo correspondente. Ao colocar o bife, verifique se ele está tocando a borda da área de corte mais próximo doutilizador, como o movimento de corte vai puxar a carne no sentido do utilizador. </li><li> Para cortar, coloque a faca de lâmina dupla na caixa de corte e puxar a faca para cortar a carne em uma faixa de 1 cm. Para evitar a ruptura da carne, usar um ligeiro movimento de corte da faca, enquanto puxa na direcção do utilizador. Mantenha as lâminas de faca contra uma borda da placa de guia para manter as proporções de corte iguais. </li><li> O resultado deve ser de 1 cm, fatia grossa quente que é de 5 cm de comprimento, com as fibras consistentemente paralela à fatia. Esta fatia é então usado para fatia da força de cisalhamento de textura na carne. </li></ol> 4. Análise da força de cisalhamento <ol><li> Para determinar a análise da força de cisalhamento, a fatia de cisalhamento protocolo vigor (SSF) usa os 1 centímetro por 5 centímetros fatia obtidos com a caixa de corte ângulo variável. Usando um equipamento de análise de textura, com uma fatia de lâmina de corte plana, a análise é realizada sobre a fatia quente. </li><li> Preparar o Analisador de Textura com uma célula de carga de 50 kg, e conjuntoa distância ao cisalhamento a 48 mm, com uma velocidade de cabeça transversal de 500 mm / min. </li><li> O corte é executado uma vez, perpendicular às fibras musculares para fornecer uma análise exacta da força máxima necessária para cortar as fibras da fatia. </li><li> Uma vez que a fatia foi cortado completamente através do centro, guardar e gravar os dados. </li></ol>

<ol>
	<li>Jayasooriya, S. D., Torley, P. J., D'Arcy, B. R., Bhandari, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+power+ultrasound+and+ageing+on+the+physical+properties+of+bovine+Semitendinosus+and+Longissimus+muscles.">Effect of high power ultrasound and ageing on the physical properties of bovine Semitendinosus and Longissimus muscles.</a> Meat Science. 75, 628-639 (2007).</li><li>Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+proteinases+and+meat+aging.">Muscle proteinases and meat aging.</a> Meat Science. 36, 93-104 (1994).</li><li>Lepetit, J., Culioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+meat.">Mechanical properties of meat.</a> Meat Science. 36, 203-237 (1994).</li><li>Honikel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+methods+supported+by+OECD+and+their+use+in+Mediterranean+meat+products.">Reference methods supported by OECD and their use in Mediterranean meat products.</a> Food Chemistry. 59, 573-582 (1997).</li><li>Monin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+methods+for+predicting+quality+of+whole+meat.">Recent methods for predicting quality of whole meat.</a> Meat Science. 49, S231-S243 (1998).</li><li>Shackelford, S., Wheeler, T., Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+slice+shear+force+as+an+objective+method+of+assessing+beef+longissimus+tenderness.">Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.</a> Journal of Animal Science. 77, 2693-2699 (1999).</li><li>Ju&#225;rez, M., Hui, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beef+Texture+and+Juiciness.">Beef Texture and Juiciness.</a> Handbook of meat and meat processing. , 177-206 (2012).</li><li>Kerth, C. R., Montgomery, J. L., Lansdell, J. L., Ramsey, C. B., Miller, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+gradient+in+longissimus+steaks.">Shear gradient in longissimus steaks.</a> Journal of Animal Science. 80, 2390-2395 (2002).</li><li>Janz, J. A. M., Aalhus, J. L., Dugan, M. E. R., Price, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mapping+method+for+the+description+of+Warner-Bratzler+shear+force+gradients+in+beef+Longissimus+thoracis+et+lumborum+and+Semitendinosus.">A mapping method for the description of Warner-Bratzler shear force gradients in beef Longissimus thoracis et lumborum and Semitendinosus.</a> Meat Science. 72, 79-90 (2006).</li><li>Derington, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+slice+shear+force+and+Warner-Bratzler+shear+force+of+beef+strip+loin+steaks+as+related+to+the+tenderness+gradient+of+the+strip+loin.">Relationships of slice shear force and Warner-Bratzler shear force of beef strip loin steaks as related to the tenderness gradient of the strip loin.</a> Meat Science. 88, 203-208 (2011).</li><li>Shackelford, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+Shear+Force.">Slice Shear Force.</a> Beef Facts. , (2009).</li><li>Henrickson, R. L., Mjoseth, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenderness+variation+in+two+bovine+muscles.">Tenderness variation in two bovine muscles.</a> Journal of Animal Science. 23, 325-331 (1964).</li><li>Ju&#225;rez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+relative+contribution+of+ante-+and+post-mortem+factors+to+the+variability+in+beef+texture.">Quantifying the relative contribution of ante- and post-mortem factors to the variability in beef texture.</a> Animal. FirstView, 1-10 (2012).</li><li>Shanks, B. C., Wulf, D. M., Maddock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+The+effect+of+freezing+on+Warner-Bratzler+shear+force+values+of+beef+longissimus+steaks+across+several+postmortem+aging+periods.">Technical note: The effect of freezing on Warner-Bratzler shear force values of beef longissimus steaks across several postmortem aging periods.</a> Journal of Animal Science. 80, 2122-2125 (2002).</li><li>Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., Dikeman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electric+belt+grill,+forced-air+convection+oven,+and+electric+broiler+cookery+methods+for+beef+tenderness+research.">Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research.</a> Meat Science. 58, 239-246 (2001).</li><li>Aalhus, J., Ju&#225;rez, M., Aldai, N., Uttaro, B., Dugan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+preparation+and+eating+quality.">Meat preparation and eating quality.</a> , 1058-1063 (2009).</li><li>Sa&#241;udo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+slaughter+weight,+breed+type+and+ageing+time+on+beef+meat+quality+using+two+different+texture+devices.">The effects of slaughter weight, breed type and ageing time on beef meat quality using two different texture devices.</a> Meat Science. 66, 925-932 (2004).</li></ol>

Não temos nada a divulgar.

A caixa de ângulo ajustável garante a lâmina SSF sempre tesouras fibras musculares em um ângulo perpendicular verdade, ao invés de aproximadamente perpendicular o que poderia acontecer com um fixo ângulo de corte de 45 ° 8. A aplicação da caixa de corte de ângulo variável permite descrições mais precisas da força de cisalhamento máxima em uma amostra, e é nesta base de melhorar técnicas objetivas de análise de qualidade de que a caixa de corte de ângulo variável foi desenvolvido. Por outro...

Ternura é um dos mais importantes atributos de qualidade em carne 1. Inconsistência na maciez da carne tem sido identificado como um dos principais problemas enfrentados pela indústria da carne 2. A força de ensaio de cisalhamento Warner-Bratzler (WBSF), caracterizado por um orifício triangular em uma placa de corte precisamente maquinado, é o método mais amplamente utilizado para indicar a tenrura da carne sensorial 3,4, como é o método instrumental que discutivelmente tem mostrado a melhor correlação com a pontuação dos provadores para carne resistência 5. No entanto, o protocolo de força de cisalhamento fatia (SSF) tornou-se uma técnica importante para a análise de textura e sensibilidade muscular 6, como uma alternativa para o protocolo padrão WBSF 7. É benéfico em casos em que a análise rápida ou um elevado número de amostras necessário para ser processado. Para o SSF, apenas um núcleo é retirado do bife quando ele ainda está quente, contra os múltiplos núcleos (3-6) feita a partir da carne geralmente réer 24 horas de refrigeração para o WBSF 6. Deste uma fatia, o SSF analisa a textura média de todo o bife de 8, como foi verificado que a tenrura varia entre os lados laterais para mediais do bife 9, com o centro e meio de carnes tendo a melhor representação para a média WBSF . A queda para o SSF é que, dentro de um bife, do lateral lados medial, há uma variação nos valores de força de cisalhamento 9, no entanto, consistentemente usando a caixa de dimensionamento para fazer 5 seções cm, pode reduzir a variabilidade que pode vir do uso de diferentes seções do bife. No entanto, uma vez que alguns bifes são de tamanhos diferentes, um Slice 5 cm pode ocorrer em diferentes locais do bife, o que poderia, então, afetam os valores de força de cisalhamento 10. Por outro lado, o quadro de corte original é concebido para obter a secção a ser analisada no protocolo SSF permite apenas dois ângulos fixos, 45 ° e 90 °, no entanto, músculomudanças de orientação da fibra dentro de bifes, e dentro do músculo 11. Shackelford e 12 Wheeler afirma que o ângulo médio longissimus foi de 43,8 °, que estava perto de 45 ° e, portanto, considerado adequado quando as amostras foram coletadas sempre no mesmo local. No entanto, Derington et al. 11 relataram uma gama do ângulo das fibras ao longo do músculo longissimus entre 33,1 ° e 53,9 °. Assim, quando, para efeitos de investigação, várias carnes do mesmo animal precisa de ser analisado, designando todos os ângulos de fibras tanto para 45 ° ou 90 ° reduz potencialmente precisão. O uso de uma caixa de corte de ângulo variável pode fornecer uma representação mais precisa da força de corte máxima, como tendo a capacidade de medir e ajustar o ângulo de corte permite a fatia para consistentemente correm paralelamente com as fibras musculares.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Vacuum Packager</td>
			<td>Koch Equipment, Kansas City, MO, USA</td>
			<td>Model UV2100</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Winpak Ltd., Winnipeg, MB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocouples and Scanning Device</td>
			<td>Agilent / Hewlett Packard, Santa Clara, CA, USA</td>
			<td>34970A Data Acquisition Switch Unit</td>
		</tr>
		<tr>
			<td>Grill</td>
			<td>Garland Commercial Ranges Ltd., Mississauga, ON, Canada</td>
			<td>Model ED-30-B</td>
		</tr>
		<tr>
			<td>Crisco Vegetable Shortening</td>
			<td>The J.M. Smucker Company, Orrville, OH, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sample sizing box</td>
			<td>G-R Manufacturing Co., Manhattan, KS, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Angle adjustable box</td>
			<td>Innovation Centre, Red Deer College, Red Deer, AB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texture Analyzer Machine</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>Model TA-XT Plus</td>
		</tr>
		<tr>
			<td>Load Cell, 50 kg</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-XT Plus</td>
		</tr>
		<tr>
			<td>USDA Warner-Bratzler knife w/guillotine block</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7</td>
		</tr>
		<tr>
			<td>Flat rectangle Blade for Slice Shear Force</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7C</td>
		</tr>
	</tbody>
</table>

introducing an angle adjustable cutting box for analyzing slice shear force in meat

A pesquisa indica o ângulo de fibra do músculo longissimus pode variar, dependendo da localização dentro de um bife e ao longo do músculo. Em vez de utilizar os originais fixos 45 ° ou 90 ° de ângulo de corte para testar a força de cisalhamento, uma caixa de corte de ângulo variável pode ser ajustada de modo que os ângulos das facas corresponder ao ângulo de fibra de cada amostra. Dentro de 2 min após o cozimento a uma temperatura interna de 71 ° C em uma grade aberta lareira fixada em 210 ° C, de 1 cm em 5 cm núcleo é cortado do bife, paralela à direção da fibra muscular, usando duas lâminas de faca definir um cm de distância. Este núcleo quente é então submetido ao protocolo da força de cisalhamento da fatia (SSF) para avaliar a textura da carne. O uso da caixa de corte de ângulo variável e o protocolo de SSF fornece uma representação exacta da força de cisalhamento máximo, como as fibras de fatia e os músculos são consistentemente paralelo. Portanto, a caixa de corte de ângulo variável, em conjunto com o protocolo de SSF, pode ser usado como um alto-throughput técnica para avaliar com precisão a maciez da carne em diferentes locais do músculo Longissimus dorsi e, possivelmente, em outros músculos.

Trinta e um final novilhos carne comercial foram abatidos e suas carcaças (528-601 kg) foram divididos. A fim de criar a variabilidade na sensibilidade, os lados direito foram imediatamente armazenados a 2 ° C, enquanto que os lados esquerdo foram realizadas a 10 ° C durante 3 horas e, em seguida, a 2 ° C. Os músculos longo dos lados direito e esquerdo foram removidos 24 horas após o abate. Amostras dos lados direito (tratamento &quot;duro&quot;) foram analisados ​​naquele dia, enquanto as amostras do...

Watch this Scientific Journal Video about Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat at JoVE.com

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Força de cisalhamento Slice é um método de referência para análise de textura de carne. Usando uma caixa de corte de ângulo ajustável poderia aumentar a sua precisão para fins de pesquisa. Os resultados obtidos a partir de diferentes locais dentro do<em&gt; Longissimus</em&gt; Muscular mostram uma alta correlação com a metodologia vigor Warner-Bratzler shear e alta adaptabilidade potencial para diferentes músculos.

Apresentando uma caixa de corte ajustável ângulo para analisar Slice força de cisalhamento na carne

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

introducing-an-angle-adjustable-cutting-box-for-analyzing-slice-shear

Research

JoVE Journal

Biology

12.8K visualizações.  Agriculture and Agri-Food Canada. Força de cisalhamento Slice é um método de referência para análise de textura de carne. Usando uma caixa de corte de ângulo ajustável poderia aumentar a sua precisão para fins de pesquisa. Os resultados obtidos a partir de diferentes locais dentro do Longissimus Muscular mostram uma alta correlação com a metodologia vigor Warner-Bratzler shear e alta adaptabilidade potencial para diferentes músculos.

Vídeo: Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Cultura Slice organotípicas de E18 cérebros de ratos

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Biologia

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

De célula inteira Recordings of Light Evocados Excitatórios Correntes Synaptic na Slice Retinal

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

Metas de identificação de microRNAs humanos com o Sistema de ensaio de luciferase lightswitch usando 3&#39;UTR repórter-Constrói e um microRNA em células aderentes Mimic

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Um método para selecionar Aptamers de comutação Estrutura Aplicado a um colorimétrico nanopartículas de ouro Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análise qPCRTag - A High Throughput, PCR em Tempo Real Ensaio para Sc2.0 Genotipagem

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

Concepção e desenvolvimento de aptâmero-nanopartículas de ouro Baseadas colorimétricos Ensaios para Aplicações In-the-campo

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

A Assembleia e Aplicação de &#39;tesoura Anéis&#39;: A endotelial novo modelo de Orbital, unidirecional e periódica fluxo de fluido e tensão de cisalhamento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Detectando e caracterizando a auto-montagem da proteína in vivo por citometria de fluxo

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

Gráfico automatizado do espaço visual dos olhos compostos housefly

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Coleta e Manutenção a Longo Prazo de Formigas Cortadeiras (Atta) em Condições de Laboratório

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...