Gostaríamos de agradecer a Srta Wu Shin-diversão pela ajuda nos experimentos. Este estudo foi suportado por uma concessão do Chang Gung Medical Research Program (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373) e IRB 100-4470A3, 104-2462A3, Taiwan.

Os testes realizados em laboratório foi realizado em conformidade com os requisitos do clínico laboratório melhoria alterações (CLIA), de 1988, os regulamentos aprovados pela institucional Review Board de Chang Gung Memorial Hospital e Universidade (licença n º 100 - 4470A3 e 2462A3-104). Foi obtido o consentimento de todos os pacientes.
1. coleta de amostras de sangue
<ol><li>Colete sangue total em tubos de EDTA-Tratado comercialmente disponíveis. Misture delicadamente e armazenar a amostra de sangue a 4 ° C até o processamento.</li>
</ol>2. o DNA extraído do sangue periférico
Usar um Kit de extração de DNA para a extração de DNA genômica (ver Tabela de materiais).
<ol><li>Adicione 10 mL de solução 1 para a amostra de sangue de 4,0 mL. Incube a amostra no gelo por 10 min.</li>
	<li>Centrifugar a amostra a 2.000 x g por 5 min a 4 ° C. Remover e descartar o sobrenadante cuidadosamente. Resuspenda o pellet em 3 mL de solução 1.</li>
	<li>Centrifugar a amostra a 2.000 x g por 5 min a 4 ° C novamente e descartar o sobrenadante. Resuspenda o pellet em 3 mL de solução 2.</li>
	<li>Adicione 10 µ l de solução-mãe pronase (225 mg/mL) para obter uma concentração final de 100 µ g/mL e misture bem. Incube a 37 ° C durante a noite.</li>
	<li>Refrigere o tubo no gelo durante 10 min. Adicionar 0,8 mL de solução 3 para a amostra e inverter a 3 - 5 vezes. Coloque a amostra no gelo por 5 min.</li>
	<li>Centrifugar a amostra a 3.000 x g por 15 min a 4 ° C. Transferi com cuidado o sobrenadante para um tubo de centrifuga conico estéril 15 mL.</li>
	<li>Adicione 6 µ l da solução estoque do RNase (10 mg/mL) para obter uma concentração final de 20 µ g/mL. Incube a 37 ° C por 15 min.</li>
	<li>Adicione 2,5 mL de álcool isopropílico e misturar completamente suavemente várias vezes por inversão para precipitar o DNA (fios de material branco, floculante formarão). Usando uma pipeta de grande diâmetro, transferi o dessensibilizador de DNA para um novo tubo de centrifugação de 1,5 mL.</li>
	<li>Centrifugar a 12.000 x g por 3 min a 4 ° C. Desprezar o sobrenadante cuidadosamente. Pellet de DNA lave adicionando 500 µ l de etanol 70%.</li>
	<li>Centrifugar a 12.000 x g por 1 min e descartar o sobrenadante. Ar seca pelota do ADN à temperatura ambiente. Adicione 100 µ l de DDQ2O e incubar a 65 ° C por 15 min Ressuspender o DNA.</li>
</ol>3. quantificação de DNA genômica
Use um espectrofotômetro (consulte a Tabela de materiais) para detectar a quantidade e a qualidade do DNA genômico.
<ol><li>Faça logon no computador ligado à máquina Espectrofotômetro. Abra o software.</li>
	<li>Inicialize a máquina espectrofotômetro:<ol>
<li>Clique em "Ácidos nucleicos" sobre o software de Espectrofotômetro.</li>
		<li>Limpe o pedestal com uma toalhita de papel descartáveis e água purificada.</li>
	</ol>
</li>
	<li>Branco o espectrofotômetro:<ol>
<li>Carrega 2 µ l de água filtrada no pedestal.</li>
		<li>Abaixe o braço superior do espectrofotómetro e clique "Em branco" para calibrá-lo.</li>
		<li>Quando estiver pronto, levante o braço e o pedestal com uma limpeza a seco.</li>
	</ol>
</li>
	<li>Medida da amostra:<ol>
<li>Clique em "DNA-50" no tipo de amostra.</li>
		<li>Carrega 2 µ l de amostra de DNA no pedestal.</li>
		<li>Abaixe o braço superior e clique em "Medida" para medir a relação A260/A280 e a concentração da amostra.</li>
		<li>Limpe o pedestal entre cada execução com uma limpeza e água purificada.</li>
	</ol>
</li>
	<li>Clique em "Print Screen" para coletar os dados.</li>
</ol>4. genéticas análises de mutações
Amplifica o ADN do alvo com a PCR4. Realizar a PCR em uma reação de 25 µ l kit de mistura usando uma DNA polimerase (ver Tabela de materiais). A tabela 1 mostra o gene TTR intrônicas cartilhas.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Gene</td>
			<td>Sequência da primeira demão</td>
		</tr>
<tr>
<td>Exon1</td>
			<td>F: 5'-TCAGATTGGCAGGGATAAGC-3' R: 5'-GCAAAGCTGGAAGGAGTCAC-3'</td>
		</tr>
<tr>
<td>Exon2</td>
			<td>F: 5'-TCTTGTTTCGCTCCAGATTTC-3' R: 5'-TCTACCAAGTGAGGGGCAAA-3'</td>
		</tr>
<tr>
<td>Exon3</td>
			<td>F: 5'-GTGTTAGTTGGTGGGGGTGT-3' R: 5'-TGAGTAAAACTGTGCATTTCCTG-3'</td>
		</tr>
<tr>
<td>Exon4</td>
			<td>F: 5'-GACTTCCGGTGGTCAGTCAT-3' R: 5'-GCGTTCTGCCCAGATACTTT-3'</td>
		</tr>
</tbody></table>Tabela 1. As primeiras demão intrônicas de gene TTR. (Sequência de referência NCBI: NC_000018.10)
<ol><li>Adicione os reagentes na tabela 2 , a um tubo PCR de 0,2 mL.</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Componentes</td>
			<td>Volume (µ l)</td>
			<td>Concentração final</td>
		</tr>
<tr>
<td>10 x Buffer</td>
			<td>2.5</td>
			<td>1 x</td>
		</tr>
<tr>
<td>MgCl2 (25 mM)</td>
			<td>1.5</td>
			<td>1.5 mM</td>
		</tr>
<tr>
<td>360 GC Enhancer</td>
			<td>1</td>
			<td>-</td>
		</tr>
<tr>
<td>360 da DNA polimerase</td>
			<td>0.2</td>
			<td>2 U/reação</td>
		</tr>
<tr>
<td>dNTP Mix (25 mM cada)</td>
			<td>2</td>
			<td>2 mM cada</td>
		</tr>
<tr>
<td>Primeira demão F (10 µM)</td>
			<td>1</td>
			<td>0,4 ΜM</td>
		</tr>
<tr>
<td>Primeira demão R (10 µM)</td>
			<td>1</td>
			<td>0,4 ΜM</td>
		</tr>
<tr>
<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
<tr>
<td>Água da PCR-classe</td>
			<td>13,8</td>
			<td>-</td>
		</tr>
<tr>
<td>Volume total</td>
			<td>25</td>
			<td>-</td>
		</tr>
</tbody></table>Tabela 2. Condições de reação de PCR.
<ol start="2"><li>Misture delicadamente e centrifugue brevemente em uma centrífuga de bancada para coletar todos os componentes para o fundo do tubo. Colocar o tubo em um thermocycler.</li>
	<li>Realize o PCR para 30 ciclos. Cada ciclo consiste de uma etapa de desnaturação por 30 s a 94 ° C, primeira demão recozimento por 30 s a 58 ° C e extensão do polymerase para 45 s a 72 ° C (ver quadro 3).</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>Passo</td>
			<td>Temperatura (° C)</td>
			<td>Tempo</td>
			<td>ciclo de</td>
		</tr>
<tr>
<td>Desnaturação inicial</td>
			<td>95</td>
			<td>5 min</td>
			<td>1</td>
		</tr>
<tr>
<td>Passo de desnaturação do DNA</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 ciclos</td>
		</tr>
<tr>
<td>Passo de recozimento da primeira demão</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 ciclos</td>
		</tr>
<tr>
<td>Etapa da extensão do polymerase</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 ciclos</td>
		</tr>
<tr>
<td>Etapa de alongamento do post</td>
			<td>72</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
<tr>
<td>Final do PCR ciclismo</td>
			<td>4</td>
			<td>Por tempo indeterminado</td>
			<td></td>
		</tr>
</tbody></table>Tabela 3. Condições de ciclismo para amplificar produtos do PCR.
<ol start="4"><li>Valide o produto do PCR visualizando com eletroforese em gel:<ol>
<li>Prepare um gel de agarose 1,2%:<ol>
<li>Misturar 1,2 g de agarose em pó com 100 mL de 0.5 x TBE num balão microwavable. Microondas por 2 min até a agarose é completamente dissolvido. Arrefecer a mistura de agarose a 55 ° C.</li>
			<li>Adicionar 2 µ l de uma solução de brometo de etídio (10 mg/mL) para a mistura de agarose e misture delicadamente. Despeje a mistura de agarose em uma bandeja de gel para cerca de 5 a 7 mm, em seguida, adicione o comb(s) no lugar. Deixe-o solidificar (pelo menos 30 min) e remova cuidadosamente comb(s).</li>
		</ol>
</li>
		<li>Carregar amostras e executar um gel de agarose:<ol>
<li>Coloque o gel e bandeja para a célula de electroforese. Preencher a célula de eletroforese com 0,5 x TBE até que o gel é coberto.</li>
			<li>Cuidadosamente, carregar 2 µ l de uma 100 bp DNA ladder (ver Tabela de materiais) para a um faixa do gel como marcador de tamanho molecular. Mistura de corante/amostra de carga (5 µ l do mix de produtos PCR com 1 µ l 6 x tintura de carregamento) em pistas adicionais.</li>
			<li>Ligue a fonte de alimentação. Funcione o gel para 25 min a 100 V.</li>
			<li>Remova o gel da célula de electroforese. Visualize os fragmentos de DNA sob UV luz em um sistema da imagem latente.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. sequenciamento de DNA
<ol><li>Purificar os produtos de PCR usando um kit comercial e sequenciar comercialmente ou com um sequenciador automático (consulte a Tabela de materiais).</li>
	<li>Envie sequências de nucleotídeos para o servidor de NCBI BLAST para comparar a consulta de bancos de dados o nucleotídeo10nucleotídeo. Depois que os resultados são exibidos, realizar alinhamentos de sequência e identificar a mutação esperada.</li>
</ol>

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Os autores não têm nada para divulgar.

Há duas etapas críticas dentro do protocolo. Em primeiro lugar, a fim de ter o número suficiente de células brancas do sangue, um espécime de hemodiluted deve ser evitada11. Em segundo lugar, o uso de primers PCR apropriados é fundamental para obter resultados fiáveis12. Usamos a ferramenta web de Primer-BLAST para projetar as primeiras demão4,13; um mínimo de 40 pares de base em cada lado dos exões TTR qu...

Amiloidose transtirretina (TTR) (ATTR) é a forma mais comum de amiloidose sistêmica hereditária1e pode ser causada por uma mutação autossômica dominante herdada no gene transtirretina (TTR)2. Mutações de TTR desestabilizam a estrutura da proteína tetramérica e levam a sua dissociação em monômeros que reagrupa em amiloides2. Mais de 100 mutações de TTR amiloidogénicos foram relatados em todo o mundo1. Análises genéticas com métodos de reação em cadeia (PCR) polimerase confirmam a presença da mutação de ponto TTR e têm vantagens, incluindo evitar a manipulação de sondas radioactivamente etiquetadas em comparação com o borrão do Sul3. PCR é uma técnica rápida, fácil, barata e confiável que tem sido aplicada a inúmeros campos em ciências modernas4.
O diagnóstico precoce desta doença progressiva e fatal é um desafio, dada a sua heterogeneidade fenotípica. Com o aumento da atenção e reconhecimento mais amplo sobre as manifestações iniciais de ATTR, bem como as emergentes de tratamentos5, estudos de diagnóstico apropriados, incluindo teste genético de TTR, portanto, são criticamente fundamentais para melhorar o prognóstico. Além disso, diferentes mutações estão associados com penetrância diferente do traço, idade de início, os padrões de progressão, severidade da doença, a sobrevida mediana, eficácia do transplante de fígado, ou TTR estabilizadores2,6, e graus variáveis de envolvimento neurológico e cardiológico, que têm grandes implicações para o aconselhamento genético7,8,9. Além disso, um teste genético altamente preciso é a única ferramenta que diferencia os dois tipos distintos de ATTR: hereditária (mutante) e tipo selvagem (formulário não mutantes, amiloidose sistêmica senil, SSA)7. É imperativo para confirmar os tipos de ATTR, porque as terapias variam amplamente2. Portanto, há uma necessidade crescente para descrever o protocolo gradual do teste genético de TTR.
A abordagem molecular para detectar a mutação será ilustrada usando o Ala97Ser, a mutação mais comum endémica em Taiwan, por exemplo. Modificações na etapa de extração de DNA, reduzir a quantidade das três soluções usadas e produz uma quantidade suficiente de DNA. Neste protocolo, todos os quatro exões TTR foram analisados, enquanto regiões incluindo 5' montante 3' a jusante, os promotores, os intrões, e regiões untranslated (UTR) não foram sequenciadas.

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		<col />
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		<tr height="21">
			<td height="21" style="height:21px;width:213px;">EDTA-treated tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:347px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA Extraction Kit</td>
			<td style="width:199px;">Stratagen</td>
			<td style="width:347px;">200600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">NanoDrop ND2000 spectrophotometer&#160;</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:347px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Delicate Task Wipers</td>
			<td style="width:199px;">Kimberly-Clark</td>
			<td style="width:347px;">Kimtech Science Kimwipes</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">AmpliTaq Gold 360 DNA Polymerase kit</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">4398823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">TTR gene intronic primers&#160;</td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">thermocycler</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">GeneAmp PCR System 9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">electrophoresis cell&#160;</td>
			<td style="width:199px;">ADVANCE</td>
			<td style="width:347px;">Mupid-2plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA ladder</td>
			<td style="width:199px;">Protech</td>
			<td style="width:347px;">PT-M1-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">dye</td>
			<td style="width:199px;">BioLabs</td>
			<td style="width:347px;">B7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">AlphaImager EC</td>
			<td style="width:199px;">Alpha Innotech</td>
			<td style="width:347px;">AlphaImager EC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">automatic sequencer&#160;</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">3730xl DNA Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

Testes genéticos são o teste mais confiável para transtirretina hereditária relacionadas com amiloidose e deve ser realizado na maioria dos casos de amiloidose transtirretina (ATTR). ATTR é uma doença rara mas fatal com fenótipos heterogêneos; Portanto, o diagnóstico é por vezes adiado. Com o aumento da atenção e reconhecimento mais amplo sobre as manifestações iniciais de ATTR, bem como tratamentos emergentes, estudos de diagnósticos adequados, incluindo a transtirretina (TTR) genética teste, para confirmar os tipos e variantes de ATTR são, portanto fundamental para melhorar o prognóstico. Análises genéticas com métodos de reação em cadeia (PCR) polimerase confirmam a presença de mutações pontuais TTR muito mais rápido e mais seguro do que métodos convencionais tais como borrão do Sul. Aqui, demonstramos a confirmação genética da ATTR Ala97Ser mutação, a mutação mais comum endémica em Taiwan. O protocolo é composto por quatro etapas principais: coleta de amostra de sangue total, a extração de DNA, a análise genética de todos os quatro exões TTR com PCR e sequenciamento de DNA.

Electroforese do gel de agarose de dois pacientes e uma saudável individual revelou bandas dos tamanhos esperados, incluindo um produto PCR bp 454 para exon 4 do gene TTR (Figura 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> Figura 1 . Eletroforese em gel retratando PCR amplificados gene TTR. Normal: um indiví...

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

Aqui, apresentamos um protocolo para confirmar a presença da mutação de ponto para o diagnóstico de amiloidose hereditária transtirretina, usando Ala97Ser, a mutação mais comum endémica em Taiwan, por exemplo.

Análise genética de hereditária transtirretina Ala97Ser relacionados a amiloidose

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

genetic-analysis-hereditary-transthyretin-ala97ser-related

Research

JoVE Journal

Medicine

7.6K visualizações.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. Aqui, apresentamos um protocolo para confirmar a presença da mutação de ponto para o diagnóstico de amiloidose hereditária transtirretina, usando Ala97Ser, a mutação mais comum endémica em Taiwan, por exemplo.

Vídeo: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH para diagnóstico pré-implantação Genética

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Medicina

Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

Examinando as características de memória episódica com potenciais relacionados a eventos em Pacientes com Doença de Alzheimer

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.

Multivariate techniques including principal component analysis (PCA) have been used to identify signature patterns of regional change in functional brain images. We have developed an algorithm to identify reproducible network biomarkers for the diagnosis of neurodegenerative disorders, assessment of disease progression, and objective evaluation of treatment effects in patient populations.

Identificação de doenças relacionadas com padrões de covariância espacial utilizando dados de neuroimagem

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

O Modelo Monoiodoacetate da osteoartrite Dor no mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Tomografia computadorizada mediada por fluorescência para a detecção e quantificação de inflamação Intestinal murino macrófago-relacionados

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Microscopia intravital de monócitos Homing e angiogênese relacionados ao Tumor em um modelo murino de Doença Arterial periférica

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly on life sciences and biomedical disciplines, whereas Scopus and Web of Science are multidisciplinary. The protocol described in the current study was used to search for publications from Jordanian authors in the years 2013-2017. In this protocol, how to use each database to conduct this type of search is explained in detail. A Scopus search resulted in the highest number of documents (11,444 documents), followed by a Web of Science search (10,943 documents). PubMed resulted in a smaller number of documents due to its narrower scope and coverage (4,363 documents). The results also show a yearly trend in: (1) the number of publications, (2) the disciplines that have the most publications, (3) the countries of collaboration, and (4) the number of open access publications. In contrast, PubMed has a sophisticated keyword optimization service (i.e., Medical Subject Heading, or MeSH), while both Scopus and Web of Science provide search analysis tools that can produce representative figures. Finally, the features of each database are explained in detail and several indices that can be extracted using the search results are provided. This study provides a base for using literature databases for bibliometric analysis.

Literature databases are commonly used to assess publications in a certain subject, discipline, country, or region of the world, a practice known as bibliometric analysis. The current protocol details how to use PubMed, Scopus, and Web of Science databases to do bibliometric analysis.

Comparando análise bibliométrica usando bancos de dados pubmed, scopus e web of science

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients subjected to axillary surgery and/or radiations. Its etiology is multifactorial and include also tumor-specific pathological features, such as lymphovascular invasion (LVI) and extranodal extension (ENE). To date, no widely employed guidelines for the early diagnosis of BCRL are available. Here, we illustrate a protocol for a digitally assisted BCRL assessment using a 3D laser scanner (3DLS) and a tablet computer. It has been specifically optimized in a discovery cohort of high-risk breast cancer patients. This study provides a proof-of-principle that augmented reality tools, such as 3DLS, can be incorporated into the clinical workup of BCRL to allow for a precise, reproducible, reliable, and cheap diagnosis.

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

Integrando ferramentas de realidade aumentada no prognóstico e diagnóstico de linfofedema relacionadaao câncer de mama

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

Um Método Não Invasivo para Gerar o Modelo de Lesão da Cartilagem Intra-Articular Induzida por Carga Cíclica do Joelho de Rato

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...