We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

declaração de ética: Antes da coleta de células de seres humanos, consentimento informado e aprovação ética deve ser obtida. 1. Prepare Stain NOTA: Prepare mancha no dia do experimento. <ol><li> Adicionar 5 uL de etídio (ou 4 mM) e 5 ul de calceína AM (ou 4 mM) a 2,5 ml de tampão fosfato salino (PBS) em 15 ml de tubo de centrifugação; misturar. Embrulhe em papel alumínio para proteger da luz e armazenar a RT até o uso. </li></ol> 2. Células Colete <ol><li> Remover inserções de cultura de células a partir de sua embalagem e rotulagem para cada pálpebra a amostrar. Marca de orientação de recolha de células, no lado do suporte de plástico membrana utilizando marcador permanente. Conduta fenda inspecção lâmpada com uma ampliação moderada (aproximadamente 20X) para confirmar a saúde adequada da região de se submeter IC. </li><li> Dispensar uma gota de anestésico tópico (0,5% de cloridrato de proparacaína) no fundo do saco conjuntival de EAch olho e instruir participante para manter os olhos fechados por um minuto. </li><li> Evert tampa e parte superior do olho manter no lugar pelos cílios; evitar qualquer contato com a área da tampa do limpador. Opcional: a ajuda de um investigador secundária pode ser necessário para executar esta etapa. </li><li> Aplicar membrana perpendicularmente sobre a parte central da região do limpador a tampa com o mínimo de pressão. </li><li> Segure membrana no local por 3-5 seg. Observe a membrana se tornar translúcida na área de contato. Neste ponto, remover suavemente a membrana e permitir tampa do participante para &quot;flip&quot; de volta no lugar. </li><li> Prontamente aplicar uma gota de PBS à amostra, para impedir que sequem. Membrana girará translúcida e opaca não deve ligar a qualquer momento, como isso indica secagem. Têm o investigador secundário prosseguir imediatamente com o processamento de amostras (secção 3). </li><li> Realizar verificação final olho para garantir a integridade da conjuntiva. Dispensar lubrificantes oculares e instruir participantepara evitar esfregar os olhos até que o efeito anestésico desgasta fora (aprox. 15 min). </li></ol> Processamento 3. Amostra <ol><li> Utilizando micro-tesoura, cortar a membrana na metade (metade de cada uma das análises a jusante), perpendicular através do meio da recolha de células. O corte ao longo da margem exterior de uma metade da membrana para separar o do suporte de plástico; evitar a interacção com as células recolhidas na membrana, incluindo assegurar que a membrana não se dobra sobre si própria. </li><li> Garantir a membrana usando uma pinça antes completamente desconectar do titular. Ponto de membrana para dentro do tubo de centrífuga de 2 ml rotulados, contendo 500 ul de 95% (v / v) de etanol e deixa-se fixar durante um mínimo de 20 min a um máximo de 2 horas antes do processamento (secção 4.2). </li><li> segunda metade imediatamente separada da membrana (como descrito na seção 3.2) e proceder imediatamente com a secção 4.1. </li></ol> Coloração 4. Amostra <ol><li> Immunocytochemical coloração <ol><li> Com cuidado, coloque membrana em 35 milímetros de vidro prato de cultura inferior com o lado coleção voltada para baixo; garantir a membrana é plana. </li><li> Pipetar 20 l de composição mancha imunocitoquímica montada na secção 1 para a membrana. Se amostra cachos, use pontas de pipeta descartáveis ​​para achatar cuidadosamente a membrana, empurrando suas bordas para baixo. Evitar o contacto com as zonas de recolha de células. </li><li> Gentilmente cobrir a membrana com tampa de vidro deslizante. Evite movimento desnecessário da amostra. Cubra a assadeira com tampa e vedação com laboratório de película em torno das bordas. Não obstrua a transparência de prato (superior e inferior) e visibilidade da amostra. Etiqueta com marcador de laboratório. </li><li> proceder imediatamente à imagem (secção 5.1), em seguida, voltar a secção 4.2 para continuar com coloração citológica da outra metade da membrana. </li></ol></li><li> citológico coloração <ol><li> Gradualmente hidratar membrana por adição lenta de 500 mL de água destilada para o tubo de retornosed para fixação na secção 3.4. conteúdo aspirado várias vezes na ponta da pipeta para misturar, em seguida, remover conteúdo. </li><li> Adicionar 500 uL de água destilada. Deixe repousar por alguns segundos, em seguida, remover. Adicionar 500 mL de mancha azul Alcian e deixe por 3 min. Remover manchas e realizar 3 consecutiva 500 mL de água destilada lavagens ou até que o líquido lavagens claras. </li><li> Adicionar 500 mL de Hematoxilina # 1 mancha e deixe por 3 min. Remover manchas e realizar três consecutivos 500 mL lavagens água destilada ou até que o líquido lavagens claras. Desidratar (inverso do ponto 4.2.1) por aspiração do líquido claro. </li><li> Adicionar 500 mL de Papanicolaou OG-6 mancha e deixe por 3 min. Remover mancha e executar um de 500 ul 95% (v / v) de lavagem etanol. </li><li> Adicionar 500 ul de corante Papanicolaou EA-65 e deixar durante 3 min. Remover mancha e executar três consecutiva de 500 ul de 95% (v / v) de etanol lavagens. </li><li> Totalmente desidratar usando 500 ul três lavagens consecutivas de 100% de etanol. usando tweezers, retirar amostra de solução; evitar áreas de coleta de células tocantes. </li><li> Coloque membrana sobre lâmina de vidro; assegurar que a linha de colheita de células é paralela ou perpendicular a deslizar margem para o alinhamento mais fácil durante o exame. </li><li> Aplique uma gota de etanol 100% e cubra com deslizamento de vidro. proceder imediatamente à imagem (secção 5.2). </li></ol></li></ol> 5. Amostra de imagem <ol><li> Imagens fluorescentes imunocitoquímicos Stains <ol><li> Use um microscópio confocal de varredura a laser (CLSM) 11 ou microscópio de fluorescência 12, equipado com uma câmera de cor digital, conectado a um computador com um software de aquisição de imagem. Devido às amostras a ser contido dentro placas de cultura, um microscópio invertido pode ser necessário. </li><li> Defina-se 11,12 microscópio de acordo com espectros de absorção e emissão de etídio homodímero-1 (528/617 nm máximos Ex / Em na presença de ADN) e calceína AM (494/517 nm Ex / Em máximos). </li><li> Selecione menor ampliação do microscópio (por exemplo, objetiva 2,5X) e ativar o sistema de imagem digital; observar a amostra indicada no monitor do computador. </li><li> Inspecione amostra para características celulares de interesse. </li><li> Selecione ampliação microscópio desejado e adquirir imagens usando software de imagem. </li><li> Salvar arquivos em qualquer formato de arquivo microscópio nativo ou um formato de arquivo não compactado (por exemplo, * .raw, * .tiff). </li></ol></li><li> Imagem citológica Stains <ol><li> Use um microscópio padrão de laboratório de campo brilhante sem filtros, equipado com uma câmera de cor digital, conectado a um computador com um software de aquisição de imagem. </li><li> Local e lâmina de vidro bloqueio (do passo 4.2.8) no palco microscópio. Rehidratar amostra com etanol a 100%, conforme necessário, ao longo do procedimento de imagem inteira. </li><li> Selecione menor ampliação do microscópio (por exemplo, objetiva 2,5X) e ativar thsistema de imagem digital e; observar a amostra indicada no monitor do computador. </li><li> Assegurar a recolha de células é alinhado com um dos eixos X ou Y de controle do estágio de microscópio. Ajuste, se necessário, pela remoção da tampa de deslizamento e rotação da amostra, utilizando uma ponta de pipeta ou pinças. </li><li> Usando a imagem (s) de captura de toda a amostra com o microscópio de ampliação menor software de imagem,. </li><li> Mudar para a ampliação do microscópio moderada (por exemplo, 10 - 20X objetiva) e ajustar fonte de luz do microscópio e parâmetros de imagem de software (exposição, contraste, balanço de brancos, etc.), e desativar a sua medição de software automatizado. Salvar essas configurações para uso futuro. </li><li> Determinar pontos inicial e final de varredura (por exemplo, superior esquerdo e direito cantos inferiores de recolha de células). </li><li> Usando a imagem (s) de captura de toda a área de colheita de células dentro dos pontos inicial e final software de imagem,. Certifique-se a 20% lado-a-lado e de cima para baixo sobreposição between todas as imagens adjacentes. </li><li> Salvar arquivos usando o formato de arquivo microscópio nativo ou um formato de arquivo não compactado (por exemplo, * .raw, * .tiff). </li><li> Gerar imagem panorâmica final usando o software automatizado costura de escolha (por exemplo, o Adobe Photoshop Elements). Use imagens de referência tomadas no passo 5.2.5) para confirmar a precisão da costura automatizada na imagem final. </li></ol></li></ol>

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The authors have no relevant funding or conflicts to disclose.

citologia de impressão é tipicamente realizada na conjuntiva bulbar, utilizando membranas de ésteres de celulose misturados. As amostras são cortadas ao tamanho a partir de folhas de material a granel, esterilizado, aplicado e removido da superfície conjuntival utilizando fórceps. Usando o mesmo material de membrana, um dispositivo de IC controlado-pistão foi recentemente concebido para coincidir com a geometria da superfície da conjuntiva bulbar, e manter a pressão constante entre 13 aplicações. E...

A pálpebra superior humano executa cerca de 10.000 pisca a cada dia 1, com apenas um 1 - região conjuntival 2 milímetros estreita opostos do globo ocular. Devido ao seu movimento de limpeza durante o pestanejar, esta porção da margem da pálpebra foi denominada a &quot;tampa do limpador&quot; região 2. Supõe-se que o atrito é aumentada nessa região durante a piscar habitual, devido à tampa limpar todo o globo. Isto pode desempenhar um papel significativo no conforto ocular. Lente de contato usuários em particular experiência de desconforto ocular e esta é uma das razões principais para cessar o uso das lentes 3. Quando uma lente de contacto é colocada sobre o olho, o coeficiente de atrito entre o material da lente e a superfície ocular foi mostrado para alterar 4. Há evidências que sugerem que os sintomas de secura pode estar relacionado a este atrito alterado 2,5,6. Esta associação também pode ser refletida em fenômenos observáveis ​​clinicamente, nomeadamente wi tampapor epiteliopatia (LWE), também chamado superior da tampa margem de coloração (ULMS) 6. LWE é um sinal precoce de irritação ocular e um preditor potencial para a doença do olho seco. Ele é observado e medido pela coloração vital das regiões superiores e inferiores margem palpebral. Embora este sistema de classificação 2 e sua validade clínica (ou seja, correlação com sintomas subjetivos) ainda estão em debate, desconforto ocular permanece um enigma para os médicos e cientistas e, mais importante, um inconveniente para os pacientes. Até à data, pouco se sabe sobre as variações clinicamente relevantes no (sub) anatomia celular e fisiologia da área do limpador a tampa 7 e apenas um relatório faz uso de métodos citológicos 8. Citologia de impressão (CI) foi utilizada há mais de 40 anos para recolher as células a partir da superfície do epitélio da conjuntiva tarsal bulbar ou por aplicação de uma membrana de 9,10. Após remoção, as células aderentes submeter Cytological coloração e de imagem microscópica. Devido às diferenças anatómicos celulares e na região do limpador a tampa, esta técnica requer a optimização.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40116-500 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
			<td>EMD Millipore</td>
			<td>PICM01250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

A membrana de cultura de células durou mais o suporte de plástico é uma ferramenta conveniente para a recolha de mão de células epiteliais da conjuntiva tampa do limpador. Este método elimina a necessidade de instrumentos esterilizados adicionais tipicamente utilizados para preparar e manipular membranas IC. A Figura 1 representa a área de IC limpador a tampa usando uma inserção de cultura de células. Nós otimizamos dois protocolos de coloração diferentes qu...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

A citologia de impressão da Área Wiper a tampa

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

impression-cytology-of-the-lid-wiper-area

Research

JoVE Journal

Medicine

10.3K visualizações.  University of Waterloo. We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. 

Vídeo: Impression Cytology of the Lid Wiper Area

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Detecção e Genogrouping de norovírus a partir de fezes das crianças, Taqman Um passo-RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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 ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH Fluorescência de Imagem biventricular isolado de coelho

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Identificação baseada em Microarray de Individual HERV Loci Expressão: Aplicação a descoberta de biomarcadores em câncer de próstata

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Coleta e Extração de DNA de saliva para Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;Patient-Like&quot; Orthotopic Singênico Rato Modelo de carcinoma hepatocelular Metástase

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Indução de Acidente Vascular Cerebral Isquêmico e isquemia-reperfusão em ratos usando a técnica de oclusão da artéria Oriente e Visualização de infarto Área

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

Automotive Radiochemical Synthesis of [<sup&gt; 18</sup&gt; F] 3F4AP: Um novo marcador de PET para imagens de doenças desmielinizantes

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Citologia tubária do tubo de Falópio como uma ferramenta promissora para a detecção precoce do câncer de ovário

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Acessando a Citotoxicidade e a Resposta Celular a Biomateriais

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...