Expressamos nosso sincero agradecimento pelo apoio financeiro recebido do Projeto de Construção de Conotação do 14º Plano Quinquenal da Universidade de Medicina Tibetana (2022ZYYGH12), as Disciplinas Abertas de 2022 do Laboratório Chave de Medicina Tibetana e Educação Básica do Ministério da Educação da Universidade de Medicina Tibetana (ZYYJC-22-04), o Programa Chave de Pesquisa e Desenvolvimento de Ningxia (2023BEG02012), e o Projeto de Promoção de Pesquisa Acadêmica Xinglin da Universidade de Medicina Tradicional Chinesa de Chengdu (XKTD2022013).

A solução composta, a solução proteica e o corante foram introduzidos em uma solução de PBS. Posteriormente, as amostras foram submetidas a aquecimento gradual usando um banho seco de aquecimento digital, e a estabilidade térmica das proteínas foi avaliada medindo a mudança na intensidade de fluorescência dentro do sistema complexo. As etapas detalhadas são descritas abaixo e a Figura 1 ilustra uma visão geral do protocolo.
1. Preparação da solução
<ol><li>Prepare uma solução de umbeliferona 1 mM adicionando 0,1 mg de umbeliferona a 616,75 μL de solução de DMSO (ver Tabela de Materiais).</li>
	<li>Prepare uma solução de proteína CD40 a 200 μg / mL adicionando 0,1 mg de CD40 a 500 μL de solução de ddH2O (consulte a Tabela de Materiais).</li>
	<li>Prepare uma solução de corante laranja 500x adicionando 1 μL de corante laranja 5000x a 9 μL de solução salina tamponada com fosfato (PBS) (consulte a Tabela de Materiais). NOTA: Realize as operações acima no gelo e, como a pipeta tem uma faixa mínima de 0.1 μL, dilua inicialmente o corante de proteína laranja por um fator de 10 para facilitar o uso subsequente.</li>
</ol>2. Teste de desempenho do corante
<ol><li>Adicione o corante laranja 500x a uma solução de PBS e dilua-o para obter corante: proporções de PBS de 1:500, 1:1000, 1:2000 e 1:4000. NOTA: Certifique-se de que a concentração final de DMSO não exceda 2% (v/v).</li>
	<li>Ligue o leitor de microplacas fluorescentes e pré-aqueça-o por 10 min.</li>
	<li>Abra o computador e o software de aquisição de dados em sequência (consulte Tabela de Materiais).</li>
	<li>Clique em Instrumento, escolha o modelo de microplaca de fluorescência correspondente e selecione OK no software de aquisição de dados. NOTA: Aguarde até que a microplaca de fluorescência termine o pré-aquecimento e exiba a temperatura no painel antes de abrir o software de aquisição de dados para garantir uma conexão bem-sucedida entre o instrumento e o software.</li>
	<li>Clique em Configurações de aquisição e selecione Fluorescência no software de aquisição de dados.</li>
	<li>Edite o programa na interface Comprimentos de onda: Lm1 = 470 nm, 570 nm e selecione OK. NOTA: O corante laranja pode ser excitado com luz UV a 300 nm ou luz visível a 470 nm, e a emissão pode ser medida a 570 nm.</li>
	<li>Adicione diferentes concentrações de corante laranja (1:500, 1:1000, 1:2000 e 1:4000) e PBS a placas de 96 poços a 100 μL/poço. NOTA: Dissolva a solução em DMSO antes de adicioná-la ao PBS e vortexe-a a 2000 x g completamente durante o processo de preparação para garantir a dissolução completa devido à tendência do corante a precipitar.</li>
	<li>Coloque a placa de 96 poços no estágio de detecção do instrumento, clique no botão Ler no software de aquisição de dados e meça a fluorescência de cada concentração de corante 13 vezes à temperatura ambiente, com um intervalo de 2 min de cada vez.</li>
	<li>Clique no botão Exportar após cada determinação, selecione Exportar para XML XLS TXT, escolha Todas as placas, selecione Placa no formato de saída e, finalmente, clique em OK para salvar os dados em uma pasta no formato "XLS" para análise posterior. NOTA: Essas etapas ajudam a determinar o impacto da medição contínua na autofluorescência do corante laranja à temperatura ambiente e identificar a concentração ideal de coloração.</li>
	<li>Ligue o banho seco de agitação de aquecimento digital, ajuste a temperatura de aquecimento para 35 °C e o tempo de aquecimento para 2 min.</li>
	<li>Preparar a taxa de diluição ideal do corante num tubo de microcentrífuga de 1,5 ml e colocá-lo no banho de aquecimento digital por 2 min.</li>
	<li>Adicionar a solução de PBS e a solução de coloração, aquecida a 35 °C, à placa de 96 poços a um volume de 100 μl por alvéolo.</li>
	<li>Coloque a placa de 96 poços no estágio de detecção do instrumento e clique no botão Ler no software de aquisição de dados.</li>
	<li>Defina a temperatura de aquecimento para 40 °C e o tempo de aquecimento para 2 min.</li>
	<li>Pipete a solução de corante na placa de 96 poços de volta para o tubo de microcentrífuga de 1,5 mL e aqueça-a no instrumento por 2 min.</li>
	<li>Repita as operações das etapas 2.12-2.15 e 2.9 para testar a absorbância da solução de corante a 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C e 95 °C, respectivamente. NOTA: Essas etapas ajudam a determinar a estabilidade da autofluorescência do corante laranja sob aquecimento contínuo em um gradiente de 35 ° C a 95 ° C. É crucial trabalhar rapidamente para evitar quedas rápidas de temperatura ou adicionar o líquido errado, o que pode levar a erros experimentais.</li>
</ol>3. Detecção da temperatura de fusão (Tm) da proteína
<ol><li>Combine a proteína CD40 e o corante laranja com a solução de PBS para atingir concentrações finais de 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL e 1:500, respectivamente.</li>
	<li>Repita as operações descritas nas etapas 2.2-2.6 e nas etapas 2.10-2.16 para avaliar a absorbância da solução de proteína CD40 em temperaturas de 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C e 95 °C, respectivamente.</li>
	<li>Reitere a etapa 2.9 para salvar e analisar os dados e, posteriormente, calcule o valor de Tm usando um software de análise de dados (consulte a Tabela de Materiais).</li>
	<li>Adicione proteína CD40, umbeliferona e corante laranja à solução de PBS para obter concentrações finais de 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 10 μM e 1:500, respectivamente. NOTA: O vórtice vigoroso durante a preparação da solução é essencial para garantir a distribuição uniforme do corante, proteína CD40 e umbeliferona na PBS.</li>
	<li>Repita as operações das etapas 3.2-3.3 para medir a absorbância da solução de complexos CD40-umbeliferona em temperaturas de 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 70 ° C, 75 ° C, 80 ° C, 85 ° C, 90 ° C e 95 ° C, respectivamente. NOTA: Essas etapas foram conduzidas para determinar os valores de Tm da proteína CD40 e dos complexos CD40-umbeliferona, com o objetivo de identificar a concentração ideal de ligação da proteína CD40 à umbeliferona.</li>
</ol>

Detecção de alta sensibilidade da ligação composto-proteína usando fluorescência

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DSF, também conhecido como ensaio de deslocamento térmico ou ensaio de fluorescência térmica, é uma técnica empregada para detectar o processo de desnaturação térmica de proteínas em amostras, monitorando mudanças no sinal de fluorescência da amostra de teste ou corante durante um aumento lento e programado de temperatura. Inicialmente estabelecido por Pantoliano14, o DSF serve como um método de alto rendimento. O procedimento principal envolve elevar a temperatura em uma placa aqueci...

Vicatia thibetica H. Boissieu, uma planta da família das umbelíferas, é comumente usada na medicina tibetana e representa um dos componentes essenciais dos cinco ingredientes básicos (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew. e Gymnadenia conopsea (L.) R. Br.) 1. Distribuído principalmente no sudoeste da China, como noroeste de Yunnan, oeste de Sichuan, Tibete e outras áreas, sua raiz seca serve como um substituto local para Angelica1. A raiz, conhecida por sua fragrância, é frequentemente utilizada como tempero de ensopado, e as folhas, conhecidas como aipo tibetano, contribuem para pratos deliciosos. Assim, Vicatia thibetica tem importância não apenas como uma planta medicinal única, mas também como fonte de alimento no Tibete.
Pesquisas nacionais e internacionais indicam que a Vicatia thibetica possui propriedades revigorantes de sangue e qi (poder ou energia), benéficas para regular a menstruação. É empregado para tratar sintomas de dismenorreia menstrual irregular causada por palpitações e deficiência sanguínea, exibindo efeitos farmacológicos, como regulação antioxidante da imunidade corporal2. O extrato alcoólico de Vicatia thibetica demonstrou a capacidade de restaurar a massa corporal e o índice de órgãos em camundongos imunocomprometidos induzidos por ciclofosfamida. Além disso, aumenta a atividade da superóxido dismutase no soro e reduz o conteúdo de malondialdeído, sugerindo uma melhora na capacidade antioxidante e um efeito de equilíbrio na peroxidação lipídica 2,3. Simultaneamente, aumenta o número de células sanguíneas e hemoglobina, o que não apenas reflete objetivamente a função hematopoiética do corpo, mas também desempenha um papel crucial no sistema imunológico 2,3.
A umbeliferona, caracterizada por cristais aciculares e sabor amargo, possui um pequeno peso molecular, é volátil e pode ser destilada com vapor. Sublima facilmente, tem baixa solubilidade em água e alta solubilidade em matéria orgânica. Como um dos principais componentes químicos da Vicatia thibetica, a umbeliferona exibe efeitos imunomoduladores na imunidade celular, imunidade humoral e imunidade inespecífica em modelos de camundongos imunossupressores induzidos por hidrocortisona 4,5.
A molécula de superfície celular CD40, membro da superfamília de receptores do fator de necrose tumoral, é amplamente expressa em células imunes6. Seu ligante homólogo, CD154, também conhecido como CD40L, é uma proteína transmembrana do tipo II expressa por linfócitos T ativados. A ativação do CD40 tem a capacidade de regular positivamente a expressão de moléculas co-estimulatórias na superfície das células dendríticas (DC) e monócitos. Esse processo promove a função de apresentação de antígenos das moléculas do complexo principal de histocompatibilidade (MHC) e ativa ainda mais as células T CD8+7.
Os macrófagos desempenham um papel crucial na formação e regulação do microambiente tumoral, e a ativação do CD40 pode aumentar a remodelação do microambiente tumoral pelos macrófagos8. A ativação do sinal CD40 influencia significativamente a proliferação e ativação das células B. As células B, quando ativadas pelo CD40, podem funcionar como células apresentadoras de antígenos eficazes. Eles apresentam antígenos, geram atividade de células T efetoras e, assim, contribuem para efeitos antitumorais9. Além disso, a ativação de CD40 em células tumorais pode induzir apoptose e inibir o crescimento tumoral10. O CD40 transduz principalmente sinais controlando a atividade de proteínas quinases de tirosina não receptoras, incluindo Lyn, Fyn, Syk e outras. Além disso, tem a capacidade de estimular as proteínas Bcl-xL, Cdk4 e Cdk6, ativar fatores de transcrição Rel/NF-kB e fosforilar CG-2 e PI3K10.
O método de Fluorescência de Varredura Diferencial (DSF) é amplamente empregado para avaliar o impacto de várias condições ambientais, como composição do tampão, temperatura e ligantes de moléculas pequenas, na estabilidade térmica das estruturas proteicas. O corante comumente utilizado para DSF é um corante laranja, ambientalmente sensível e hidrofóbico. Em condições normais, a estrutura da proteína é dobrada, ocultando seu segmento hidrofóbico internamente. À medida que a temperatura aumenta, a região hidrofóbica da proteína fica mais exposta, resultando em uma quebra gradual da estrutura natural da proteína. O corante se liga seletivamente a essa porção proteica exposta, amplificando sua fluorescência. Os valores de Tm são então calculados monitorando as mudanças na detecção do sinal de fluorescência11. Até certo ponto, variações nos valores de Tm podem medir mudanças na estabilidade da proteína devido a mutações, mudanças nos tampões ou ligação do ligante. Além disso, pode indicar alterações estruturais durante o processo de enovelamento proteico12. Essa abordagem oferece dados precisos, uma ampla faixa de temperatura, alta sensibilidade e perda mínima de amostra de proteína13.
Neste estudo, a determinação da fluorescência foi realizada usando um leitor de microplacas fluorescentes em vez de um aparelho de reação em cadeia da polimerase quantitativa (PCR) de fluorescência. Essa modificação permite a detecção de DSF em laboratórios que não possuem um instrumento de PCR quantitativo fluorescente, tornando o método menos complexo e reduzindo as etapas necessárias para a configuração do instrumento, simplificando assim o processo experimental. No entanto, existem certas desvantagens nessa abordagem. Enquanto a complexidade é reduzida, o procedimento se torna mais complicado. A detecção manual de fluorescência em diferentes temperaturas é necessária, e a coleta automática e contínua de fluorescência do sistema não pode ser alcançada. Assim, este estudo utilizou a técnica DSF para explorar a interação entre a proteína CD40 e a umbeliferona, fornecendo novos insights sobre os mecanismos moleculares da medicina tibetana.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD40 protein</td>
			<td>MedChemExpress</td>
			<td>HY-P75408</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>FlexStation 3 multifunctional microplate reader</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>FlexStation 3</td>
			<td></td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 7.1</td>
			<td>Shanghai Meigu Molecular Instruments Co., LTD</td>
			<td>SoftMax Pro 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>SSYPRO orange dye</td>
			<td>Sigma</td>
			<td>S5942</td>
			<td></td>
		</tr>
		<tr>
			<td>Umbelliferone</td>
			<td>Shanghai Yuanye Biotechnology Co.</td>
			<td>B21854</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

A investigação das interações entre diferentes moléculas é um aspecto crucial para a compreensão da patogênese da doença e a triagem de alvos de drogas. A umbeliferona, um ingrediente ativo da medicina tibetana Vicatia thibetica, exibe um efeito imunomodulador com um mecanismo desconhecido. A proteína CD40 é um alvo chave na resposta imune. Portanto, este estudo emprega o princípio da tecnologia de fluorescência de varredura diferencial para analisar as interações entre a proteína CD40 e a umbeliferona usando marcadores enzimáticos fluorescentes. Inicialmente, a estabilidade do corante laranja fluorescente proteico foi verificada experimentalmente e a razão de diluição ótima de 1:500 foi determinada. Posteriormente, observou-se que o valor de fusão da temperatura (Tm) da proteína CD40 tendeu a diminuir com o aumento da concentração. Curiosamente, descobriu-se que a interação entre a proteína CD40 e a umbeliferona aumenta a estabilidade térmica da proteína CD40. Este estudo representa a primeira tentativa de detectar o potencial de ligação de compostos e proteínas de moléculas pequenas usando microplacas de fluorescência e corantes fluorescentes. A técnica é caracterizada por alta sensibilidade e precisão, prometendo avanços nos campos de estabilidade de proteínas, estrutura de proteínas e interações proteína-ligante, facilitando assim pesquisas e explorações futuras.

O corante laranja exibiu consistentemente excitação de fluorescência estável em Ex = 470 nm e Em = 570 nm, tanto à temperatura ambiente quanto a temperaturas elevadas. Determinou-se uma razão de diluição ótima de 1:500 (Figura 2A,B). A detecção do valor de Tm mostrou-se desafiadora quando a concentração da proteína CD40 estava abaixo de 15 μg/mL (Figura 3A,B). No entanto, na concentração de 15 μg/mL, foi detec...

Watch this Scientific Journal Video about Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency at JoVE.com

Autor em destaque: simplificando os métodos de PCR para maior acessibilidade e eficiência

Este protocolo descreve um método exclusivo para detectar a ligação entre compostos e moléculas de proteína, oferecendo as vantagens de perda mínima de amostra de proteína e alta precisão de dados.

Detecção da interação proteína-umbeliferona CD40 por fluorescência de varredura diferencial

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

Nosso protocolo oferece uma vantagem significativa ao permitir o uso sem equipamento especializado. Ao contrário dos métodos tradicionais de PCR quantitativo de fluorescência, ele simplifica o processo experimental, exigindo menos etapas e reduzindo a complexidade geral, tornando-o mais seguro e fácil de conduzir. Este experimento requer que o operador esteja muito familiarizado com o procedimento experimental e o leitor de microplacas fluorescentes, e não perca muito tempo durante o aquecimento da solução e a detecção de fluorescência.Vamos nos concentrar no mecanismo molecular pelo qual a umbeliferona se liga à proteína CD14 para produzir efeitos imunológicos.

detection-cd40-protein-umbelliferone-interaction-via-differential

Research

JoVE Journal

Medicine

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

371 visualizações.  University of Tibetan Medicine. Nosso protocolo oferece uma vantagem significativa ao permitir o uso sem equipamento especializado. Ao contrário dos métodos tradicionais de PCR quantitativo de fluorescência, ele simplifica o processo experimental, exigindo menos etapas e reduzindo a complexidade geral, tornando-o mais seguro e fácil de conduzir. Este experimento requer que o operador esteja muito familiarizado com o procedimento experimental e o leitor de microplacas fluorescentes, e não perca muito tempo durante o aqu...

Vídeo: Autor em destaque: simplificando os métodos de PCR para maior acessibilidade e eficiência

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Imagens em tempo real multiespectrais de fluorescência para detecção intra-operatória do linfonodo sentinela em Oncologia Ginecológica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Medicina

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Análise de biofilme oral de Palatal Expansores por fluorescência de hibridização in situ e microscopia confocal a laser Scanning

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Geração Experimental de Carcinoma Associado fibroblastos (FAC) de Fibroblastos Humanos mamária

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescência de Cerenkov Imaging (CLI) para o Monitoramento Terapia de Câncer

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms. Oriented structures are analyzed using Gabor filters and phase portraits to detect sites of radiating tissue patterns. Each site is characterized and classified using measures to represent spiculating patterns. The methods should assist in the detection of breast cancer.

Detecção de distorção arquitetural em mamografias anteriores<em&gt; Via</em&gt; Análise de Padrões Orientada

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of ...

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 ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Efeitos diferenciais de drogas hipolipemiantes na modulação da morfologia das partículas de colesterol

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

Scanning Skeletal Remains for Bone Mineral Density in Forensic Contexts

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. BMD is an important component of bone&#39;s nutritional status and in skeletal remains of both juveniles and adults, and it can provide information about bone quality. For adults remains, it can provide information on pathological conditions or when bone insufficiency may have occurred. In juveniles, it provides a useful metric to elucidate cases of fatal starvation or neglect, which are generally difficult to identify. This paper provides a protocol for the anatomical orientation and analysis of skeletal remains for scanning via dual-energy X-ray absorptiometry (DXA). Three case studies are presented to illustrate when DXA scans can be informative to the forensic practitioner. The first case study presents an individual with observed longitudinal fractures in the weight bearing bones and DXA is used to assess bone insufficiency. BMD is found to be normal suggesting another etiology for the fracture pattern present. The second case study employed DXA to investigate suspected chronic malnutrition. The BMD results are consistent with results from long bone lengths and suggest the juvenile had suffered from chronic malnutrition. The final case study provides an example where fatal starvation in a fourteen-month infant is suspected, which supports autopsy findings of fatal starvation. DXA scans showed low bone mineral density for chronological age and is substantiated by traditional assessments of infant health. However, when dealing with skeletal remains taphonomic alterations should be considered before applying this method.

Bone mineral density (BMD) is an important factor in understanding nutritional intake. For human skeletal remains, it is a useful metric to assess quality of life in both juveniles and adults, particularly in fatal starvation and neglect cases. This paper provides guidelines for scanning human skeletal remains for forensic purposes.

Escaneamento de restos esqueléticos para densidade mineral óssea em contextos forenses

The purpose of this paper is to introduce a promising, novel method to aid in the assessment of bone quality in forensically relevant skeletal remains. ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Visualizando a formação de babados de membrana usando microscopia eletrônica de varredura

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Detection and Removal of Tooth-Colored Composite Resin Using the Fluorescence-Aided Identification Technique

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) is a noninvasive tool to facilitate the distinction of composite resin material from sound tooth substance. Compared to conventional illumination, FIT is a very accurate, reliable, and fast diagnostic method. When composite resin is illuminated with a wavelength of approximately 398 &plusmn; 5 nm, certain fluorescent components make the composite resin appear brighter than the tooth structure. Any fluorescence-inducing light source with the appropriate wavelength can be used for this method. Optimally, this technique is used without additional natural or artificial lighting. The application of FIT can be used for diagnostic purposes, for example, dental charts, and additionally for the complete and minimally invasive removal of composite resin restorations, bracket debonding, and trauma splint removal. The assessment of volumetric changes after composite removal can be provided by overlapping pre- and postoperative scans and subsequent calculation using suitable software.

The Fluorescence-aided Identification Technique is a practicable, fast, and reliable approach for the differentiation of composite resin restorations from tooth substance, and facilitates the minimally invasive and complete removal of composite resin restorations and composite bonded trauma splints.

Detecção e Remoção de Resina Composta Cor de Dente utilizando a Técnica de Identificação Auxiliada por Fluorescência

The detection and removal of tooth-colored filling materials is a major challenge for every dentist. The Fluorescence-aided Identification Technique (FIT) ...