This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

Experimentos com amostras de sangue foram realizadas em conformidade com as leis e diretrizes institucionais. As amostras foram as amostras de sangue residuais que tinham sido adquiridas e processadas em testes clínicos na instituição. Configuração óptica 1. PT-AS NOTA: Pode-se usar um tubo de micro-capilar vazio para uma configuração inicial PT-AS. <ol><li> Montar um tubo de micro-capilar vazio com diâmetros interior e exterior de 200 e 330 uM, respectivamente, e um comprimento superior a cinco centímetros ~ em uma fixação de tubo capilar. Chaves de fibras comercialmente disponíveis podem ser usados ​​como a fixação de tubo. </li><li> Ancorar firmemente um ponteiro laser 650 nm, isto é, a sonda fonte de luz, para iluminar o tubo capilar. O feixe de sonda deve ser maior que o tubo capilar. Coloque uma tela (por exemplo, papel branco) por trás do tubo capilar para observar um padrão periódico angular. </li><li> Para a parte de detecção, remova quaisquer lentes em uma webcam para capturar diretamente o scattrando padrão. Posicionar o webcam atrás do tubo capilar com um ângulo de 25-35 ° em relação à direcção do feixe sonda. Assegure-se que o padrão periódico angular produzida pelo tubo capilar pode ser medida com o detector (Figura 1). Observar o padrão periódico angular no meio do sensor de imagem quando o sensor de imagem está correctamente posicionado. </li><li> Posicionar uma fonte de luz de excitação PT 532-nm para iluminar o tubo capilar. Posicionar a fonte de luz PT em qualquer ângulo, desde que a luz de excitação PT sobrepõe com feixe de sonda no tubo capilar e não atinge o detector directamente. PT excitação das amostras de sangue utilizando alta potência óptica tipicamente melhora o PT-Como a sensibilidade, uma vez que conduz a uma variação maior no IR. <ol><li> Utilizar a maior potência óptica da fonte de luz de excitação PT empregue. Além disso, garantir que a luz PT excitação sobrepõe a luz sonda no tubo capilar. Use um tamanho feixe da luz de excitação PTpelo menos duas vezes a da sonda de luz para aquecer todo o volume da sonda. </li></ol></li><li> Coloque um filtro passa longo em frente ao detector para bloquear a luz de 532 nm, e medir apenas a luz da sonda 650-nm. </li><li> Instalar um chopper óptica no caminho da luz de excitação PT antes de iluminar o tubo capilar. O helicóptero óptico é utilizado para modular a intensidade de luz de excitação PT. </li></ol> Preparação da Amostra 2. Sangue <ol><li> Desenhar 6 mL de sangue inteiro fresco em estado anémico em ácido etilenodiaminotetracético tubos de recolha de sangue, e misturar bem as amostras. Nenhum outro tratamento é necessário. </li><li> Medem-se as amostras de sangue utilizando o sensor de PT-AS dentro de 24 horas de extracção para impedir a coagulação. </li></ol> 3. PT-como protocolos de medição <ol><li> Coloque um tubo de micro-capilar com uma amostra de sangue para medir. Encher o tubo capilar com o sangue através de uma acção capilar, colocando o tubo no sangue samplo. O volume mínimo de amostra necessário para a medição é determinada pelo diâmetro interior do tubo capilar e o tamanho do feixe de sonda. <ol><li> Empregar um tubo com um diâmetro interior de 200 um. O tamanho do feixe de sonda foi de 2 mm nos resultados representativos, sugerindo que a medição pode ser realizada com um volume de amostra de&gt; 63 nl. </li></ol></li><li> Montar o tubo capilar na posição designada no suporte. </li><li> Ligue o laser sonda 650 nm para iluminar o tubo de micro-capilar carregado de sangue. O padrão periódico angular devem ser observadas com a webcam. </li><li> Ligue o 532-nm do laser de excitação PT para iluminar o tubo. </li><li> Executar o helicóptero óptico para modular a intensidade da luz de excitação PT a 2 Hz. NOTA: O critério para a selecção de esta condição de operação é descrita em Discussão e Kim et al. 12. <ol><li> Montar uma roda de corte no conjunto do cabeçote do motor do helicóptero ópticasistema. </li><li> Ligue a caixa de controle helicóptero, e use o botão de controle no console para definir a frequência de modulação. </li><li> Execute o helicóptero usando o botão de controle. </li></ol></li><li> Gravar o padrão de espalhamento flutuante através da webcam para 5 segundos no formato MPEG-4 (mp4). </li></ol> 4. Processamento de Sinais NOTA: processamento de sinal PT-AS foi realizada utilizando um código MATLAB desenvolveu-lab. <ol><li> Carregar o arquivo de vídeo para extrair as imagens. Para cada imagem [ver Figura 2 (a), para uma imagem representativa], obter o padrão de dispersão média calculando a média dos valores de pixel ao longo da direcção vertical [Figura 2 (b, c)]. </li><li> Avaliar a transformada de Fourier do padrão de dispersão em média, e calcular a fase no pico de frequência espacial. Executar essas operações para todos os quadros de todas as imagens gravadas. </li><li> Utilizando os valores de fase obtidos a partir de todas as imagens, traçar a fase temporaisflutuação [Figura 2 (d)]. Note-se que a fase varia com a frequência de modulação PT. Aqui a transformada de Fourier a flutuação de fase no domínio do tempo, e obter a amplitude na frequência de modulação. Este sinal é referido como o PT-Como o sinal [Figura 2 (e)]. </li><li> Meça a [Hb] de uma amostra de sangue, convertendo seu sinal PT-AS para o correspondente [Hb], utilizando a curva de calibração que é obtido no Protocolo 5. </li></ol> 5. PT-AS Calibração <ol><li> Preparar as amostras de sangue, tendo [Hb] valores que estão uniformemente distribuídas no intervalo de detecção do sensor PT-AS (por exemplo, 0-18 g / dl). </li><li> Antes da calibração, quantificar os valores de [Hb] das amostras utilizando um analisador de hematologia referência. Medir os sinais PT-AS das amostras. </li><li> Derivar uma curva de calibração relacionando [Hb] para o sinal PT-AS realizando uma linear de mínimos quadrados encaixam, [Hb] = A [PT-AS Signal] + B, das experimental resultados. Para as condições de funcionamento especificados no quadro 1, a relação entre [Hb] e o sinal PT-AS foi encontrado para ser [Hb] = 5,13 [sinal PT-AS] - 0,09. Use o código MATLAB para realizar o ajuste linear. </li></ol>

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No conflict of interest is declared.

O sensor PT-AS representa um método totalmente óptica capaz de medição direta [Hb] de amostras de sangue não transformados. O método quantifica [Hb] no sangue, utilizando a resposta PT intrínseca de moléculas de hemoglobina em eritrócitos. Sob iluminação pela luz de 532 nm, as moléculas de hemoglobina absorver a energia da luz e produzir calor. O aumento da temperatura resultante altera o RI da amostra de sangue. A sensibilidade elevada RI da BSI foi explorada para medir esta mudança RI no sangue. Anteriorm...

Um exame de sangue é comumente realizada para avaliar a saúde humana em geral e para detectar biomarcadores relacionados a certas doenças. Por exemplo, a concentração de colesterol no sangue serve como um critério para a hiperlipidemia, que está estreitamente relacionada com doenças cardiovasculares e pancreatite. O conteúdo de glicose no sangue deve ser medida com frequência, pois o nível de glicose está associada a complicações tais como cetoacidose diabética e síndrome hiperosmolar hiperglicêmicos. doenças graves como a malária, o vírus da imunodeficiência humana e síndrome da imunodeficiência adquirida são diagnosticadas através de exames de sangue, e quantificação dos componentes do sangue, incluindo eritrócitos, trombócitos, leucócitos e permite o rastreio de pâncreas e doenças renais. A hemoglobina (Hb), um componente crítico de sangue, torna-se cerca de 96% dos eritrócitos, e transporta o oxigénio para órgãos humanos. alteração significativa da sua concentração de massa ([Hb]) pode indicar-memudanças tabolic, doença hepatobiliar, e distúrbios neurológicos, cardiovasculares e endocrinológicos 1. [Hb] é, portanto, rotineiramente medidos em exames de sangue. Em particular, pacientes anêmicos, pacientes de diálise, e as mulheres grávidas são fortemente recomendados para monitorar [Hb] como uma tarefa vital 2. Vários métodos de detecção [Hb] foram assim desenvolvidos. O método da hemoglobina cianeto, uma das técnicas mais comuns para [Hb] quantificação, emprega cianeto de potássio (KCN), para destruir a bicamada lipídica de eritrócitos 3. A hemoglobina cianeto produzido pelo exposições químicas alta absorção em torno de 540 nm; Assim, [Hb] medições podem ser feitas através de análise colorimétrica. Este método é amplamente utilizado devido à sua simplicidade, mas os produtos químicos utilizados (por exemplo, KCN e óxido dimethyllaurylamine) são tóxicos para os seres humanos eo meio ambiente. O regime de hematócrito mede a relação do volume de células vermelhas do sangue em comparação com o volume total de sangueume através da separação centrífuga; no entanto, exige um volume relativamente grande de sangue (50-100 ul) 4. Espectrofotometria de métodos de medida [Hb] precisamente sem produtos químicos, mas medições em vários comprimentos de onda e um volume de sangue grande são necessárias 5,6. Da mesma forma, vários métodos ópticos para medir [Hb] têm sido propostos, incluindo os métodos de detecção baseadas em luz-se espalhando, mas as suas precisões de medição dependem fortemente da precisão do modelo de sangue teórica. Para superar estas limitações, [Hb] métodos de detecção baseados no efeito fototérmico (PT) de Hb recentemente têm sido propostos 7. Hb, que é composto principalmente de óxidos de ferro, absorve a luz a 532 nm e converte a energia da luz em calor 8-10. Este aumento de temperatura PT pode ser detectado opticamente por medição de uma alteração no índice de refracção (RI) de amostras de sangue. Yim et ai. empregada espectral-domain reflectometr de coerência ópticaY para medir a alteração óptica caminho de comprimento PT numa câmara contendo 11 sangue. Embora o método permite a livre de produtos químicos e directa [Hb] medição, a utilização de um espectrómetro e um arranjo de interferometria pode dificultar a sua miniaturização. Recentemente, apresentado um método de detecção alternativo [Hb], denominado sensor de fotoï¿½tï¿½rmica angular de espalhamento de luz (PT-AS), que é mais adequado para a miniaturização do dispositivo 12. O sensor PT-AS explora a sensibilidade elevada RI da interferometria de dispersão de volta (BSI) para medir as mudanças PT no RI de uma amostra de sangue dentro de um tubo capilar. BSI têm sido utilizados para medir RI de várias soluções de 13-15 e para monitorar interacções bioquímicas em solução livre 16. O sensor PT-AS emprega disposição óptica semelhante à do BSI, mas combina configuração de excitação fototérmica para medir aumento PT de RI em amostras de sangue. Princípios de funcionamento do BSI e os sensores de PT-AS são descritos em detalhe noutro lugar <sup&gt; 12,15. PT-demonstrado como sensor de medição de alta sensibilidade [Hb] ao longo de um largo intervalo de detecção (0,35-17,9 g / dl) e é capaz de funcionar com volumes de amostra de &lt;100 nl. Sem o pré-condicionamento de amostra de sangue é necessário, e o tempo de medição é apenas ~ 5 seg. Aqui, a montagem experimental e um protocolo de medição detalhada são descritos. Os resultados representativos PT-AS são fornecidos usando amostras de sangue de pacientes anêmicos, e os resultados são comparados com os de um analisador hematológico para avaliar a precisão do sensor PT-AS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

Fotoï¿½tï¿½rmica angular de espalhamento de luz (PT-AS) é um método óptico de novo para a medição da concentração de hemoglobina ([Hb]) de amostras de sangue. Com base na resposta fototérmico intrínseca de moléculas de hemoglobina, o sensor permite alta sensibilidade, medida livre de produtos químicos de [Hb]. [Hb] capacidade de detecção com um limite de 0,12 g / dl ao longo do intervalo de 0,35-17,9 g / dl foi demonstrada anteriormente. O método pode ser facilmente implementada usando dispositivos electrónicos de consumo de baixo custo, tais como um ponteiro laser e uma webcam. O uso de um tubo de micro-capilar como um recipiente para o sangue também permite que o ensaio de hemoglobina com um volume de sangue escala nanolitros e um baixo custo de operação. Aqui, são apresentados instruções detalhadas para os procedimentos de processamento de instalação e de sinal PT-AS óptica. Protocolos experimentais e resultados representativos para as amostras de sangue em condições anémicas ([Hb] = 5,3, 7,5, e 9,9 g / dL) são também fornecidos, e as medições são comparadas com as froma analisador hematológico. Sua simplicidade na implementação e operação deve permitir a sua ampla adoção em laboratórios clínicos e contextos de recursos limitados.

Um ensaio de hemoglobina foi realizada utilizando o sensor de PT-AS, e suas medições foram comparadas com aquelas de um analisador de hematologia. O experimento foi realizado com uma intensidade de luz PT excitação de 1,4 W / cm 2, PT modulação de frequência de 2 Hz, e medição do tempo de 5 segundos. A Tabela 1 resume as condições experimentais. Os tamanhos de feixe da luz da sonda e excitação PT foram de 5,5 e 2 mm, respectivamente. A webcam grav...

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

A rápida e sem produtos químicos dosagem de hemoglobina com Photothermal angular de espalhamento de luz

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

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9.6K visualizações.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Vídeo: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

Padronização Subtractive rápida de camadas de células vivas com uma Probe microfluídicos

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Bioengenharia

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Rápido Mix Preparação de Bioinspirada Nanoscale hidroxiapatita para aplicações biomédicas

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

Construção e configuração de um Bioreactor Fotossintético Algal com Escala de Banco com Monitoramento de Temperatura, Luz e pH para testes de crescimento cinético

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

Protocolo para transferência de MicroRNA na medula óssea-derivado hematopoiéticas células-tronco adultas para habilitar celular engenharia combinada com alvo magnético

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Um ensaio rápido de virulência bacteriana baseada em imagem usando ameba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a rapid and cost-effective method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes.

Caracterização Rápida de Partes Genéticas com Sistemas Livres de Células

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Fermentações controladas por luz para produção de produtos químicos e proteicos microbianos

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Imagem de tecidos biológicos com fluorescência combinada de dois fótons e microscopia de dispersão de Raman estimulada

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

A Nonsequencing Approach for the Rapid Detection of RNA Editing

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger sequencing and RNA sequencing techniques. However, these methods can be costly and time-consuming. In this protocol, a portable microtemperature gradient gel electrophoresis (&#181;TGGE) system is used as a nonsequencing approach for the rapid detection of RNA editing. The process is based on the principle of electrophoresis, which uses high temperatures to denature nucleic acid samples as they move across a polyacrylamide gel. Across a range of temperatures, a DNA fragment forms a gradient of fully double-stranded DNA to partially separated strands and then to entirely separated single-stranded DNA. RNA-edited sites with distinct nucleotide bases produce different melting profiles in &#181;TGGE analyses. We used the &#181;TGGE-based approach to characterize the differences between the melting profiles of four edited RNA fragments and their corresponding nonedited (wild-type) fragments. Pattern Similarity Scores (PaSSs) were calculated by comparing the band patterns produced by the edited and nonedited RNAs and were used to assess the reproducibility of the method. Overall, the platform described here enables the detection of even single base mutations in RNAs in a straightforward, simple, and cost-effective manner. It is anticipated that this analysis tool will aid new molecular biology findings.

Rapid detection and reliable quantification of RNA editing events at a genomic scale remain challenging and currently rely on direct RNA sequencing methods. The protocol described here uses microtemperature gradient gel electrophoresis (&#181;TGGE) as a simple, quick, and portable method of detecting RNA editing.

Uma abordagem não-sequencial para a detecção rápida da edição de RNA

RNA editing is a process that leads to posttranscriptional sequence alterations in RNAs. Detection and quantification of RNA editing rely mainly on Sanger ...