Este trabalho foi financiado em parte pelo Alan A. e Edith L. Wolff Charitable Trust, St. Louis, e da Fundação Barnes-Jewish Hospital. L. Shmuylovich e E. Ghosh foram parcialmente apoiado por bolsas de estudo Predoctoral do Heartland de afiliados da American Heart Association. S. Zhu receberam apoio parcial do Compton Scholars Programa Universidade de Washington e da Faculdade de Artes e Verão Prêmio de Pesquisa de Graduação em Ciências &#39;. S. Mossahebi recebeu o apoio parcial do Departamento de Física.

O procedimento para a aquisição de imagens diagnósticas e analisá-los para obter os parâmetros de PDF é detalhado abaixo. Apesar de cateterismo cardíaco é mencionado na porção seleção assunto abaixo, a metodologia descrita aplica-se apenas à parte ecocardiográfico. A descrição da parte cateterismo foi incluído para validação independente de previsões baseadas em modelos e não está relacionado com a análise de E-ondas através do formalismo PDF. Antes da aquisição de dados, todos os indivíduos fornecem assinado, o consentimento informado para participação no estudo, de acordo com o Conselho de Revisão Institucional (Proteção Human Research Office) da Escola de Medicina da Universidade de Washington. NOTA: Todos os programas de software (junto com tutoriais sobre como usá-los) descritos nesta secção podem ser baixados do <a href="http://cbl1.wustl.edu/SoftwareAgreement.htm" target="_blank">http://cbl1.wustl.edu/Softw</a>areAgreement.htm Seleção 1. Assunto NOTA: Todos os indivíduos na base de dados Cardiovascular Biofísica Laboratório teve ecocardiografia simultânea e cateterismo cardíaco realizado e foram encaminhados por seus médicos para cateterismo cardíaco diagnóstico. Os critérios de inclusão do banco de dados são: 1) ausência de quaisquer anormalidades valvares significativas, 2) ausência de anormalidades de movimento de parede ou bloqueio do ramo no ECG, 3) presença de uma janela ecocardiográfica satisfatória com E- claramente identificável e A-ondas. 2. Ecocardiográfica Aquisição de Dados <ol><li> Gravar um estudo completo / eco-Doppler 2D para todas as disciplinas, de acordo com a Sociedade Americana de Ecocardiografia 16 critérios. NOTA: Os ecocardiogramas de triagem foram registrados em uma câmara clínico padrão por um ultra-sonografista. Se desejado, a gravação ecocardiográfica transtorácica adicional pode ser realizada para fins de verificaçãos depois de um cateter adequado, de alta fidelidade é avançado no LV para medir simultaneamente a hemodinâmica LV. </li><li> Assuntos da imagem em decúbito dorsal. Numa configuração nonresearch, posicionamento lateral esquerda padrão pode ser utilizado sem perda de generalidade do método. Obter visualizações apical de quatro câmaras, utilizando um transdutor de 2,5 MHz, com o volume de amostra fechado em 1,5-5 mm dirigidos entre as pontas dos folhetos da válvula mitral e ortogonal ao plano MV (para minimizar os efeitos de alinhamento, como visto na cor-mode M Doppler ), a parede de filtro fixado em 1 (125 Hz) e 2 (250 Hz), a linha de base adaptado para tomar vantagem de toda a altura do ecrã e da velocidade de escala ajustado para explorar a gama dinâmica de saída sem serrilhado. </li><li> Realize Doppler tecidual com o volume da amostra fechado em 2,5 mm e posicionados nas poções lateral e septal do anel mitral. </li><li> Salve exames Doppler em formato DICOM na máquina de eco e registro em DVD com simulteletrocardiograma aneously registada (ECG). </li></ol> 3. Doppler Processamento de Imagem e Análise convencional NOTA: Esta seção descreve dois programas personalizados MATLAB. O primeiro programa é descrito no passo 3.1 e o segundo programa é descrito nos passos 3,2-3,5. Todos os programas de software (junto com tutoriais sobre como usá-los) pode ser baixado do <a href="http://cbl1.wustl.edu/SoftwareAgreement.htm" style="font-size: 14px; line-height: 28px;" target="_blank">http://cbl1.wustl.edu/SoftwareAgreement.htm</a> <ol><li> Converta imagens do formato DICOM e vídeo para arquivos de bitmap (bmp) (usando um programa MATLAB costume). NOTA: O procedimento descrito abaixo para caber Doppler E-ondas e Doppler tecido E&#39;- ondas é mostrado na Figura 1. </li><li> Coloque os arquivos de imagem bitmap em outro programa MATLAB personalizado para medir parâmetros de fluxo transmitral convencionais, tais como E pico, de pico, E dur </sub&gt;, &#39;pico, A&#39; E de pico, etc. e recortar as imagens para análise PDF. Escolha imagens com contorno fluxos através discernível e ciclo cardíaco completo como indicado pelo ECG para análise. </li><li> Marcar a taxa de amostragem de tempo (medido em pixels / s no eixo horizontal) e taxa de amostragem de velocidade (medida em pixels / (m / s) ao longo do eixo vertical) nas imagens. Identificar o ciclo cardíaco completo observando e marcação picos consecutivos (R ou qualquer característica distinta do ECG) na imagem. </li><li> Mark transmitral Doppler E e A-onda ou tecido e Doppler? -E? A&#39;- onda do ciclo cardíaco seleccionado. <ol><li> Selecione o Doppler E-ondas ponto de pico ie. E pico, (ou pico E &#39;) e marcar o início da onda usando a linha que liga o pico para o início como uma guia para coincidir com a aceleração da inclinação da onda E (ou E&#39;-ondas). O início da onda é utilizado para calcular o intervalo do início ao pefluxo ak denotado como o tempo da onda E (ou E&#39;-wave) aceleração (AT). </li><li> Marca o fim da onda E (ou E&#39;-ondas), utilizando a linha que liga o pico para o final como um guia para corresponder ao declive de desaceleração. Isto é usado para calcular o intervalo de pico para a linha de base como indicado o tempo de desaceleração (DT). O intervalo do início ao fim da onda é a duração da onda E (E dur = NO + DT). O programa guia o usuário através de todo o processo com instruções apropriadas. </li></ol></li><li> Marcar a onda A usando um processo semelhante ao do E-ondas. Com ambos os E-e A-ondas marcado o programa calcula o pico E / A proporção de pico. NOTA: O programa salva as ondas marcadas como imagens recortadas contendo o E e apenas A-ondas. O programa também cria um ficheiro de dados com os parâmetros de corte e medidos para cada batimento. </li></ol> 4. montagem automatizada de transmitral Fluxo Usando o formalismo PDF <ol&gt; <li> A montagem automatizada de Doppler E e A-ondas e Doppler tecidual e os contornos? -E? Onda A&#39;- é feito usando um programa LabView costume 18,19. <ol><li> Coloque a imagem cortada, eo programa calcula automaticamente o envelope velocidade máxima (MVE). Seleccione o MVE, definindo o limite de tal forma que se aproxima de fluxos através MVE como mostrado na Figura 1. O início e terminação dos pontos que definem o MVA pode ser seleccionada ao longo do eixo de tempo por parte do operador de modo a que apenas o MVE pontos que proporcionem boa correspondência para a parte seleccionada real da onda são usados ​​como entrada para a montagem posterior. </li></ol></li><li> NOTA: Os pontos MVE selecionadas pelo usuário são a entrada para o programa de computador que se adapta automaticamente a solução do modelo PDF para a velocidade em função do tempo, utilizando um Marquardt Levenberg (iterativo) algoritmo. A montagem é realizada com a exigência de que o erro quadrático médio entre a clínica (de entrada)dados (MVE) e o modelo de PDF contorno previsto ser minimizada. Uma vez que o modelo é linear, um único conjunto de parâmetros é obtida para cada Doppler de onda E derivada MVE utilizado como entrada. Assim numericamente k, C e valores X O exclusivos são gerados para cada onda E e K &#39;, c&#39;, e x o &#39;para cada onda E&#39;. </li><li> No caso do ajuste é obviamente subóptima quando o ajuste é sobreposta sobre a onda E (ou E&#39;-ondas) imagem (isto é. Algoritmo tentou encaixar ruído incluídos no MVE por exemplo) modificar o MVE usando mais / menos pontos, modificando assim, o modelo previu contorno com conseqüente modificação dos parâmetros de PDF para conseguir um melhor ajuste. </li></ol> Salve os dados quando o ajuste apropriado PDF foi gerado. NOTA: O programa é escrito para salvar automaticamente os dados de imagem e arquivos de texto contendo os parâmetros PDF ea informação de contorno. Os parâmetros obtidos PDF a partir do procedimento acima descrito pode ser utilizado para elucidar novo fisiologia e distinguir entre a fisiologia normal e patológica, como detalhado na secção de resultados representativos abaixo.

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S., Kov&#225;cs, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Increasing+Heart+Rate+on+Deceleration+Time,+Velocity+Time+Integral,+and+E/A.">Consequences of Increasing Heart Rate on Deceleration Time, Velocity Time Integral, and E/A.</a> Am. J. Cardiol. 97, 130-136 (2006).</li></ol>

The authors have no competing financial interests.

De acordo com nosso foco metodológico, os principais aspectos dos métodos que facilitem a obtenção de resultados precisos e significativos são destacados. ECOCARDIOGRAFIA A Sociedade Americana de Ecocardiografia (ASE) tem diretrizes para a realização de estudos transtorácicas 16. Durante um exame de eco, há uma infinidade de fatores que afetam a qualidade da imagem. Os fatores que estão além do controle do ultra-sonografista incluem: capacid...

Estudos pioneiros de Katz 1 em 1930 revelou que o ventrículo esquerdo mamíferos inicia enchimento por ser uma bomba de sucção mecânica, e muito esforço, desde então, tem se dedicado a desvendar o funcionamento da diástole. Por muitos anos, os métodos invasivos eram as únicas opções disponíveis para a avaliação clínica ou de investigação da função diastólica (DF) 2-16. Na década de 1970, no entanto, os avanços técnicos e desenvolvimentos na ecocardiografia finalmente deu cardiologistas e fisiologistas ferramentas práticas para a caracterização não invasiva do DF. Sem uma teoria causal unificação ou paradigma para a diástole a respeito de como o coração funciona quando se enche, os pesquisadores propuseram vários índices fenomenológica com base na correlação com características clínicas. A curvilínea, rapidamente subindo e descendo forma do fluxo sanguíneo velocidade contorno transmitral durante cedo, enchimento rápido, por exemplo, foi aproximada como um triângulo e diastólica fuíndices nction foram definidos a partir de características geométricas (altura, largura, área, etc.) desse triângulo. Avanços técnicos na ecocardiografia permitiram movimento do tecido, tensão e taxa de deformação durante o enchimento a ser medida, por exemplo, e cada avanço técnico trouxe com ele uma nova safra de índices fenomenológicas para ser correlacionados com características clínicas. No entanto, os índices permanecem correlativo e não causal e muitos índices são diferentes medidas do mesmo fisiologia subjacente. Não é surpreendente, portanto, que os índices clínicos atualmente empregados do DF têm sensibilidade e especificidade limitadas. Para superar essas limitações o recheio formalismo (PDF) parametrizada diastólica, a cinemática causal, modelo parâmetro amontoado de enchimento do ventrículo esquerdo, que é motivado por e incorpora a fisiologia de sucção da bomba de diástole foi desenvolvido e validado 17. Ele modela a função diastólica (que se manifesta pelas formas curvilíneasde contornos fluxo transmitral), de acordo com as regras do movimento oscilatório amortecido harmônica. A equação para o movimento oscilatório amortecido harmónica é baseado na segunda lei de Newton, e podem ser escritos, por unidade de massa, como: <img alt="Equação 1" fo:content-width="1in" src="/files/ftp_upload/51471/51471eq1.jpg" /> Equação 1 Esta equação diferencial de 2 ª ordem linear tem três parâmetros: k - rigidez câmara, c - viscoelasticidade / relaxamento, e x o - inicial de deslocamento / pré-carga do oscilador. O modelo prevê que os diferentes padrões de enchimento diastólica clinicamente observados são o resultado de variação no valor numérico destes três parâmetros do modelo. Com base no formalismo PDF e mecânica clássica, E-ondas pode ser classificado como sendo determinada por ou sobre-amortecida regimes sub-amortecidos do movimento. Numerosos estudos <sup&gt; 17-21 validaram que contornos E-onda clinicamente gravadas e modelo PDF previsto contornos mostram excelente concordância e elucidaram os hemodinâmicos / análogos fisiológicas dos três parâmetros PDF 21. O processo para a extração de parâmetros do modelo a partir de dados da onda E clinicamente registrada é detalhada nos métodos abaixo. Ao contrário índices típicos de DF em uso clínico atual, três parâmetros do modelo PDF são causalidade baseado. Como discutido nos métodos a seguir, de índices adicionais de fisiologia diastólica podem ser derivadas a partir destes parâmetros fundamentais e da aplicação do formalismo PDF para os aspectos da diástole excepto fluxos através. Neste trabalho, métodos de análise baseados em PDF das relações fisiológicas fluxo mitral e que podem ser extraídas a partir da abordagem PDF, seus parâmetros e os índices derivados são descritos. Além disso, mostra-se que os parâmetros PDF ou índices derivadas deles podem provocarAlém propriedades intrínsecas da câmara dos efeitos externos da carga pode fornecer correlações com parâmetros definidos tradicionais invasiva e pode diferenciar entre os grupos normal e patológica.

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			<td height="42" style="height:42px;width:216px;">Name of Equipment/ Software</td>
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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">Philips iE33</td>
			<td style="width:159px;">Philips (Andover, MA.)</td>
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			<td height="21" style="height:21px;width:216px;">LabVIEW 6.0</td>
			<td style="width:159px;">National Instruments</td>
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			<td>Version 6.0.2</td>
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			<td height="21" style="height:21px;width:216px;">MATLAB</td>
			<td style="width:159px;">MathWorks</td>
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			<td>Version R2010b</td>
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quantification of global diastolic function by kinematic modeling-based analysis of transmitral flow via the parametrized diastolic filling formalism

Avaliação da função cardíaca quantitativa continua a ser um desafio para os fisiologistas e médicos. Embora os métodos invasivos têm historicamente constituído o único meio disponível, o desenvolvimento de métodos de imagem não invasivos (ecocardiograma, ressonância magnética, tomografia computadorizada) com alta resolução temporal e espacial proporcionar uma nova janela para a avaliação quantitativa da função diastólica. O ecocardiograma é o acordado padrão para avaliação da função diastólica, mas os índices de uso clínico atual simplesmente utilizar recursos de dimensão câmara (M-mode) ou movimento de sangue / tecido (Doppler) formas de onda selecionado sem incorporar os determinantes causais fisiológicas do próprio movimento. O reconhecimento de que todos os ventrículos esquerdo (VE) iniciado o enchimento, servindo como bombas de sucção mecânica permite que a função diastólica global a ser avaliada com base em leis do movimento que se aplicam a todas as câmaras. O que diferencia um coração de outro são os parâmetros da equação de movimento que govenchimento Erns. Assim, o desenvolvimento do enchimento parametrizada diastólica (PDF) formalismo tem mostrado que toda a gama de fluxo transmitral precoce clinicamente observados (Doppler E-Wave) padrões são extremamente bem em forma, as leis do movimento oscilatório amortecido. Isto permite a análise de E-ondas individuais de acordo com um mecanismo causal (sucção iniciada pelo recuo) que rende três (numericamente) parâmetros concentrados únicas cujos análogos fisiológico são câmara de rigidez (k), viscoelasticidade / relaxamento (c) e carga (x o). A gravação do fluxo transmitral (Doppler E-ondas) é uma prática comum em cardiologia clínica e, portanto, o método de gravação ecocardiográfico é apenas brevemente revisto. Nosso foco é a determinação dos parâmetros de PDF a partir de dados E-onda rotineiramente registrados. Conforme os resultados, destacam indicar, uma vez que os parâmetros de PDF foram obtidos a partir de um número adequado de carga variável E-ondas, os investigator é livre para usar os parâmetros ou construir índices a partir dos parâmetros (como a energia armazenada meia kx o 2, pressão máxima AV gradiente kx o, índice de carga independente da função diastólica, etc.) e selecionar o aspecto da fisiologia ou fisiopatologia ser quantificado.

As formas de onda de Doppler representante dos quatro diferentes tipos de padrões de enchimento (, pseudormal relaxamento retardada normal, constritiva restritiva) usando o método descrito acima são mostrados na Figura 2. Figura 2A mostra o padrão normal, o que, por si só, é indistinguível da pseudonormal padrão. Figura 2B mostra um relaxamento retardada e a Figura 2C mostra um padrão constritiva restritiva associada com disfunção diastólica grave. Para maio...

Watch this Scientific Journal Video about Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism at JoVE.

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Accurate, quantificação baseada em causalidade da função diastólica global tem sido alcançado por meio de análise baseada na modelização cinemática do fluxo transmitral através do parametrizada enchimento diastólico formalismo (PDF). PDF gera rigidez, relaxamento e parâmetros de carga únicas e elucida &#39;novo&#39; fisiologia, proporcionando índices sensíveis e específicos de disfunção.

Quantificação da função global diastólica por análise baseada em Modelagem cinemática de transmitral fluxo através do formalismo de enchimento parametrizada diastólica

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

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JoVE Journal

Bioengineering

11.1K visualizações.  Washington University in St. Louis.  Accurate, quantificação baseada em causalidade da função diastólica global tem sido alcançado por meio de análise baseada na modelização cinemática do fluxo transmitral através do parametrizada enchimento diastólico formalismo (PDF). PDF gera rigidez, relaxamento e parâmetros de carga únicas e elucida 'novo' fisiologia, proporcionando índices sensíveis e específicos de disfunção. 

Vídeo: Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Avaliação de Entrega Gene Polimérico Nanopartículas por Análise Nanoparticle Rastreamento e alta capacidade de Citometria de Fluxo

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Bioengenharia

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigando a separação de fluxo tridimensional induzida por um polip de dobra vocal modelo

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Elétrica Cell-substrato impedância Sensing para a quantificação do endotelial Proliferação, Barreira Function, e Motilidade

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

A lignina Down-regulação da<em&gt; Zea mays</em&gt; Via dsRNAi e Klason Análise lignina

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Limite externo assistido segmentação e quantificação dos ossos Trabecular por um Plugin do Imagej

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Medição de metaloproteinase matriz celular Global e atividade metabólica em hidrogel 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantificação simultânea de kynureninas selecionadas analisadas por espectrometria de cromatografia líquida-massa em média coletada de culturas celulares cancerosas

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

A Quantificação da Injeção por Testes Mecânicos

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.