As técnicas mostradas aqui foram desenvolvidos com o financiamento por doações do Departamento de Energia (Ciências da Bioenergia) e da National Science Foundation.

Parte 1: Crescendo e armazenamento de material vegetal adequado <ol><li> Em nossa experiência, hipocótilo jovens de mudas de pepino estiolados servir como uma fonte conveniente de material da parede celular para estas experiências. Sementes de pepino são semeadas em papel molhado em uma caixa à prova de luz, que é manter-se em um armário em uma sala escura temperatura constante fixada em 26 ° C. A temperatura exata não é crítica, como qualquer coisa entre 22 ° e 30 ° C deve ser fino, mas a temperatura vai determinar o quão rápido as mudas atingem um grau adequado de desenvolvimento. A temperatura mais quente, mais rápido as mudas irão se desenvolver. Normalmente usamos mudas quando eles têm crescido para cerca de 5 cm de comprimento, que é alcançado 3-4 dias após a semeadura. É importante que a muda ser cultivada no escuro, já que mesmo pequenas quantidades de luz afetam tanto a taxa de desenvolvimento de plântulas e as propriedades da parede celular que medimos com esta técnica. No dia 3, você pode espreitar na caixa, usando uma luz verde dim filtrada, para verificar o desenvolvimento das plântulas. </li><li> Mudas são rapidamente cortado e embalado em pequenas caixas de plástico, 100-150 mudas por caixa, e armazenadas a -80 ° C. A esta temperatura que permanecem úteis por semanas. </li></ol> Parte 2: As amostras de parede celular Preparando <ol><li> Pequenos grupos (8-10) de mudas de corte congelados são transferidos do freezer a um recipiente isolado contendo um bloco freezer -80. </li><li> A cutícula que cobre o hipocótilo é abrasiva com carborundum. Isso é feito por várias vezes o desenho hipocótilo entre o polegar eo indicador, que são revestidos com uma pasta espessa de pó de carborundum molhado. Leva um pouco de experiência para saber a quantidade correta de pressão para o uso: muita pressão e da camada epidérmica começa a ser desfiado e rasgado; muito pouca pressão ea cutícula não será permeabilizadas. A pessoa também tem que trabalhar rápido, pois como o degelo congelados hipocótilo, torna-se flácido e difícil de gerenciar. </li><li> O hipocótilo abraded é mergulhado em água gelada para remover a maior parte do carborundum aderir e depois armazenados em água com gelo, enquanto o hipocótilo restantes são preparadas de uma forma similar. </li><li> O hipocótilo são cortadas no comprimento desejado, geralmente 1,2 centímetros, com uma lâmina de barbear novo single gumes e, em seguida, alinhados em uma lâmina de vidro. </li><li> Agora precisamos de achatar as paredes para remover seiva celular e para facilitar a fixação. A segunda lâmina de vidro é colocado em cima do grupo de 8-10 amostras, formando um sanduíche. Um peso (400-500 g) é colocado em cima da lâmina de vidro por 5 min. Para o peso que usam rotineiramente um copo contendo uma quantidade adequada de água. </li><li> Etapa opcional: Dependendo do experimento, o hipocótilo pode ser inactivado com um tratamento térmico breve a este ponto. Para fazer isso, vincular a lâminas de vidro, juntamente com um par de faixas de borracha, coloque o conjunto em um recipiente com 100 mL de água desionizada à temperatura ambiente e colocá-lo em um forno de microondas na potência máxima. Com o nosso forno de microondas a água começa a ferver em cerca de 50 s e paramos no microondas 15 s após a ebulição começa. A água quente é derramada rapidamente fora e substituída por água fria para parar a desnaturação. Você pode ter de variar esses horários, como seu micro pode ser diferente da nossa. Com o aquecimento excessivo das amostras tornam-se fracos e quebram com facilidade. Com o aquecimento insuficiente expansin endógena não é inativada e as amostras de parede manter alguma capacidade de resposta ao pH ácido. </li></ol> Parte 3: setup Extensômetro <ol><li> As amostras de parede são agora fixada em um extensômetro força constante. Este é um dispositivo feito sob medida, que consiste de uma cuba de acrílico para a realização da final da amostra basal de parede e uma braçadeira móvel unida à extremidade apical da amostra. O grampo móvel é montado na extremidade de uma haste que passa através das bobinas aberto de um sensor de posição, um LVDT ou &quot;Linear Variable Differential Transformer &#39;, que eletronicamente detecta a posição de um pequeno cilindro de metal, ou&#39; core &#39;, que é anexado à haste. A extremidade superior da haste é ligada a uma alavanca com um contrapeso ajustável. Esta alavanca exerce um valor ajustável de força para cima sobre a amostra parede. A força é ajustado pela adição ou remoção de pesos de metal calibrada até o final da alavanca. </li><li> Voltar para a amostra de parede - o fim basal da amostra hipocótilo é pego com uma pinça fina e ~ 2-3 mm da extremidade apical é colocado entre as mandíbulas abertas do grampo móvel. Este grampo é um grampo jacaré de mola de metal cujas mandíbulas são revestidas com plástico para evitar o contato direto entre a superfície do metal ea solução-tampão ou a amostra parede. Em nossa experiência de íons metálicos pode lixiviar do grampo e inibir a capacidade da parede para estender, e assim mantemos o metal coberto. </li><li> Segurando o conjunto de grampo móvel em uma mão, o fim basal da amostra parede agora é manobrado entre as duas peças articuladas na célula, Plexiglas e cuvetepeças são reunidas e aparafusadas, garantindo com isso a extremidade inferior da amostra parede na tina. </li><li> O conjunto de grampo móvel é agora lançado suavemente, permitindo que a força total do contrapesos para ser transferido para a amostra parede. Rotineiramente usamos um contrapeso total de 20 g para as amostras de parede preparados a partir de hipocótilos de pepino. Materiais da parede ou outros experimentos podem requerer diferentes pesos. </li><li> A cuvete é preenchido com tampão (200 uL) ea posição da cubeta movido para cima ou para baixo com um parafuso de ajuste, de modo que o grampo móvel é levada ao extremo inferior da faixa de medição LVDT. Nosso LVDT é conectado a uma unidade de aquisição de dados de um microcomputador, e por isso monitorar a posição LVDT através do computador. Temos oito assembléias LVDT ligado em paralelo com um computador, o que nos permite executar oito amostras de parede simultaneamente. O computador registra a posição de cada um dos conjuntos LVDT uma vez a cada 30 s. </li></ol> Parte 4: Medição de extensão resposta ao pH ácido ou expansin - Resultados Representante <ol><li> [Primeira experiência] Para a medição de ácido induzida por extensão, nós começamos com amostras de parede nativa (isto é, não inativado pelo calor) e tampão neutro é adicionado à cubeta. As amostras de parede se estendem por alguns minutos, em resposta à tensão adicionado, mas o decai extensão para uma baixa taxa depois de alguns minutos. Nosso computador nos permite monitorar a variação no comprimento da amostra (isto é, a mudança de posição do grampo móvel, após o início do experimento) ou podemos monitorar a derivada temporal da posição, em outras palavras, a taxa de extensão . A taxa de extensão estabiliza a um baixo valor com o tempo. </li><li> Depois de ~ 20 minutos, o tampão neutro é removido. Nós usamos um tubo de metal fino, feito de uma agulha hipodérmica calibre grande, conectado a uma bomba de vácuo para remover o tampão rapidamente, com o mínimo de perturbação da parede ou a montagem mecânica. Tampão ácida é então adicionada, às vezes com 1-2 trocas rápidas para garantir a troca completa de tampão ácido. Então sentar e ver a resposta da parede. Tipicamente, podemos detectar a taxa mais rápida de extensão em poucos minutos. Após 60 min geralmente temos informações suficientes para avaliar a resposta de extensão, embora em alguns casos, as medições pode se estender para períodos mais longos. </li><li> [Segundo experimento] Para a medição de expansin induzida extensão da parede celular, começamos com amostras inativado pelo calor parede e tampão ácido é adicionado à cubeta. Como no primeiro experimento, a taxa de extensão da parede celular estabiliza gradualmente para um valor baixo por causa da falta de expansin funcional. </li><li> Depois de ~ 20 minutos, proteína expansin é adicionado à cubeta por &#39;spiking&#39; com o buffer na cuvete com 10-20 uL de uma solução expansin. O expansin penetra rapidamente a amostra da parede celular e em poucos minutos, vemos que a taxa de extensão aumentou. Esta resposta de extensão pode ser seguido por uma hora ou mais. </li></ol><img src="/files/ftp_upload/1263/fig1.png" alt="" width="600" height="469" /> Figura 1: Esquema de procedimentos para a preparação de paredes celulares para avaliar induzida por ácido ou expansin induzida extensão da parede.

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+long-term+extension+of+isolated+cell+walls+from+growing+cucumber+hypocotyls.">Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls.</a> Planta. 177, 121-130 (1989).</li><li>Rayle, D. L., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acid+Growth+Theory+of+auxin-induced+cell+elongation+is+alive+and+well.">The Acid Growth Theory of auxin-induced cell elongation is alive and well.</a> Plant Physiol. 99, 1271-1274 (1992).</li><li>Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin-induced+growth+of+Avena+coleoptiles+involves+two+mechanisms+with+different+pH+optima.">Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima.</a> Plant Physiol. 99, 1556-1561 (1992).</li><li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loosening+of+plant+cell+walls+by+expansins.">Loosening of plant cell walls by expansins.</a> Nature. 407, 321-326 (2000).</li><li>McQueen-Mason, S., Durachko, D. M., Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+endogenous+proteins+that+induce+cell+wall+expansion+in+plants.">Two endogenous proteins that induce cell wall expansion in plants.</a> Plant Cell. 4, 1425-1433 (1992).</li></ol>

Para esta demonstração foram utilizados paredes celulares de hipocótilo do pepino porque eles provaram ser uma fonte confiável de amostras de parede que são fáceis de manusear e que respondem com boa sensibilidade. Nós também tivemos um bom sucesso com paredes de mudas de outras, bem como alguns materiais do supermercado, tais como folhas de espinafre jovens e talos de aipo. Basicamente, o jovem, macio, tecidos vegetais de rápido crescimento tendem a ser facilmente medido com esta técnica, mas resistente, velh...

measuring plant cell wall extension (creep) induced by acidic ph and by alpha-expansin

Paredes celulares de plantas crescendo caracteristicamente apresentam uma propriedade conhecida como &quot;crescimento ácido&quot;, pelo qual significa que elas são mais extensíveis em pH baixo (&lt;5) 1. O hormônio vegetal auxina rapidamente estimula o alongamento celular em caules jovens e tecidos semelhantes ao menos em parte por um mecanismo de crescimento ácido-2, 3. Auxina ativa uma bomba H + na membrana plasmática, causando a acidificação da solução da parede celular. Acidificação da parede ativa expansins, que são endógenas afrouxamento da parede celular-4 proteínas, fazendo com que a parede celular para produzir as tensões de parede criado por pressão de turgor celular. Como resultado, a célula começa a aumentar rapidamente. Esse fenômeno de crescimento ácido &quot;é facilmente medido em isolada (não vivas) espécimes da parede celular. A capacidade das paredes das células se submeter induzida por ácido extensão não é simplesmente o resultado do arranjo estrutural dos polissacarídeos da parede celular (pectinas, por exemplo), mas depende da atividade de expansins 5. Expansins não têm qualquer atividade enzimática e conhecida a única forma de ensaio para a atividade expansin é medir sua indução da extensão da parede celular. Este relatório detalha o video fontes e técnicas de preparação para a obtenção de materiais de parede adequado para ensaios expansin e passa a mostrar induzida por ácido extensão e expansin induzida extensão da parede amostras preparadas a partir de hipocótilos de pepino crescimento. Para obter amostras de células da parede adequados, mudas de pepino são cultivados no escuro, o hipocótilo são cortadas e congeladas a -80 ° C. Hipocótilo congelados são desgastados, achatados, e depois preso na tensão constante em uma tina especial para medições extensômetro. Para medir induzida por ácido de extensão, as paredes são inicialmente tamponada em pH neutro, resultando em baixa atividade de expansins que são componentes das paredes das células nativas. Após a troca de buffer para pH ácido, expansins são ativados e as paredes celulares estender rapidamente. Nós também demonstram atividade expansin em um ensaio de reconstituição. Para esta parte, nós usamos um tratamento térmico breve a desnaturar a expansins nativas nas amostras de parede celular. Estas paredes das células inativadas não se estendem até mesmo em tampão ácido, mas além de expansins para as paredes das células rapidamente restaura a sua capacidade de estender.

Watch this Scientific Journal Video about Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin at JoVE.com

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

Nós demonstramos o uso de um extensômetro de força constante para medir a extensão de longo prazo (creep) de espécimes de plantas parede celular induzida por buffers ácidas e proteína expansin.

Medição Usina Extensão parede celular (Creep) induzida pelo pH ácido e por Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

measuring-plant-cell-wall-extension-creep-induced-acidic-ph-alpha

Research

JoVE Journal

Biology

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

13.9K visualizações.  Penn State University. Nós demonstramos o uso de um extensômetro de força constante para medir a extensão de longo prazo (creep) de espécimes de plantas parede celular induzida por buffers ácidas e proteína expansin.

Vídeo: Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

Title Cell Encapsulation by Droplets

Encapsulation título celular por Droplets

Biologia

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Induzida por vírus silenciamento gênico (VIGS) em Nicotiana benthamiana Tomate e

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identificação de fenótipos inibição do crescimento induzido pela expressão de Effectors Tipo III bacteriana em Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

Agar-Microcosmos Bloco de decomposição controlada de Tecidos Vegetais por fungos aeróbicos

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

Derramando e executando um gel de proteína através da reutilização Cassetes comerciais

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profiling glicana de Polímeros da planta da parede celular usando Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...