Os autores gostariam de agradecer Dr. Paul Witting e Ms. Genevieve Fong por sua assistência com extração de mRNA e PCR, eo Garnett Passe e Rodney Williams Memorial Foundation para o apoio.

Declaração de Ética: Todos os procedimentos descritos a seguir foram aprovados pelo Comitê de Ética da Universidade de Sydney animal. 1. Animais NOTA: 1 e 8-9 meses de idade camundongos (C57 / BL6) foram obtidos a partir do Centro de Recursos Animal (Perth, Austrália). Os ratinhos foram alojados no mecanismo Bosch Roedor na Universidade de Sydney. <ol><li> Casa ratos em gaiolas-padrão do mouse em um ciclo claro / escuro de 12/12 horas de luz com acesso a comida e água ad libitum. </li><li> Camundongos Divida em cada faixa etária em infravermelho próximo (NIR), tratados, ou sham grupos tratados (controle) para comparação. </li></ol> 2. Infravermelho Próximo (NIR) Irradiação e Tratamento Sham <ol><li> Raspar a pele na região da cabeça e pescoço do mouse com um barbeador elétrico, tanto quanto possível para evitar a rebrota rápida de cabelo antes da realização do regime de tratamento. A fim de manter o status de livre de patógenos dos ratos, use 70% de etanolpara limpar instrumentos entre animais para maquiagem e desinfetante F10 veterinário entre animais de gaiolas separadas. Faça isto 2-3 dias antes do início do tratamento para que os animais não estão sobre-tratadas. </li><li> Conter rato com a extremidade proximal da cauda e permitir que ele relaxe na palma de uma mão ou de bancada, de modo a minimizar o stress, que pode levar a resultados falsos. </li><li> Segure o NIR (670 nm) LED (light emitting diode) dispositivo de 1-2 cm de distância da área exposta (raspada) e ligar o aparelho durante 90 segundos (Figura 1A). NOTA: A mudança de temperatura como resultado de exposição de 90 segundos foi medida como &lt;0,2 ° C em 100 ml de água. </li><li> Repita os passos de 2,2-2,3 para o grupo de tratamento simulado de animais, mas deixar o aparelho desligado (Figura 1B). </li><li> Repita os passos de 2,2-2,3 para o grupo de tratamento Nir-bloqueado de animais, mas cubra o aparelho com papel alumínio. </li><li> Repita os passos 2,2-2,5 diariamente às approximate 24 intervalos de RH durante 5 dias consecutivos. </li><li> Extraia o epitélio vestibular sensorial (3 cristas e uma mácula utricular) de ambas as orelhas no quinto dia pós-tratamento (ver secção 3 abaixo). </li></ol> 3. Extracção de tecido 20 <ol><li> Preparar 300 ml de um fluido cerebrospinal artificial (ACSF) à base de glicerol que consistem em (em mM): 26 de NaHCO3, 11 de glucose, 250 glicerol, 2,5 de KCl, 1,2 de NaH 2 PO 4, 1,2 de MgCl2, 2,5 e CaCl 2. Antes da adição de CaCl2, gás a solução com carbogénio (95% O 2 e 5% CO 2) para estabelecer um pH de 7,4 e evitar a precipitação do cálcio (turvação). Arrefeça a solução num congelador a -80 ° C durante 45 minutos de modo que uma pasta de gelo é formada. </li><li> Prepare o tampão de lise isolamento do RNA em microtubos parafuso superior marcados (pára o popping de tampas quando as alterações de pressão tubo entre congelamento e descongelamento em nitrogênio líquido), de acordocom as instruções do fabricante ou práticas de laboratório habituais. Já nitrogênio líquido pronto em um frasco de Dewar de alumínio. NOTA: Use roupas de proteção e eyeware ao manusear nitrogênio líquido. Certifique-se de que o nitrogênio líquido é usado em um quarto bem ventilado como o volume da sua forma de gás é aproximadamente 700 vezes maior do que a sua forma líquida e pode causar asfixia. </li><li> Profundamente anestesiar os ratos com cetamina (400 mg / kg) através de uma injecção intra-peritoneal. Permitir que o reflexo posterior de membros para desaparecer completamente como indicação de que os ratos são totalmente anestesiado. </li><li> Decapitar ratos com tesoura de aço inoxidável afiadas e fazer uma incisão ao longo da pele sagital do crânio usando uma lâmina de barbear (arredondado # 22). Neste momento e em toda as etapas 3,5-3,9, manter a calota craniana, cérebro e aparelho vestibular subjacente o mais fresco possível pela aplicação regular de ACSF gelada sobre o tecido. </li><li> Usando o braço pontas de tesouras padrão standard fazer uma small incisão no crânio no Lambda e cortar ao longo da sutura sagital. </li><li> Deslize suavemente um braço de um fórceps rasas debaixo do osso parietal mantendo a lâmina tão próximo quanto possível da superfície inferior do osso sem arrastar o cérebro. Seguros e afastar o osso parietal lateral e posterior do osso occipital até que o cérebro está exposto. </li><li> Use uma pequena espátula de aço inoxidável e levante o cérebro de distância do anterior e fossa craniana média para expor o nervo vestibular (CN VIII). Transecto o nervo para evitar tensão desnecessária sobre os axônios aferentes primários que inervam diretamente as células ciliadas vestibulares. </li><li> Remover o cérebro in toto após transecção do CN VIII. </li><li> Observe o labirinto ósseo contendo a cóclea e os órgãos vestibulares periféricas da fossa craniana média. Faça duas pequenas incisões ao lado de cada labirinto ósseo e extirpar toda a estrutura, mantendo o canal semicircular anterior e puxando mais tardealiado. </li><li> Mergulhar imediatamente labirintos excisadas em um prato de dissecação contendo solução ACSF gelado (como descrito no passo 3.1), enquanto que irriga continuamente com carbogéneo. </li><li> Sob um microscópio estereoscópico, mantenha pressionado o labirinto pela cóclea e fixá-lo ao fundo do prato com uma pinça. <ol><li> Use uma pinça reta fina para riscar uma pequena abertura no osso acima do canal semicircular (SSC) ampulla anterior. </li><li> Gentilmente ampliar esta abertura, alcançando imediatamente abaixo do osso e sacudindo para fora ampulífuga. Tenha cuidado aqui para não empurrar pinça na abertura e danificar o labirinto membranoso frágil abaixo. Continuar dessa maneira até que o utrículo, anterior e ampolas laterais estão todos expostos. Se possível remover a ampola posterior também. </li></ol></li><li> Usando uma pinça fina, levantar delicadamente o utrículo e ampolas de distância do labirinto ósseo até que estejam completamente isolada. Sempre que possível, mantê-lospor seus canais semicirculares associados para evitar danos ao epitélio sensorial. Em alguns casos, a parte proximal da conduta membranoso semicircular pode ter de ser cortado com tesoura de íris para libertar as ampolas a partir do osso. </li><li> Firmemente compreender os órgãos vestibulares entre a ponta de uma pinça e lugar para o tampão de lise preparado anteriormente na etapa 3.2. Agite suavemente a pinça em torno do tampão para garantir órgãos vestibulares têm destacado a pinça. Confira este for o caso, trazendo a pinça, ao microscópio estereoscópico. <ol><li> Parafuso na tampa de microtúbulos e congelar a amostra em nitrogênio líquido imediatamente. </li></ol></li></ol> 4. Extracção de ARN e RT-PCR <ol><li> Siga métodos padrão de RNA mensageiro (mRNA) extracção de acordo com as instruções do fabricante ou protocolos de laboratório preferenciais. NOTA: Um kit comercial que utilizou ARN de suporte para ajudar a isolar pequenos rendimentos de ARNm foi utilizada nesteprotocolo. Volumes de eluição inferiores podem ser utilizados para aumentar as concentrações finais de ARNm. NOTA: Negativo controles &quot;não enzima&quot; (CNE) e controles &quot;nenhum modelo de DNA&quot; (NTC) também deve ser concluído para garantir a validade dos efeitos observados. </li><li> Aplicar os métodos padrão de transcrição reversa do ARNm de ADN complementar (ADNc) e amplificação de genes alvo 21-23. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Os resultados representativos descritos aqui mostram que a entrega breve transcraniana de luz NIR (90 seg / dia durante 5 dias) é suficiente para elevar os níveis de expressão de antioxidantes em ratos mais velhos quando comparados com os ratos tratados por simulação. Enquanto calor emitido poderia representar uma fonte de mitocondrial e / ou ativação neuronal, como relatado para vestibular rato aferentes 24 - nossa medição do calor emitido pelo NIR LED dispositivo foi &lt;0,2 ° C durante 90 segundo...

O declínio da performance de equilíbrio e quedas subsequentes são comuns, e, infelizmente, muitas vezes definem características de envelhecimento natural 1. O impacto desse declínio pode ser tanto física e social, e reduz significativamente a qualidade de vida das pessoas mais velhas. Em resposta, terapias físicas e reabilitação têm sido o foco da investigação sobre as quedas, mas não têm sido associadas com redução consistente na prevalência de quedas repetidas. Ao mesmo tempo, o trabalho investiga as mudanças no sistema vestibular periférico ou central (o sistema responsável por manter o equilíbrio) é escassa, e potenciais estratégias terapêuticas dirigidas a esses sistemas e as causas subjacentes de desequilíbrio limitado. Trabalhos recentes sobre doenças neurodegenerativas associadas com a idade relacionada com a idade, incluindo degeneração macular 2-4, modelos da doença de Alzheimer, 5-8 e 9-12 doença de Parkinson têm mostrado efeitos neuroprotectores de simple aplicação não invasiva do infravermelho próximo (NIR) luz. Além disso, no sistema vestibular, NIR foi usada para aumentar a actividade dos neurónios aferentes primários vestibulares in vitro 13. Embora o mecanismo de NIR luz não é bem compreendido, a maioria dos estudos que usam NIR sugeriram que estimula NIR mitocôndrias complexo IV (citocromo c oxidase) 14-17 para facilitar o metabolismo celular. No epitélio sensorial vestibular da placa subcuticular do tipo células ciliadas I é denso em mitocôndrias 18 e, como tal, pode representar um local de ação para o tratamento terapêutico NIR. Aqui, uma breve regime de tratamento, não-invasivo de transcraniana aplicado NIR que podem ser utilizadas para medir o metabolismo celular (e implicitamente, a função mitocondrial) no rato epitélio sensorial vestibular é descrito. Também é discutida a preparação de um epitélio sensorial vestibular e é mostrado que o NIR aumenta a expressão de um ubiquitonos anti-oxidante (superóxido dismutase 1) no epitélio sensorial - anteriormente mostrado ser importante para a sobrevivência de células do cabelo cóclea 19.

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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Estratégias para atenuar o declínio na função equilíbrio com o aumento da idade são predominantemente focado em terapias físicas, incluindo tarefas de equilíbrio e exercícios. No entanto, estas abordagens não abordam as causas subjacentes do declínio do equilíbrio. Usando ratos, o impacto da luz de infravermelho próximo (NIR) sobre o metabolismo de células do epitélio sensitivo vestibular foi avaliada. Os dados coletados mostram que essa intervenção simples e segura pode proteger essas células vulneráveis ​​dos efeitos deletérios do envelhecimento natural. ARNm foi extraído a partir do epitélio sensitivo vestibular periférico isolado (ampulares crista, e mácula utricular) e posteriormente transcrita para uma biblioteca de ADNc. Esta biblioteca foi então sondadas para a expressão de antioxidante ubíqua (SOD-1). A expressão do gene antioxidante foi então usado para quantificar o metabolismo celular. Usando entrega transcraniana de Nir em jovens (4 semanas) e mais velhos (8-9 meses), ratos, e um regime de tratamento breve (90 seg / dia para 5 days), este trabalho sugere NIR sozinho pode ser suficiente para melhorar a função mitocondrial no epitélio sensitivo vestibular. Uma vez que existem atualmente não há métodos disponíveis, acessíveis e não-invasivos de terapia para melhorar a função das células ciliadas vestibular, a aplicação de radiação NIR externa fornece uma estratégia potencial para neutralizar o impacto do envelhecimento sobre o metabolismo celular inthe epitélio sensorial vestibular.

Para comparar o impacto do tratamento NIR em jovens (4 semanas) e mais velhos (8-9 meses), camundongos medimos a expressão de antioxidante superóxido dismutase 1 (SOD-1) em jovens (n ​​= 16) e mais velhos (n = 20) os ratos que foram tratados Nir, tratamento simulado, ou NIR-bloqueados. A Figura 2 mostra um aumento significativo na normalizada SOD-1 expressão β-actina de mais de 2 vezes em jovens tratados com NIR animais jovens em comparação com os animais tratados de forma simulada (p &lt;0,01...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

A disfunção mitocondrial é uma característica da senescência celular. Este trabalho usa não-invasivo do infravermelho próximo (NIR) de tratamento para melhorar a função mitocondrial no rato epitélio sensorial vestibular de envelhecimento.

Infravermelho Próximo (NIR) Luz aumenta a expressão de um marcador da função mitocondrial no mouse Vestibular Sensorial Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.8K visualizações.  University of Sydney.  A disfunção mitocondrial é uma característica da senescência celular. Este trabalho usa não-invasivo do infravermelho próximo (NIR) de tratamento para melhorar a função mitocondrial no rato epitélio sensorial vestibular de envelhecimento. 

Vídeo: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Explorando Funções Cognitivas em Bebés, Crianças e Adultos com Espectroscopia no Infravermelho Próximo

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Biologia

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Oi-C: um método para estudar a arquitetura tridimensional de Genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células NAMALWA

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Na reconstituição in vitro da atividade T. Telomerase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Usando tomografia de tórax para visualizar a estrutura tridimensional e Função da Usina Vasculatura

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

Perfis de expressão de microRNA iPS Human Cells, Retinal Pigment Epithelium Derivado de Ips, e Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método de Avaliação de efeitos de uma gama de comprimentos de onda e intensidades de Terapia da Luz Vermelha / near-infrared sobre o estresse oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudar mitocondrial estrutura e função em<em&gt; Drosophila</em&gt; ovários

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Culturas de células primárias do Mouse epithelium Retinal do Pigment

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...