Gli autori desiderano ringraziare il Dr. Paul Witting e la signora Genevieve Fong per la loro assistenza con estrazione mRNA e PCR, e Garnett Passe e Rodney Williams Memorial Foundation per il supporto.

Etica Dichiarazione: Tutte le procedure descritte qui di seguito sono stati approvati dalla University of Sydney Comitato etico degli animali. 1. Gli animali NOTE: 1 e 8-9 mesi topi (C57 / BL6) sono stati ottenuti dalla Animal Resources Centre (Perth, Australia). I topi sono stati alloggiati in Facility Bosch Roditore presso l&#39;Università di Sydney. <ol><li> Casa topi in gabbie standard del mouse su un 12/12 luce hr / ciclo scuro con accesso a cibo e acqua ad libitum. </li><li> Topi Divide in ogni fascia d&#39;età in vicino infrarosso (NIR) trattati, o sham trattati (controllo) gruppi per il confronto. </li></ol> 2. vicino infrarosso (NIR) irradiazione e Sham trattamento <ol><li> Accorciare il pelo sulla regione della testa e del collo del mouse con un rasoio elettrico più vicino possibile impedire rapida ricrescita dei capelli prima del completamento del regime di trattamento. Per mantenere lo stato privo di patogeni dei topi, utilizzare etanolo al 70%per pulire strumenti tra gli animali da trucco e F10 veterinaria disinfettante tra animali di gabbie separate. Fare questo 2 - 3 giorni prima l&#39;inizio del trattamento in modo gli animali non sono più manico. </li><li> Trattenere il mouse tenendo l&#39;estremità prossimale della coda e permettono di rilassarsi nel palmo di una mano o superiore del banco di modo da ridurre al minimo lo stress che potrebbe portare a risultati falsi. </li><li> Tenere il NIR (670 nm) LED (light emitting diode) Dispositivo 1 - 2 cm di distanza dal (rasato) area esposta e accendere il dispositivo per 90 secondi (Figura 1A). NOTA: La variazione di temperatura a seguito di esposizione 90 sec stata misurata come &lt;0,2 ° C in 100 ml di acqua. </li><li> Ripetere i passaggi 2,2-2,3 per il gruppo di trattamento sham degli animali, ma lasciare l&#39;apparecchio spento (Figura 1B). </li><li> Ripetere i passaggi 2,2-2,3 per il gruppo di trattamento Nir-bloccato di animali, ma coprire il dispositivo con un foglio di alluminio. </li><li> Ripetere i passaggi 2,2-2,5 giorni alle approximate 24 intervalli hr per 5 giorni consecutivi. </li><li> Estrarre l&#39;epitelio vestibolare sensoriale (3 creste e 1 macula utricular) da entrambe le orecchie per il quinto giorno dopo il trattamento (vedere paragrafo 3). </li></ol> 3. Tissue estrazione 20 <ol><li> Preparare 300 ml di un liquido cerebrospinale artificiale (ACSF) basato glicerolo-costituiti (in mM): 26 NaHCO3, 11 glucosio, 250 glicerolo, 2,5 KCl, 1,2 NaH 2 PO 4, 1.2 MgCl 2, 2,5 e CaCl 2. Prima dell&#39;aggiunta di CaCl 2, gas la soluzione con CarboGen (95% O 2 e 5% CO 2) per stabilire un pH di 7,4 e di evitare la precipitazione di calcio (nuvolosità). Raffreddare la soluzione in un -80 ° C freezer per 45 minuti in modo da formare una sospensione di ghiaccio. </li><li> Preparare il tampone di lisi isolamento RNA etichettati microtubi tappo a vite (ferma il popping dei coperchi, quando le variazioni di pressione del tubo tra congelamento e scongelamento in azoto liquido) in basele istruzioni del fabbricante o usuali pratiche di laboratorio. Avere azoto liquido pronto in un matraccio dewar alluminio. NOTA: Usare indumenti protettivi e eyeware durante la manipolazione di azoto liquido. Assicurarsi che l&#39;azoto liquido viene utilizzato in una stanza ben ventilata come il volume della sua forma di gas è di circa 700 volte quella della sua forma liquida e può causare asfissia. </li><li> Profondamente anestetizzare topi con ketamina (400 mg / kg) tramite una iniezione intra-peritoneale. Lasciare che il riflesso arti posteriori a placarsi del tutto come indicazione che i topi sono completamente anestetizzati. </li><li> Decapitare topi con le forbici in acciaio inossidabile affilate e fare un&#39;incisione lungo la pelle sagittale del cranio con una lama di rasoio (arrotondato # 22). A questo punto e tutto passi 3,5-3,9, mantenere il più fresco possibile, l&#39;applicazione regolare di ACSF ghiacciato sopra il tessuto della volta cranica, cervello e apparato vestibolare sottostante. </li><li> Utilizzando il braccio punta di forbici modello standard fare un small &#39;incisione nel cranio a Lambda e tagliare lungo la sutura sagittale. </li><li> Scorrere delicatamente un braccio di un superficiale rongeurs sotto l&#39;osso parietale mantenendo la lama più vicino possibile alla superficie inferiore dell&#39;osso senza trascinare il cervello. Sicuro e tirare via l&#39;osso parietale laterale e l&#39;osso occipitale posteriormente fino a quando il cervello è esposto. </li><li> Utilizzare una piccola spatola di acciaio inox e sollevare il cervello dal anteriore e centrale fossa cranica per esporre il nervo vestibolococleare (CN VIII). Transetto il nervo per evitare inutili tensioni sui assoni afferenti primari che innervano direttamente le cellule ciliate vestibolari. </li><li> Rimuovere il cervello in toto dopo resezione del CN VIII. </li><li> Osservare il labirinto osseo contiene la coclea e gli organi vestibolari periferici nella fossa cranica media. Fare due piccole incisioni accanto a ogni labirinto osseo e asportare l&#39;intera struttura tenendo il canale semicircolare anteriore e tirare tardialleato. </li><li> Immergere immediatamente labirinti escisse in un piatto dissezione contenente soluzione ACSF ghiacciata (come descritto al punto 3.1), mentre continua perfusione con CarboGen. </li><li> Sotto uno stereomicroscopio, tenere premuto il labirinto della coclea e fissarla al fondo del piatto con una pinza. <ol><li> Utilizzare pinze dritto fini graffiare una piccola apertura nel midollo sopra anteriore canale semicircolare (SSC) ampolla. </li><li> Allargare delicatamente questa apertura raggiungendo immediatamente sotto l&#39;osso e sfogliando verso l&#39;esterno lontano dal ampolla. Fare attenzione qui per non spingere pinze nell&#39;apertura e danneggiare il labirinto membranoso fragile sotto. Continuare in questo modo fino a quando il utricolo, anteriore e ampolle laterale sono tutti esposti. Se possibile rimuovere anche l&#39;ampolla posteriori. </li></ol></li><li> Utilizzando una pinza sottile, sollevare delicatamente il utricolo e ampolle dal labirinto osseo fino a quando non sono completamente staccati. Ove possibile, tenerlidai canali semicircolari associati per evitare di danneggiare l&#39;epitelio sensoriale. In alcuni casi, la parte prossimale del condotto membranoso semicircolare può avere bisogno di essere tagliato con forbici iris per rilasciare le ampolle dall&#39;osso. </li><li> Saldamente cogliere gli organi vestibolari tra la punta di una pinza e posto nel buffer di lisi preparata in precedenza al punto 3.2. Mescolare delicatamente la pinza intorno nel buffer per garantire organi vestibolari hanno staccato dalla pinza. Controllare questo è il caso per portare le pinze con lo stereomicroscopio. <ol><li> Avvitare sul coperchio microtubuli e congelare il campione in azoto liquido immediatamente. </li></ol></li></ol> 4. RNA Estrazione e RT-PCR <ol><li> Seguire metodi standard di RNA messaggero (mRNA) di estrazione secondo le istruzioni del fabbricante o protocolli di laboratorio preferito. NOTA: un kit commerciale che utilizzava RNA carrier per aiutare isolare le piccole rese di mRNA è stato utilizzato in questoprotocollo. Volumi di eluizione inferiori possono essere usati per aumentare le concentrazioni finali di mRNA. NOTA: Controlli negativo &quot;no enzima&quot; (NEC) e controlli &quot;non modello DNA&quot; (NTC) devono essere completati per garantire la validità degli effetti osservati. </li><li> Applicare metodi standard di trascrizione inversa di mRNA a DNA complementare (cDNA) e l&#39;amplificazione di geni bersaglio 21-23. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

I risultati rappresentativi qui descritti mostrano che breve consegna transcranica di NIR luce (90 sec / giorno per 5 giorni) è sufficiente per aumentare i livelli di espressione antiossidante nei topi anziani rispetto ai topi sham trattati. Mentre calore emesso potrebbe rappresentare una fonte di mitocondriale e / o l&#39;attivazione neuronale, come riportato per ratto vestibolare afferenti 24 - nostra misurazione del calore emesso dal LED NIR dispositivo era &lt;0,2 ° C superiore al 90 sec, e come tale è...

Il calo delle prestazioni equilibrio e cadute successive sono comuni, e le caratteristiche, purtroppo, spesso definiscono di invecchiamento naturale 1. L&#39;impatto di questo declino può essere sia fisica che sociale, e riduce in modo significativo la qualità della vita per le persone anziane. In risposta, terapie fisiche e riabilitazione sono stati al centro della ricerca di cadute, ma non sono stati associati con la riduzione consistente della prevalenza di cadute ripetute. Allo stesso tempo, il lavoro indagando i cambiamenti nel sistema periferico o centrale vestibolare (il sistema responsabile per mantenere l&#39;equilibrio) è scarso, e potenziali strategie terapeutiche destinate a questi sistemi e le cause di squilibrio limitato. Un recente lavoro su malattie neurodegenerative legate all&#39;età, tra cui età-correlate degenerazione maculare 2-4, modelli di malattia di Alzheimer 5-8, e il morbo di Parkinson 9-12 hanno mostrato effetti neuroprotettivi di simple applicazione non invasiva del vicino infrarosso (NIR) luce. Inoltre, nel sistema vestibolare, NIR è stato usato per aumentare l&#39;attività di neuroni vestibolari afferenti primari in vitro 13. Mentre il meccanismo di Nir luce non è ben compreso,, maggior parte degli studi che utilizzano NIR hanno suggerito che Nir stimola mitocondri complesso IV (citocromo c ossidasi) 14-17 per facilitare il metabolismo cellulare. Nel epitelio sensoriale vestibolare piastra sottocuticolare delle cellule ciliate che tipo è denso in mitocondri 18 e come tale può rappresentare un luogo di azione per il trattamento terapeutico Nir. Qui, una breve, regime di trattamento non invasivo transcranially applicato NIR che può essere utilizzato per misurare il metabolismo cellulare (e di conseguenza, la funzione mitocondriale) nel topo vestibolare dell&#39;epitelio sensoriale descritto. Anche discusso è una preparazione dell&#39;epitelio sensoriale vestibolare e si dimostra che NIR aumenta l&#39;espressione di un ubiquitonoi anti-ossidante (superossido dismutasi 1) nell&#39;epitelio sensoriale - precedentemente dimostrato di essere importante per la sopravvivenza delle cellule dei capelli coclea 19.

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			<td height="63" style="height:63px;">Screw top microtubules</td>
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			<td height="63" style="height:63px;width:216px;">Ketamine</td>
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			<td height="21" style="height:21px;width:216px;">Friedman-Pearson Rongeurs</td>
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			<td height="42" style="height:42px;width:216px;">Stereo microscope</td>
			<td style="width:101px;">Leica Microsystems</td>
			<td style="width:119px;">A60S</td>
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			<td height="21" style="height:21px;width:216px;">Dumont #5 SF Forceps</td>
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			<td height="21" style="height:21px;width:216px;">Isolate II RNA Micro Kit</td>
			<td style="width:101px;">Bioline</td>
			<td style="width:119px;">BIO-52075</td>
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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Strategie per attenuare il declino in funzione di bilanciamento con l&#39;aumentare dell&#39;età sono prevalentemente concentrati su terapie fisiche, tra cui le attività di bilancio e l&#39;esercizio fisico. Tuttavia, questi approcci non affrontano le cause alla base dell&#39;equilibrio declino. Usando topi, è stato valutato l&#39;effetto di luce vicino infrarosso (NIR) sul metabolismo di cellule dell&#39;epitelio sensoriale vestibolare. I dati raccolti mostrano che questo intervento semplice e sicuro può proteggere queste cellule vulnerabili dagli effetti deleteri di invecchiamento naturale. mRNA è stato estratto dalla isolato epitelio periferico vestibolare sensoriale (ampollare crista e macula utricular) e successivamente trascritto in una libreria di cDNA. Questa libreria è stato poi indagato per l&#39;espressione di antiossidanti onnipresente (SOD-1). Antiossidante espressione genica è stato poi utilizzato per quantificare il metabolismo cellulare. Utilizzando consegna transcranica di Nir in giovane (4 settimane) e anziani (8-9 mesi) i topi, e un regime di trattamento breve (90 sec / giorno per 5 days), questo lavoro suggerisce Nir sola può essere sufficiente per migliorare la funzione mitocondriale nell&#39;epitelio sensoriale vestibolare. Dal momento che non ci sono attualmente disponibili, a prezzi accessibili, i metodi non invasivi di terapia per migliorare la funzione delle cellule dei capelli vestibolare, l&#39;applicazione delle radiazioni NIR esterna fornisce una strategia potenziale per contrastare l&#39;impatto dell&#39;invecchiamento sul metabolismo cellulare inthe vestibolare epitelio sensoriale.

Per confrontare l&#39;impatto del trattamento Nir nei giovani (4 settimane) e anziani (8-9 mesi) topi abbiamo misurato l&#39;espressione di antiossidanti superossido dismutasi 1 (SOD-1) nei giovani (n = 16) e anziani (n = 20) i topi che erano NIR-trattata, sham-trattati, o Nir-bloccato. figura 2 mostra un aumento significativo normalizzato SOD-1 β-actina di oltre 2 volte nei giovani Nir-animali trattati rispetto ai giovani animali sham trattati (p &lt;0.01) e animali giovani NIR-bloccati (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

Disfunzione mitocondriale è una caratteristica della senescenza cellulare. Questo documento utilizza nel vicino infrarosso (NIR) trattamento non invasivo per migliorare la funzione mitocondriale nel invecchiamento del mouse vestibolare epitelio sensoriale.

Vicino infrarosso (NIR) Luce aumenta l&#39;espressione di un marcatore di mitocondriale Funzione nel mouse vestibolare sensoriale 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 Views.  University of Sydney.  Disfunzione mitocondriale è una caratteristica della senescenza cellulare. Questo documento utilizza nel vicino infrarosso (NIR) trattamento non invasivo per migliorare la funzione mitocondriale nel invecchiamento del mouse vestibolare epitelio sensoriale. 

Video: 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.

Esplorare le funzioni cognitive in neonati, bambini e adulti con Near Infrared Spectroscopy

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

Ricerca

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

Hi-C: un metodo per studiare l&#39;architettura tridimensionale dei genomi.

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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.

Utilizzando ad alta risoluzione Tomografia Computerizzata Per visualizzare la struttura tridimensionale e funzione delle piante Vasculature

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.

MicroRNA Expression Profili di cellule iPS umane, dell&#39;epitelio pigmentato retinico derivate da iPS, e fetale dell&#39;epitelio pigmentato retinico

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.

Metodo per la valutazione degli effetti di un intervallo di lunghezze d&#39;onda e intensità di / vicino-infrarosso di terapia della luce rossa sullo stress ossidativo<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.

Studiare mitocondriale Struttura e funzione in<em&gt; Drosophila</em&gt; ovaie

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.

Colture cellulari primarie dall'epitelio retinico del pigmento del Mouse

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