Yazarlar mRNA çıkarma ve PCR ile yardım için Dr. Paul Witting ve Bayan Genevieve Fong kabul etmek isteyen, ve destek için Garnett Passe ve Rodney Williams Memorial Vakfı.

Etik Beyanı: Aşağıda belirtilen tüm işlemler Sydney Hayvan Etik Kurulu Üniversitesi tarafından onaylanmıştır. 1. Hayvanlar NOT: 1 ve 8 - 9 aylık fareler (C57 / BL6) Hayvan Kaynakları Merkezi (Perth, Avustralya) elde edilmiştir. Fareler Sydney Üniversitesi&#39;nde Bosch Kemirgen Tesisinde yerleştirildiler. <ol><li> Gıda ve su ad libitum erişimi olan bir 12/12 saat ışık / karanlık döngüsünde standart fare kafesleri Ev fareleri. </li><li> Yakın kızılötesi her bir yaş grubunda Böl fareler (NUR) tedavi, ya da sahte karşılaştırma için (kontrol) gruplar muamele. </li></ol> 2. yakın kızılötesi (NIR) Işınlama ve Sham Tedavisi <ol><li> Tedavi rejiminin tamamlanmasından önce saç hızlı yeniden büyümesini önlemeye mümkün olduğunca yakından bir elektrikli ustura ile fare baş ve boyun bölgesinde kürk Tıraş. Farelerin patojensiz durumunu muhafaza etmek için,% 70 etanol kullanımıtıraş hayvanlar ve ayrı kafeslere gelen hayvanlar arasındaki F10 veteriner dezenfektan arasındaki aletleri temizlemek için. Hayvanlar üzerinde saplı değildir bu yüzden tedaviye başlamadan 3 gün önce - bu 2 yapın. </li><li> Kuyruk proksimal ucunu tutarak fare dizginlemek ve yanlış sonuçlara yol açabilir stresi en aza indirecek şekilde o bir el ya da tezgah üstünde avuç içinde dinlenmek için izin verir. </li><li> LED Nir (670 nm) (ışık yayan diyot) 1 Cihazı tutun - 2 cm uzakta maruz (traş) alan ve 90 saniye (Şekil 1A) için cihazı açın. NOT: 90 saniye maruz kalmanın bir sonucu olarak sıcaklık değişimi, 100 ml su içinde 0.2 ° C &lt;olarak ölçülmüştür. </li><li> Hayvanların sahte tedavi grubu için 2.3 ancak aygıt kapalı (Şekil 1B) bırakın - Tekrar 2.2 adımları. </li><li> Hayvanların Nir-bloke tedavi grubu için 2.3 ama alüminyum folyo ile cihazı kapak - yineleyin 2.2 adımları. </li><li> Approximat günlük 2.5 - 2.2 tekrarlayın adımlarıbirbirini takip eden 5 gün süre ile 24 saat aralıklarla, e. </li><li> Beşinci gün tedavi sonrası her iki kulaklarından vestibüler duyu epitel (3 krista ve 1 utricular makülayı) Extract (aşağıdaki Bölüm 3&#39;e bakın). </li></ol> 3. Doku Ekstraksiyon 20 <ol><li> 26 NaHCO 3, 11 glukoz, 250 gliserol, 2.5 KCl, 1.2 NaH 2 PO 4, 1.2 MgCl2, ve 2.5 CaCl2 (mM olarak) oluşan, bir gliserol bazlı yapay serebrospinal akışkan (ACSF) 300 ml hazırlayın. CaCI2 ilave edilmeden önce, 7.4 bir pH değeri ve kalsiyum çökelmesi (bulanıklık) önlemek için karbojen (% 95 O2 ve% 5 CO2) ile çözelti gaz. Bir buz bulamaç oluşturulur, böylece, 45 dakika için -80 ° C dondurucu içinde bir çözüm soğutun. </li><li> Etiketli vida üst mikrotüplerde RNA izolasyonu lizis tamponu hazırlayın (kapak haşhaş durduğunda donma ve sıvı azot içinde çözülme arasındaki boru basınç değişiklikleri) göreüretici veya olağan laboratuar uygulamalarının talimatlarına. Bir alüminyum Dewar şişede sıvı azot hazır bulundurun. NOT: Sıvı azot kullanırken koruyucu kıyafet ve Eyeware kullanın. Doğal gaz formunda hacmi yaklaşık olarak sıvı halde 700 katıdır ve boğulmaya neden olabilir gibi sıvı nitrojen iyi havalandırılmış bir odada kullanılan emin olun. </li><li> Derin bir intra-peritonal enjeksiyon yoluyla ketamin (400 mg / kg) ile fareler uyuşturan. Arka-bacak refleks tamamen fareler tamamen anestezi olduğu göstergesi olarak düşmesi. </li><li> Keskin paslanmaz çelik makas ile fareler başını kesmek ve bir jilet kullanarak kafatasının sagital cilt boyunca bir kesi yapmak (# 22 yuvarlanmış). Bu noktada ve genelinde 3.5 adımları - 3.9, doku üzerinde buz soğuk ACSF düzenli uygulanması ile mümkün olduğunca serin kafatası tonoz, beyin, ve altta yatan vestibüler aparat tutun. </li><li> Bir hızlı s yapmak standart desen makas sivri kolunu kullanmal lambda kafatası insizyon ve sagital sütür boyunca kesilir. </li><li> Yavaşça beyin sürükleyerek olmadan kemik alt yüzeyine mümkün olduğunca yakın bıçak tutarak parietal kemikte altında sığ rongeurs bir kolunu kaydırın. Güvenli ve beyin maruz kadar posteriora yanal parietal kemik ve oksipital kemik uzak çekin. </li><li> Bir küçük paslanmaz çelik spatula kullanın ve vestibülokohlear sinir (CN VIII) maruz uzakta anterior ve orta kranial fossa beyni kaldırın. Doğrudan vestibüler saç hücrelerini innerve primer afferent aksonlar gereksiz gerilimi önlemek için sinir transect. </li><li> CN VIII kesisi sonrasında toto beyin çıkarın. </li><li> Koklea ve orta kranial fossada periferik vestibüler organları içeren kemik labirent gözlemleyin. Her kemik labirent yanında iki küçük kesiler yapmak ve ön semisirküler kanal tutan ve daha sonra çekerek tüm yapıyı tüketimmüttefik. </li><li> Hemen sürekli karbojen ile perfüze ise (aşama 3.1&#39;de tarif edildiği gibi), buz soğukluğunda ACSF çözeltisini ihtiva eden bir kesme çanak kesilerek çıkarılmış labirentler bırakın. </li><li> Steromikroskopla, koklea ile labirenti basılı tutun ve forseps ile çanak altına sabitleyin. <ol><li> Ön semisirküler kanal (SSC) ampulla üstünde kemik küçük bir açıklık kazımak için düz ince forseps kullanın. </li><li> Yavaşça hemen kemik altına ulaşan ve dışa uzak ampulla gelen iterek bu açılış büyütmek. Ağzına forseps itin ve aşağıda kırılgan membranöz labirent zarar değil burada dikkatli. Kesecik, ön ve yan ampülleri tüm maruz kadar bu şekilde devam ediniz. Mümkünse de arka ampulla çıkarın. </li></ol></li><li> Ince forseps kullanarak, hafifçe kaldırın ve kırbacık tamamen müstakil kadar kemik labirent uzak ampülleri. Mümkünse, onları tutmakilişkili semisirküler kanallar tarafından duyusal epitel zarar vermemek için. Bazı durumlarda, yarım daire membranöz kanalın proksimal kısmı kemikten ampülleri serbest bırakmak için iris makas ile kesilebilir gerekebilir. </li><li> Güvenli adımda 3.2 önceki hazırlanan lizis tamponu içine forseps ucu ile yer arasındaki vestibüler organları kavramak. Yavaşça vestibüler organları forseps kopuk emin olmak için tampon etrafında forseps girdap. Bu stereomicroscope altında forseps getirerek durumda kontrol edin. <ol><li> Mikrotübül kapağındaki vida ve hemen sıvı nitrojen içinde örnek dondurma. </li></ol></li></ol> 4. RNA Ekstraksiyonu ve RT-PCR <ol><li> Üreticinin talimatlarına veya tercih edilen laboratuvar protokollerine göre mesajcı RNA (mRNA) ekstre standart yöntemleri takip edin. NOT: mRNA küçük verim yalıtmak için taşıyıcı RNA&#39;lar kullanılan bir ticari kit bu kullanıldıprotokol. Alt elüsyon hacimleri mRNA nihai konsantrasyonlarını arttırmak için kullanılabilir. NOT: Negatif &quot;hayır enzim&quot; denetimleri (UEM) ve &quot;hayır DNA şablonu&quot; denetimleri (NTSC) da gözlenen etkilerin geçerliliğini sağlamak için tamamlanması gereken. </li><li> Hedef genlerin 21-23 tamamlayıcı DNA (cDNA) ve büyütme için mRNA&#39;nın ters transkripsiyon standart yöntemler uygulanır. </li></ol>

<ol>
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Yazarlar hiçbir rakip mali çıkarlarını var beyan.

Burada açıklanan temsili sonuçlar NIR ışık kısa transkraniyal dağıtım (5 gün boyunca 90 sn / gün) plasebo ile tedavi edilen fareler ile karşılaştırıldığında, daha yaşlı farelerin antioksidan ifade seviyelerini yükseltmek için yeterli olduğunu göstermektedir. Nir yaydığı ısı bizim ölçüm cihazı &lt;0,2 ° C 90 sn bitmişti LED ve gibi neden düşüktür - yayılan ısı mitokondriyal ve / veya nöronal aktivasyon kaynağı temsil edebilir ederken, sıçan vestibüler için bildirilen <su...

Denge performansını Azalan ve sonraki düşüşler yaygındır ve doğal yaşlanma 1 maalesef sıklıkla tanımlayan özellikler. Bu düşüşün etkisi fiziksel hem de sosyal olmak, ve önemli ölçüde yaşlı insanlar için yaşam kalitesini düşürür yapabilirsiniz. Buna karşılık, fizik tedaviler ve rehabilitasyon düşme içine araştırma odağı olmuştur ama tekrarlanan düşme yaygınlığı tutarlı azalma ile ilişkili değil. Aynı zamanda, iş periferik veya santral vestibüler sistemi (dengesini korumak için sorumlu sistem) kıt olduğu ve bu sistemleri hedefleyen potansiyel tedavi stratejileri değişiklik ve sınırlı dengesizliğin altında yatan nedenleri araştırmak. Yaşla ilgili maküler dejenerasyon, 2-4, Alzheimer hastalığı modelleri 5-8, ve Parkinson hastalığı dahil olmak üzere, 9-12 yaş ile ilişkili nörodejeneratif hastalıkların üzerine yapılan son çalışmalar si nöro-koruyucu etkiler göstermiştiryakın kızılötesi (NIR) ışık mple non-invaziv bir uygulama. Bundan başka, denge sisteminin, NIR in vitro 13 vestibüler birincil aferent nöronların etkinliğini geliştirmek için kullanılmıştır. Nir ışık mekanizması iyi anlaşılamamakla birlikte, Nir kullanarak çalışmaların çoğu Nir hücresel metabolizmayı kolaylaştırmak için mitokondri karmaşık IV (sitokrom-c oksidaz) 14-17 uyardığını ileri sürmüşlerdir. Vestibüler duyu epitel tip I saç hücrelerinin subkutiküler plaka mitokondri 18 yoğun olduğu ve gibi terapötik Nir tedavisi için bir eylem siteyi temsil edebilir. Burada, transcranially kısa bir invazif olmayan bir tedavi rejimi hücresel metabolizmayı ölçmek için kullanılabilir (ve dolaylı mitokondriyal fonksiyonu olarak), fare vestibüler duyusal epitel anlatılan NIR uygulanır. Ayrıca ele alınan vestibüler duyusal epitel bir preparattır ve NIR bir ubiquito ekspresyonunu arttırdığı gösterilmiştirBize Anti-oksidanın, duyusal epitel (süperoksit dismutaz 1) - daha önce koklea saç hücre hayatta kalma 19 için önemli olduğu gösterilmiştir.

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

Artan yaş ile denge fonksiyonu düşüş zayıflatmaya yönelik stratejiler denge görevleri ve egzersiz gibi fiziksel tedaviler ağırlıklı odaklanmış bulunmaktadır. Ancak, bu yaklaşım denge düşüş yatan nedenleri ele yoktur. Fareler kullanarak, vestibüler duyu epitel hücrelerin metabolizması üzerine yakın kızılötesi ışığın etkisi (NUR) değerlendirildi. Toplanan veriler bu basit ve güvenli bir müdahale doğal yaşlanma zararlı etkilerinden bu hassas hücreleri koruyan olabileceğini göstermektedir. mRNA izole vestibüler duyusal epiteli (krista ampullaris ve utricular makula) elde edilir ve daha sonra, bir cDNA kitaplığında transkribe edilmiştir. Bu kütüphane, daha sonra her yerde antioksidan (SOD-1) ifadesi için problanmıştır. Antioksidan gen ifadesi hücre metabolizmasını ölçmek için kullanıldı. Genç Nir ve transkranial teslim (4 hafta) ve üzeri kullanma - 5 d (8 9 ay) fareler ve kısa bir tedavi rejimi (90 sn / günays), bu çalışma vestibüler duyusal epitel mitokondriyal fonksiyonu geliştirmek için yeterli olabilir ve yalnız NIR göstermektedir. Vestibüler saç hücre fonksiyonunu artırmak için tedavinin hiçbir mevcut, uygun fiyatlı, non-invaziv yöntemler şu anda olduğundan, dış NIR radyasyon uygulaması vestibüler duyu epiteli inthe hücresel metabolizma üzerinde yaşlanma etkilerini ortadan kaldırmak için potansiyel bir strateji sağlar.

Genç NIR tedavi etkisini (4 hafta) ve daha büyük karşılaştırılması - fareler (8 9 ay) (n = 20) (n = 16), genç antioksidan süperoksit dismutaz 1 (SOD-1) ifadesini ölçülür ve eski plasebo ile tedavi edilmiş, NIR-muamele ya da NIR-bloke fareler. Şekil 2, genç plasebo ile tedavi edilen hayvanlara kıyasla NIR tedavi genç hayvanlarda en fazla 2 misli β-aktin normalize SOD-1 ekspresyonunda önemli bir artış olduğunu göstermektedir (p &lt;0.01) ve genç Nir-bloke hayvanlar (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

Mitokondriyal disfonksiyon hücresel yaşlanma bir özelliğidir. Bu kağıt yaşlanma fare vestibüler duyu epitel mitokondriyal fonksiyon geliştirmek için non-invaziv yakın kızılötesi (NIR) tedavi kullanır.

Yakın Kızılötesi (NIR) Işık Fare Vestibüler Duyu Epitelinin Mitokondriyal Fonksiyonun bir Marker Anlatım artırır

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 Görüntüleme.  University of Sydney.  Mitokondriyal disfonksiyon hücresel yaşlanma bir özelliğidir. Bu kağıt yaşlanma fare vestibüler duyu epitel mitokondriyal fonksiyon geliştirmek için non-invaziv yakın kızılötesi (NIR) tedavi kullanır. 

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.

Yakın Kızılötesi Spektroskopisi ile bebekler, Çocuk ve Yetişkin Bilişsel Fonksiyonları keşfetmek

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

Biyoloji

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.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

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: genomların Üç boyutlu Mimari Eğitim Bir Yöntem.

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.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

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.

Active T. in vitro Sulandırma castaneum Telomeraz

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.

Bitki damarları ve Üç Boyutlu Yapı ve Fonksiyon Visualize&#39;a Yüksek Çözünürlüklü Bilgisayarlı Tomografi Kullanımı

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.

İnsan iPS Hücreleri MicroRNA İfade Profilleri, iPS Kaynaklanan Retina pigment epiteli ve Fetal Retina pigment epiteli

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.

Dalga boyundaki ve Oksidatif Stres Kırmızı / yakın-kızılötesi ışık Terapisi şiddetler bir Range Etkilerinin Değerlendirilmesi için Yöntemi<em&gt; İn 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.

Mitokondriyal Yapısı ve İşlevi incelenmesi<em&gt; Drosophila</em&gt; Yumurtalıklar

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.

Fare retina Pigment epiteli birincil hücre kültürleri

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