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Rotorod yürüme aparatı Rotorod dönen bir silindir, 4 cm, çapı 35 cm yerden sabit ve şeffaf pleksiglas çevrili idi. Rotorod silindir çekiş kolaylaştırır dokulu kauçuk kaplama, kaplıydı. Cihazların katında hayvanların düştü sırasında yaralanmaları önlemek için köpük tabakası ile kaplıydı. Küçük bir elektrikli motor, lastik bir bant ile rotorod açmak için güç sağladı. Rotorod her 5 saniyede bir kez döndürmek için kuruldu Video kayıtları ve kinematik 1000 ikinci bir deklanşör hızı kullanarak yüksek hızlı bir dijital video kamera, hayvanlar film kullanıldı. Hayvan rotorod yürürken ön, yan ve arka görünümleri çekildi. Iki kol Nikon, MII soğuk ışık kaynağı ve fiber optik ışıkların bir set kayıt sırasında yeterli ışık sağlamak için kullanılmıştır. Sony DSR 20 DV CAM güverte bantlar kullanarak kare kare incelendi. Video kareleri bir frame grabber ve Macintosh bilgisayar kullanarak ele geçirildi. Test prosedürü ve analiz 6 OHDA ve kontrolü sıçanlarda yürüme hareketleri arasındaki farklılıkları değerlendirmek için, ameliyat sonrası gün 15 video kayıtları - 30 (lezyon herhangi bir kurtarma büyük ölçüde tamamlanmış olması beklenebilir sonra), iki bağımsız tarafından incelenmiştir deneysel koşullara kör gözlemciler. 6-OHDA nigrostriatal lezyon, sol ön ve arka bacaklarda kalıcı bozuklukları sağ hemisfer sağlandı. Bir derecelendirme ölçeği oranı sıçan &#39;duruş ve ön ayakları atlama hareketleri için geliştirilmiştir (Whishaw ve ark, 2003). Step beş bileşen değerlendirildi: <ol><li> duruş, duruş derece tambur üzerinde sıçan konumunu inceleyerek tarafından yapıldı; </li><li> asansör ve açıklaması, pençe davul kaldırıldı adım döngüsünün kısmı; </li><li> sıçan ileriye taşındı uzuv tambur üzerinde gerçekleştirildi adım döngüsü kısmını taşımak; </li><li> avans, uzuv tambur ile temas yeniden uzatıldı erişemeyeceği bir kısmını; </li><li> yerleştirme, pençe davul yerleştirilmiş olduğu. </li></ol> Aşağıdaki kriterlere normal bir hareketi tanımlamak için kullanılmıştır: <ul><li> Postür: sıçan vücut kaldırdı kafa ile davul üzerine odaklı. </li><li> Asansör ve sürüm: davul yüzeyinden pençe yükselterek omuz, dirsek ve el bileği fleksiyonda. </li><li> Carry: pençe yüzeyi üzerinde davul düzenlenen basamak gevşek fleksiyonda, haneli ipuçları vücut orta hat üzerinde yoğunlaşmış. Avans: ekstremite semiflexed bir pozisyondan basamak uzun ve açık pozisyon hareketli ileri uzatılır. </li><li> Yerleştirme: bacak, omuz, dirsek, ve bilek uzatılmış olan ve rakamları farenin yaklaşık merkezinde bulunan haneli 2 (orta hat haneli ikinci), ileri ve tambur yüzeyine pençe yerleştirmek için açıldı . gövde. </li></ul> Her hareketi 3-noktalı bir ölçekte Oy: 1 = / hareket / hareketi biraz anormal ortaya çıkan bir kontrol sıçan, 0.5 benziyordu ve 0 = / hareketi açıkça anormal (Whishaw ve ark, 2003). Bu derecelendirme ölçeği, sonra beş adım postoperatif günlerde her sıçan hareketleri 1, 3, 5, 7, 9, ve 15 değerlendirmek için kullanılmıştır. Beş önlemler puanlar, her gün her bir sıçan için ortalama gün tekrarlanan bir tedbir olarak bir ANOVA sonuçlar tabi. 6-OHDA ve kontrolü sıçanlarda yürüme rotorod Genel Bakış Niteliksel hareket analizi için postoperatif test oturumları için hareket derecelendirme ölçeği uygulandı. Test oturumları sırasında, 6-OHDA lezyon grubu F (1,8) = 33.2, p &lt;0.001 kontrol grubuna göre anlamlı ölçüde daha düşük puanlar aldı, ama Test Günü anlamlı bir etkisi vardı, ne de önemli bir Grubu Test Gün etkileşim. Step puanlar daha sonra bir değişken (kontrol sıçanlar için, sol, sağ pençe vs pençe 6-OHDA sıçanlarda lezyona ipsilateral ön ayakları karşısında lezyona kontralateral ön ayakları) olarak ön ayakları ile ayrı ayrı analiz edildi. Ön ayakları, F (1,40) = 15.8, p &lt;0.01 önemli bir etkisi vardı ve ön ayakları Grup, F (1,40) = 15.0, p &lt;0.01 önemli bir etkileşim vardı. Takip t-testi, 6-OHDA grubunun kontralateral-lezyon ön ayakları ipsilateral lezyon ön ayakları veya farklı değildi kontrol sıçanlarda, ön ayakları göre anlamlı derecede düşük puanlar aldığını göstermiştir. Hareketin bileşenleri, 6-OHDA grup tarafından elde edilen yoksul puanları, duruş önlemler katkıda belirlemek için6-OHDA grup ipsilateral ve kontralateral ön ayakları adım döngüsü bileşenleri tek tek kontrol sıçanların elde edilen değerler ile karşılaştırılmıştır. 6-OHDA grup duruş ve kontralateral bacak tarafından yapılan atlama bileşenleri tüm farklıydı, ama ipsilateral ekstremite bileşenlerinin hiçbiri (p &lt;0.05). Niteliksel ve tanımlayıcı farklılıklar kontrolü ve 6-OHDA sıçanlarda duruş Bir kontrol sıçan, baş yatay konumda düzenlenen ve destekleyici ön ayakları ve arka bacaklarda, destekleyici bir kemer oluşturan davul binme. 6-OHDA sıçanlar genellikle kontrol sıçanların ipsilateral bacaklarda veya kol ve bacaklarda meydana gelen daha drum yüzeyine daha yukarıda engelli birbirlerine daha yakın konumlandırılmış ön ayakları (izleyici bakan) ve (bkz. kontrol sıçan video ile baş aşağı yürüdü Skor duruş için &quot;1&quot; ve &quot;0&quot; puan ile 6-OHDA lezyon hayvan). Tipik olarak, duruş anormallik, bir fare kafası göreli konumunu bir bakışta görülebilir. Ön ayakları step Kontrol dizisi, adım ön ayakları omuz, dirsek ve el bileği esneme kaldırdı. Bacak kaldırdı olduğu gibi, rakamlar gevşek fleksiyonda ve uzuv tambur yüzeyi üzerinde kaldırdı. Ekstremite gelişmiş, omuz, dirsek ve el bileği uzatılır ve yerleştirilir rakam genişletilmiş ve açık. Kontrol sıçanlara kıyasla, lezyon uzuv kontralateral DA-tükenmiş sıçanlarda tüm bileşenlerinin genlik zayıflatılmış. Uzuv, daha sonra davul bültenleri az kaldırdı ve daha kısa bir mesafe kontrolü ön ayakları daha ileri düzeydedir. Buna ek olarak, bacak kaldırıldığında, basamak daha fleksiyonda ve bacak yerleştirilir, kontrol sıçan göre daha az basamak genişletilmiş ve açılır. Davul üst ulaştığında kontrolü ön ayakları kaldırdı, bilek pençe ileri yukarı kaldırın ve fleksiyonda ve bilek, bacak gelişmeler olarak genişletilmiştir. Buna karşılık, 6-OHDA sıçan lezyon ön ayakları kontralateral tambur hareketi ile daha kaudal yapılan ve bu nedenle hala uzatılmış bilek ile daha sonraki sürümlerde. Bilek fleksiyon pasif olarak görünür ve tambur hareketi ile basamak geriye doğru yapılır ve ön ayakları öne çıkarılıyor, pençe ve önkol drum yüzeyine kapalı açıkça kaldırdı değildir oluşur. Bir kontrol sıçan ön ayakları iyice ileriye doğru uzatır ve rakamları ekstremite ilerledikçe açık. Bacak yerleştirilir, 4, 3, ve 2 basamak art arda takip, tambur ilk yüzey ile iletişime rakam 5, arpej hareketi ile konur. Buna karşılık, 6-OHDA sıçan bacak kaldırdı ve uzun süreli, daha az basamak daha genişletilmiş ve bir &#39;tokat&#39; ile hemen hemen aynı anda davul yüzey teması. Video &quot;1&quot; puan, ağırlıklı olarak &quot;0&quot; olarak skorlandı 6-OHDA lezyon hayvan ön ayakları adım hareketleri kontrol hayvan ön ayakları adım hareketleri gösterir. Adım Hind ekstremite 6-OHDA sıçanların kontralateral-lezyon arka bacaklarda engelli de vardı. Kontrolü arka bacak geriye doğru hareket ettikçe, dönüm davul karşı itmek için görünür ve öyle olduğu gibi, ayak parmakları dışa doğru döndürün. Bacak ileriye doğru hareket ettikçe, ayağı dışa doğru salladı. 6-OHDA sıçan arka bacak davul rotasyon ile doğrudan geriye doğru hareket eder ve eğer bir şey, biraz içe döner. Bacak ileriye doğru hareket ettikçe, ayak doğrudan ileriye yapılır. Kontrol sıçan salıncaklar dışa ve gelişmiş olarak, ayak, ayak bileği daha yüksek tutulur, ayak ve ayak uzatmak. Ilk davul ve kilo kontrolü sıçan yerleştirme, ayak 5 temaslarında yüzey, avuç içi ve ayak parmakları arasında dağıtılır. Buna karşılık, taşıma sırasında 6-OHDA sıçan ayak parmakları fleksiyonda ve yerleştirme arpej bir hareket olmadan yapılır. Kontrol sıçan ayak parmakları, ayak 5 ilk kaldırdı ve ayak 1 son kaldırdı ters arpej desen serbest bırakılır. Bu hareket, ayak bileği, taşıma hareketi sırasında duruş fleksiyondayken devam ederken, 6-OHDA lezyon rat eksikti. Yine 6-OHDA sıçan önceden hareketleri, az exorotated uzuv, zayıflatılmış az kaldırdı ve kontrol sıçan daha az gelişmiş. Kontrolü sıçan, ayak parmakları yer olarak, onlar sıçan vücut için 5 puan yanal göreceli gibi ayak genişletilmiş ve açılır. Toe 5 kontak ilk olarak, sıçan, ayak uçları ağırlık desteklediği gibi kalan ayak parmakları ardından davul. Ayak taşınan 6-OHDA sıçan, ayak parmakları biraz fleksiyonda, ayak parmakları, ayak bileğine kadar yatay düşük, ayak ayak tambur üzerine yerleştirilir az açılışı var. Adım uzunluğu da kontrol sıçanların biraz daha kısadır. Devam arasındaki farklılıkları ölçmek içinrol ve 6-OHDA arka bacak hareketleri, asansör ve serbest bileşenleri, terfi taşıma ve yerleştirme 3-noktalı bir ölçekte değerlendirildi, ön ayakları. Bu önlemler, cerrahi sonrası 20 günde yapılan video kayıtları yapılmış, ve üç adım döngüsü, her bir sıçan için değerlendirildi. Önemli Grup farklılıkları, F (1,8) = 47.6, p &lt;0.001 vardı. Bileşenleri takip t-testi, 6-OHDA sıçanların bileşenleri daha kontrol sıçanların tüm bileşenlerinin önemli ölçüde daha yüksek puan aldıklarını gösterdi. Video &quot;1&quot; olarak skorlandı kontrol hayvan arka bacak adım hareketleri gösterir. Video gösterilen 6-OHDA hareketleri adım Hind bacak ağırlıklı olarak &quot;0&quot; puan ile değerlendirildi.

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Bu çalışmanın amacı, kemirgen lokomosyon beceri rotorod testi duruş niteliksel inceleme, ön ayakları adımlama ve sinir sistemi yaralı sıçanlarda arka bacak adım için kullanılabilir olup olmadığını incelemektir. Analizi için, tek taraflı DA tükenişleri ve kontrol sıçanlarda fareler, ön, yan ve arka görünümleri kaydedilen video. Duruş ve ön ayakları hareketleri bir derecelendirme ölçeği adım hareketleri kronik cerrahi sonrası engelli olduğunu belirtti. Bacak hareketlerinin incelenmesi DA-tükenmiş sıça...

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified, and another reference was included.
Additional reference:
15. Whishaw IQ, Li K, Whishaw PA, Gorny B, Metz GA. Distinct forelimb and hind limb stepping impairments in unilateral dopamine-depleted rats: use of the rotorod as a method for the qualitative analysis of skilled walking. J Neurosci Meth 126, 13-23 (2003).
Revised Abstract:
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Additional Discussion Paragraph:
A more detailed presentation of the present study has been made (Whishaw et al, 2003), but the present study presents the video support describing the stepping movement in the good and affected limbs of unilateral dopamine-depleted rats. For the analysis, rats with unilateral DA depletions and control rats were video recorded from front, lateral and posterior views. A rating scale of posture and forelimb movements indicated that stepping movements were chronically impaired following surgery. Examination of limb movements indicated that whereas the DA-depleted rats could use the limbs contralateral to the lesion for support, they received minimal use for shifting weight. The results of this study indicate that the rotorod task, in addition to providing quantitative measures of motor impairments, can also provide insights into the qualitative impairments.
Original Abstract:
The rotorod test, in which animals walk on a rotating drum, is widely used to assess motor status in laboratory rodents. Performance is measured by the duration that an animal stays up on the drum as a function of drum speed. Here we report that the task provides a rich source of information about qualitative aspects of walking movements. Because movements are performed in a fixed location, they can readily be examined using high-speed video recording methods. The present study was undertaken to examine the potential of the rotorod to reveal qualitative changes in the walking movements of hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). Beginning on the day following surgery and then periodically over the next two months, the rats were filmed from frontal, lateral, and posterior views as they walked on the rotorod. Behavior was analyzed by frame-by-frame replay of the video records. Rating scales of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in their posture and in the use of the limbs contralateral to the DA-depletion. The contralateral limbs not only displayed postural and movement abnormalities, they participated less in initiating and sustaining propulsion than did the ipsilateral limbs. These findings not only reveal new deficits secondary to unilateral DA-depletion, but also show that the rotorod can provide a robust tool for the qualitative analysis of movement.

A correction was made to: <a href="http://www.jove.com/video/1030/">Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat</a>. An additional paragraph was added to the discussion, the abstract was modified and another reference was included.

Erratum: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

use of rotorod as a method for the qualitative analysis of walking in rat

Hayvanlar dönen bir tambur üzerinde yürümek rotorod testi, yaygın laboratuar kemirgenler motor durumunu değerlendirmek için kullanılır. Performans, bir hayvan, davul hızının bir fonksiyonu olarak davul üzerinde kalmasını süresi ile ölçülür. Burada görev yürüme hareketleri nitel yönleri hakkında bilgi zengin bir kaynak sağladığını bildirmektedir. Hareketleri sabit bir yerde yapılır çünkü, onlar hemen yüksek hızda video kayıt yöntemleri kullanılarak incelenir olabilir. Bu çalışmada (nigrostriatal dopamin tüketmek için, sağ nigrostriatal paket içine enjeksiyon, 6 hydroxydopamine (6-OHDA) tarafından üretilen hemi-Parkinson analog sıçan, yürüme hareketleri niteliksel değişiklikler ortaya çıkarmak için rotorod potansiyelini incelemek için yapıldı DA .) Gün cerrahi sonrası başlayan ve onlar rotorod üzerinde yürürken daha sonra periyodik olarak önümüzdeki iki ay içinde, fareler, ön, yan ve arka kez çekildi. Davranış kare-kare video kayıtları tekrar analiz edildi. Davranış adım Derecelendirme ölçekleri, hemi-Parkinson sıçanlarda kronik kendi duruş ve DA-tükenmesi kontralateral kol ve bacaklarda kullanımı engelli olduğunu belirtti. Sadece postural ve hareket anormallikleri görüntülenmez kontralateral bacaklarda, ipsilateral bacaklarda daha tahrik başlatılması ve sürdürülmesi daha katıldı. Bu bulgular, sadece tek taraflı DA tükenmesi ikincil yeni açıkları ortaya çıkarmak değil, aynı zamanda rotorod hareketin nitel analiz için güçlü bir araç sağlayabilir olduğunu göstermektedir.

Watch this Scientific Journal Video about Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat at JoVE.com

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Rotorod test hemi-Parkinson analog sıçanlarda yürüme hareketi motor durumunu değerlendirmek için kullanılır.

Rotorod Rat Yürüyüş Kalitatif Analiz için bir yöntem olarak kullanımı

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

use-rotorod-as-method-for-qualitative-analysis-walking

Research

JoVE Journal

Biology

12.9K Görüntüleme.  University of Lethbridge. Rotorod test hemi-Parkinson analog sıçanlarda yürüme hareketi motor durumunu değerlendirmek için kullanılır.

Video: Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Doğru Genotiplendirme için Basit Bir Yöntem

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biyoloji

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Görev Yürüyüş Merdiven Basamak: Puanlama Sistemi ve Pratik Uygulama.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Model Genetik Organizma olarak kullanmak için Medaka Embriyolar, Mikroenjeksiyon

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

Görüntüleme için Basit Bir Yöntem Arabidopsis Infiltratif Görüntüleme Orta Perfluorodecalin kullanarak bırakır.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Mikrobiyal Systems Biyoloji Chemostats Kullanımı

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Kanatlı Üretim Örneklerden Bakteriyel Topluluklar Nitel ve Nicel Değerlendirme Karma DNA Ekstraksiyon Yöntemi

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...