Bu çalışma bir FAPESP hibesi (15/06477-9) tarafından desteklendi.

1. Maya toplam özü hazırlığı
<ol><li>1 L YNB-glu ortam (%2 m/v glikoz ile desteklenmiş Maya Nitrojen Temeli)9'da TAP etiketine kaynaşmış birleştirme faktörlerinden birini uygun amino asitler veya çekirdek bazlarla ifade eden maya hücrelerini büyütün, Bu durumda, adenin (20 μg/mL), lösin (30 μg/mL), triptofan (30 μg/mL), OD600 = 1.0'a kadar.</li>
	<li>1 L kültüründen hücreleri 17.000 x g'da üç adet 500 mL santrifüj şişesinde 4 °C'de 10 dakika santrifüjle toplayın ve 10 mL soğuk steril su ile iki kez yıkayın.</li>
	<li>Toplanan maya hücrelerini soğuk tampon A hücre hacminin 1/10'unda yeniden depolar (10 mmol L-1 HEPES pH = 7.9, 1.5 mmol L-1 MgCl2,50 mmol L-1 KCl, %5 v/v gliserol, 0.5 mmol L-1 DTT, EDTA içermeyen Proteaz inhibitör Kokteyli).</li>
	<li>Sıvı nitrojende küçük hücre süspansiyon damlalarını dondurun. Küçük damlalar, yeniden sulandırılmış hücre çözeltisinin pipetleilmesi ve 50 μL'nin doğrudan sıvı nitrojene düşmesiyle elde edilebilir. DİkKAT: Sıvı nitrojen cilt veya gözlerle temas halinde yaralanmalara neden olabilir. Uygun güvenlik ekipmanlarını kullanarak sapla. NOT: Protokol burada duraklatılabilir. Donmuş hücre süspansiyon damlaları -80 °C'de saklanabilir.</li>
	<li>3 dakika boyunca 20 Hz/s'de altı döngü boyunca bir Ball Mill cihazında öğüterek maya hücreleri lysates hazırlayın. NOT: Her döngüden sonra, erimeyi önlemek için donmuş hücreleri barındıran kabı sıvı nitrojene batırın. Protokol burada duraklatılabilir. Dondurulmuş özler -80 °C'de saklanabilir. Maya birleştirme özleri, sferoplastları10'u yutacak homojenizatörler kullanılarak veya harç ve pestle11,12kullanılarak da hazırlanabilir.</li>
	<li>İçerdiği tüpleri oda sıcaklığında suya yerleştirerek, ara sıra sallayarak özü eritin.</li>
	<li>Santrifüj, 4 °C'de 1 saat boyunca 45.000 x g'da santrifüj çıkarır.</li>
	<li>Temizlenen süpernatantın protein içeriğini BCA yöntemi ile ölçün13.</li>
	<li>Özlerin aliquotlarını hazırlayın, sıvı nitrojende hızlı bir şekilde dondurun ve -80 ° C'de saklayın. NOT: Protokol burada duraklatılabilir.</li>
</ol>2. Gliserol gradyan hazırlığı
<ol><li>Tampon A'da biri %10 v/v, diğeri %30 v/v gliserol içeren iki gliserol çözeltisi hazırlayın.</li>
	<li>Çapraz bağlama maddesi glutaraldehitini %30 v/v gliserol çözeltisine %0,1 v/v'ye ekleyin ve homojenize etmek için karıştırın. DİkKAT: Uygun güvenlik ekipmanlarını kullanarak duman kaputunun içindeki glutaraldehiti kullanın.</li>
	<li>12 mL santrifüj tüpünün (14 x 89 mm) altına 6 mL soğuk %10 v/v gliserol çözeltisi ekleyin.</li>
	<li>Tüpün altındaki Degrade Master cihazı ile sağlanan uzun bir iğneye bağlı bir şırınga ile glutaraldehit ile desteklenmiş soğuk 30% v / v gliserol çözeltisinin 6 mL'sini ekleyin, % 10 v / v gliserol çözeltisinin hemen altında. NOT: Alternatif olarak, %10 v/v gliserol çözeltisi% 30 v/v gliserol/glutaraldehit çözeltisinin üstüne dikkatlice pipetlenebilir.</li>
	<li>Sürekli yoğunluk degradesi oluşturmak için Degrade Ana aygıtını kullanın. NOT: Degrade Master cihazında, tüpler uygun bir rafa yerleştirilir ve parametrelerin belirlenmesi için üreticinin önerilerine (zaman/açı/hız) uyularak kısa bir süre döndürülür. Bu çalışmada kullandık: 2:25 dk/81.5°/11 rpm.</li>
	<li>Hücre özlerinin eklenmesinden hemen önce tepeye 200 μL% 7 v / v gliserol / tampon A yastığı ekleyin. NOT: Minderin gliserol konsantrasyonu, doğrusal gradyanı oluşturmak için kullanılan daha az konsantre gliserol çözeltisinden daha düşük olmalıdır.</li>
</ol>3. Santrifüjü çıkarır
<ol><li>Yastık ile her 12 mL lineer gliserol gradyanı 10% –30% glutaraldehitin üzerine yaklaşık 2 mg toplam protein yükleyin.</li>
	<li>Tüpleri önceden soğutulmuş bir salıncak kovası rotorasına yerleştirin. NOT: Ultrasantrifüjlemeden önce karıştırmayı önlemek için tüpleri hafifçe takın.</li>
	<li>4 °C'de 16 saat için 194.000 x g'da santrifüj.</li>
	<li>Aliquot her 12 mL tüp yirmi dört 500 μL fraksiyonlarda uyarlanmış bir EconoSystem kullanarak veya dikkatlice pipetleme ile. NOT: Uyarlanmış bir EconoSystem, peristaltik bir pompa, UV dedektörü ve tüp delinme cihazına bağlı fraksiyon toplayıcıdan oluşur. Çökeltme profili, emiciliğin 280 nm'de ölçülebilmesiyle izlenebilir.</li>
	<li>Glistaltik pompadan %40 v/v gliserol çözeltisi kullanarak gliserol%10-%30 gradyanı tüpten fraksiyon toplayıcıya itin. NOT: Protokol burada duraklatılabilir. Fraksiyonları kullanıma kadar -80 °C'de saklayın.</li>
	<li>Her kesirden 50 μL'yi doğrudan bir nokta lekesi veya yuva lekesi cihazına nitroselüloz zarlarına yükleyin. CBP'ye karşı antikor kullanarak immünoblot ile proteini tespit edin (TAP etiketinin CBP kısmını tespit etmek için 1:6.000) ve ikincil antikor olarak Horseradish Peroxidase (1:15.000) ile konjuge edilen tavşan karşıtı IgG.</li>
</ol>

<ol>
	<li>Kastner, B., Will, C. L., Stark, H., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+nuclear+pre-mRNA+splicing+in+higher+eukaryotes.">Structural insights into nuclear pre-mRNA splicing in higher eukaryotes.</a> Cold Spring Harbor Perspectives in Biology. 11 (11), 032417 (2019).</li><li>Yan, C., Wan, R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+pre-mRNA+splicing+through+structural+biology+of+the+spliceosome.">Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome.</a> Cold Spring Harbor Perspectives in Biology. 11 (1), 032409 (2019).</li><li>Cordin, O., Hahn, D., Beggs, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+regulation+of+spliceosomal+RNA+helicases.">Structure, function and regulation of spliceosomal RNA helicases.</a> Current Opinion in Cell Biology. 24, 431-438 (2012).</li><li>Kastner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GraFix:+sample+preparation+for+single-particle+electron+cryomicroscopy.">GraFix: sample preparation for single-particle electron cryomicroscopy.</a> Nature Methods. 5 (1), 53-55 (2008).</li><li>Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafix:+stabilization+of+fragile+macromolecular+complexes+for+single+particle+cryo-EM.">Grafix: stabilization of fragile macromolecular complexes for single particle cryo-EM.</a> Methods in Enzymology. 481, 109-126 (2010).</li><li>Yan, C., Wan, R., Bai, R., Huang, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+yeast+activated+spliceosome+at+3.5+&#197;+resolution.">Structure of a yeast activated spliceosome at 3.5 &#197; resolution.</a> Science. 353 (6302), 895-904 (2016).</li><li>Ohi, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+revelas+stable+multiprotein+complexes+in+both+fission+and+budding+yeasts+containing+Myb-related+Cdc5p/Cef1p,+novel+pre-mRNA+splicing+factors,+and+snRNAs.">Proteomics analysis revelas stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.</a> Molecular and Cellular Biology. 22 (7), 2011-2024 (2002).</li><li>Lourenco, R. F., Leme, A. F. P., Oliveira, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+yeast+mutant+RNA+exosome+complexes.">Proteomic analysis of yeast mutant RNA exosome complexes.</a> Journal of Proteome Research. 12 (12), 5912-5922 (2013).</li><li>Rigaut, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterizatioin+and+proteome+exploration.">A generic protein purification method for protein complex characterizatioin and proteome exploration.</a> Nature Biotechnology. 17, 1030-1032 (1999).</li><li>Cheng, S. C., Newman, A., Lin, R. J., McFarland, G. D., Abelson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+fractionation+of+yeast+splicing+extract.">Preparation and fractionation of yeast splicing extract.</a> Methods in Enzymology. 181, 89-96 (1990).</li><li>Umen, J. G., Guthrie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+a+U5+snRNP+protein+in+3'+splice+site+selection.">A novel role for a U5 snRNP protein in 3' splice site selection.</a> Genes &amp; Development. 9, 855-868 (1995).</li><li>Dunn, E. A., Rader, S. D., Hertel, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+yeast+whole+cell+splicing+extract.">Preparation of yeast whole cell splicing extract.</a> Spliceosomal Pre-mRNA Splicing: Methods and Protocols. Methods in Molecular Biology. 1126, 123-135 (2014).</li><li>Hill, H. D., Straka, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+determination+using+bicinchoninic+acid+in+the+presence+of+sulfhydryl+reagents.">Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.</a> Analalytical Biochemistry. 170 (1), 203-208 (1988).</li><li>Cepeda, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+assembly+factor+Nop53+controls+association+of+the+RNA+exosome+with+pre-60S+particles+in+yeast.">The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.</a> The Journal of Biological Chemistry. 294 (50), 19365-19380 (2019).</li><li>Lin, R. J., Newman, A. J., Cheng, S. C., Abelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mRNA+splicing+in+vitro.">Yeast mRNA splicing in vitro.</a> The Journal of Biological Chemistry. 260 (27), 14780-14792 (1985).</li></ol>

Yazarların çıkar çatışmaları yok.

Protein-protein ve ribonik asitler-protein etkileşimleri çapraz bağlama ajanları kullanılarak stabilize edilebilir. Elde eden kompleksin gliserol gradyanı üzerinde ultrasantrifüjasyona dayanacak şekilde kararlı olması önemlidir. Ayrıca, arabellek koşulları etkileşime izin vermeli, ancak belirli olmayan bağlamayı önlemek için yeterince katı olmalıdır. Burada gösterilen deneylerde, in vitro birleştirme reaksiyonları için önceden kurulmuş bir tampon çözeltisi kullandık15</su...

Birleştirme, çok sayıda faktörün koordineli bir şekilde bağlanmasını ve serbest bırakılmasını gerektiren son derece dinamik bir işlemdir. Bu birleştirme faktörleri arasında RNA bağlayıcı proteinler, ATPases, helicases, protein kinazları ve fosfatazlar, ubiquitin ligases, diğerleri arasında1,2,3; ve moleküler yeniden düzenlemelerin gerçekleşmesine izin vermek için, bu faktörlerin bazıları çok geçici olarak spliceosome alt komitelerine bağlanır ve bu RNP ara komplekslerinin izolasyonu ve tanımlanmasını çok zorlaştırır.
İşte, Grafix yöntemi4,5'i, maya birleştirme faktörü Cwc24'ün Bact complex6 ile etkileşimini stabilize etmek için kullandık, bu alt sıkıştırıcıya birlikte bağlı diğer faktörlerin tanımlanmasına izin vermek ve ubiquitin ligase Prp19, Cwc24'ün on dokuz (NTC) kompleksine bağlanmasında veya serbest bırakılmasında ve aktivasyondan önce intronun 5' ucuna kadar herhangi bir rol oynar ve ilk transesterifikasyon reaksiyonu alır yer. Makromolekülleri gliserol gradyanı boyunca artan bir çapraz bağlantı konsantrasyonuna maruz kalmanın avantajı, kompleksler arası çaprazbağlantılardan 4,5ve dolayısıyla agregaların oluşumunu önlemesidir.
Bu yöntem, büyük komplekslerin izolasyonuna izin vermesine rağmen, büyük dinamik kompleksler içinde geçici etkileşimleri sürdürmek için güvenilir olmayabileceği protein koimmunoprecipitation ve çekme tahlillerinin tamamlayıcısı olarak kullanılmıştır7,8. Gliserol gradyanındaki fiksasyon reaktiflerinin kullanımı, bu faktörlerin bağlanmasını stabilize ederek, belirli proteinlerin birleştirme altcomplexes ile etkileşimlerinin onaylanmasını sağlar. Seçilen çapraz bağlantı kimyasal olarak geri döndürülemez olduğundan, geri kazanılan fraksiyonlarda bulunan proteinler gradyan santrifüjlemeden sonra nokta lekesi ile analiz edildi.

<table>
	<tbody>
		<tr>
			<td>Anti-Calmodulin Binding Protein Epitope</td>
			<td>Millipore</td>
			<td>07-482</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-Rabbit IgG</td>
			<td>GE Healthcare</td>
			<td>NA934</td>
			<td></td>
		</tr>
		<tr>
			<td>EconoSystem</td>
			<td>Bio-Rad</td>
			<td>1-800-424-6723</td>
			<td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td>
		</tr>
		<tr>
			<td>EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Recovery System</td>
			<td>Beckman Coulter</td>
			<td>270-331580</td>
			<td>Tube-perforating device that was connected to the parts of the EconoSystem</td>
		</tr>
		<tr>
			<td>Gradient Master Model 107ip</td>
			<td>Biocomp</td>
			<td>107-201M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 200</td>
			<td>Retsch</td>
			<td>207460001</td>
			<td>Ball Mill device</td>
		</tr>
		<tr>
			<td>Rotor F12-6x500Lex</td>
			<td>Thermo Scientific</td>
			<td>096-062375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>36-101-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor P40ST</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge CP 80 NX</td>
			<td>Hitachi</td>
			<td>901069</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

Pre-mRNA birleştirme, karmaşık bileşenlerin montajı, RNA işlemesi ve serbest bırakılması sırasında spliceosome alt komkomplexlerinin birçok moleküler yeniden düzenlenmesini içeren çok dinamik bir süreçtir. Gliserol gradyan santrifüjleme fonksiyonel ve yapısal çalışmalar için protein veya RNP (RiboNucleoProtein) komplekslerinin ayrılması için kullanılmıştır. Burada, ilk olarak tek parçacıklı kriyo-elektron mikroskopisi için makromoleküler kompleksleri arındırmak ve stabilize etmek, spliceosome kompleksine geçici olarak bağlanan birleştirme faktörleri arasındaki etkileşimleri tanımlamak için geliştirilen Grafix'in (Gradyan Fiksasyonu) kullanımını açıklıyoruz. Bu yöntem, kompleksleri stabilize etmek için numunelerin artan bir sabitleme reaktif konsantrasyonuna santrifüjlenmesine dayanmaktadır. Gliserol gradyanlarına yüklenen maya toplam özlerinin santrifüjlenmesinden sonra, geri kazanılan fraksiyonlar spliceosome alt komplekslerinin tanımlanması ve bireysel birleştirme faktörlerinin varlığının belirlenmesi için nokta lekesi ile analiz edilir.

Cwc24-TAP'ın sedimantasyon profilini analiz etmek ve Grafix yönteminin birleştirme altkompanlarına bağlanmasını stabilize etmek için etkili olup olmadığını belirlemek için, Cwc24-TAP'ı ifade eden hücrelerin toplam maya özlerini gliserol gradyanları üzerinde santrifüjleme yoluyla, çapraz bağlama ajanı olarak glutaraldehitin varlığında veya yokluğunda ayırdık. Yirmi dört 500 μL fraksiyon örnekleri daha sonra TAP etiketinin CBP kısmına karşı antikor içeren yuva lekesi ile analiz edildi. S...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Burada, bir çapraz bağlayıcı varlığında gliserol gradyan santrifüjleme olan Grafix'in (Gradyan Fiksasyonu), spliceosome kompleksine geçici olarak bağlanan birleştirme faktörleri arasındaki etkileşimleri tanımlamak için kullanımını açıklıyoruz.

Saccharomyces cerevisiae Spliceosome Subcomplexes Geçici Interactors Tespiti için Grafix kullanımı

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

utilization-grafix-for-detection-transient-interactors-saccharomyces

Research

JoVE Journal

Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

3.5K Görüntüleme.  University of Sao Paulo. Burada, bir çapraz bağlayıcı varlığında gliserol gradyan santrifüjleme olan Grafix'in (Gradyan Fiksasyonu), spliceosome kompleksine geçici olarak bağlanan birleştirme faktörleri arasındaki etkileşimleri tanımlamak için kullanımını açıklıyoruz.

Video: Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against human leukocyte antigens (HLA). Histocompatibility guidelines are based on laboratory tests of anti-HLA immunity, which presents either as pre-existing or de novo generated HLA antibodies that constitute a major transplantation barrier. Current tests are built on a single-antigen beads (SAB) platform using a fixed set of ~100 preselected recombinant HLA antigens to probe transplant sera. However, in humans there exist a far greater variety of HLA types, with no two individuals other than identical twins who can share the same combination of HLA sequences. While advanced technologies for HLA typing and direct sequencing can precisely capture any mismatches in DNA sequence between a donor's and recipient's HLA, the SAB assay, due to its limited variety in sequence representation, is unable to precisely detect alloantibodies specifically against the donor HLA mismatches. We sought to develop a complementary method using a different technology to detect and characterize anti-donor HLA antibodies on a personalized basis. The screening tool is a custom peptide array of donor HLA-derived sequences for probing post-transplant sera of the organ recipient to assess the risk for antibody-mediated rejection. On a single array for one donor-recipient pair, up to 600 unique peptides are made based on the donor's HLA protein sequences, each peptide carrying at least one mismatched residue in a 15-amino acid sequence. In our pilot experiments to compare antigen patterns for pre- and post-transplant sera on these arrays, we were able to detect anti-HLA signals with the resolution that also allowed us to pinpoint the immune epitopes involved. These personalized antigen arrays allow high-resolution detection of donor-specific HLA epitopes in organ transplantation.

Mismatches in human leukocyte antigen (HLA) sequences between organ donor and recipient pairs are the major cause of antibody-mediated rejection in organ transplantation. Here we present the use of custom antigen arrays that are based on individual donors' HLA sequences to probe anti-donor HLA alloantibodies in organ recipients.

Peptid diziler HLA Alloantibodies Organ nakli olarak algılanması için kişiselleştirilmiş

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Biyokimya

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

RNA Interactors Protein kinaz memeli hücre döngüsü sırasında RNA-harekete geçirmek, eğitim

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

Çay (Camellia sinensis L.) türetilmiştir yeni kurulan hücre süspansiyon kültürü altı sistemik Böcek Ilacı metabolizması üzerinde çalışma Yaprak -ları

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

Rho kinase aktivitesinin geçici Inhibisyonu ile ınsan kaynaklı pluripotent kök hücreli türev Kardiyomiyositlerin engraftment artırılması

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Tandem Kütle Spektrometresi Ile Birleşen Sıvı Kromatografi kullanarak Saccharomyces Cerevisiae'nin Hücresel Lipidomukan Analizi

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

Bitki MikroRNAlarının Saptanması ve Ölçülmesi için RNA Leke Analizi

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Fırıncı Maya saccharomyces cerevisiae Mitokondriyal Genom İfade Ürünlerinin Etiketlenmesi ve Görselleştirilmesi

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

Sıvı KromatografiSi Kullanan Saccharomyces Cerevisiae'nin Nicel Metabolomikleri Tandem Kütle Spektrometresi ile Birleştiğinde

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Metabolik Etiketleme ve İnce Tabaka Kromatografisi ile Saccharomyces serevizisinde Nötr Lipid Sentezinin Analizi

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

Kriyo-Elektron Tomografisi için Saccharomyces cerevisiae'nin Hazırlanması ve Kriyo-FIB mikromakinesi

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...