Duennwald laboratuvarda İş Yaşlanma Araştırma (uzaktan), Kalıtsal Hastalık Vakfı (HDF) ve William Wood Vakfı Amerikan Federasyonu hibe ile desteklenmektedir.

1. Maya toksik PolyQ-genişleme Proteinlerin Anlatım Sistematik analizi maya 7 toksisite üretmek için gerekli olan bir polyQ-genişletme proteinin amino asit sekansı hassas oluşturmuştur. Bu toksik polyQ-genişletme proteini Şekil, Huntinğtin protein, bir polyQ bölgeye ve bir karboksi-terminal füzyon orijinal bir flüoresan sekansı protein (GFP veya CFP ya ila 17 amino asit, ardından bir amino-terminal bayrak-etiketi içerir 1. a). Indüklenebilir ve nispi kuvvetli GAL1 promotör 7, 9 kontrolü altında eksprese olduğunda glutamines 46 ya da daha fazla polyQ açılımı olan proteinlerin ifade (72 ve 103 glutamines gibi) maya toksisite üretir. Daha önce açıklandığı gibi, maya polyQ toksisitesi olan prion konformasyonundadır proteini Rnq1p taşıyan hücrelerin sadece açıktır, [RNQ +], örneğin, maya suşu W303 9. Toksik polyQ-genişleme protein rBöyle polyQ boyu bağımlı toksisite (aşağıya bakınız) ve kümeleme (Şekil 1 b) maya polyQ-biyoloji ecapitulates merkezi yönleri. Özellikle, toksik polyQ-genişleme proteinler hemen maya hücrelerini öldürmek değil; onlar yerine bozan veya hücre döngüsü ve hücre devision tutuklu, böylece plaklar veya sıvı kültürler (bizim yayınlanmamış bulgular) maya kolonisi büyümenin yavaşlaması ya da engellenmesi. 2. Maya Hücreleri ile Potansiyel Sorunları Toksik PolyQ-genişleme Proteinler ifade Aksi takdirde zehirli polyQ genişleme proteinleri Maya hücreleri herhangi bir büyüme kusuru 9 göstermeyebilir. PolyQ-toksisite bu bastırıcılarının genetik yapısı basit Mendel mutasyonlara dayalı görünmüyor ve dolayısıyla nispeten yüksek frekans (bizim yayımlanmamış sonuçlar) ile oluşabilir. Biz bu spontan bastırıcılarının benzer için [RNQ +] polyQ toxicity.Thes belirlenmesinde işlev tanımlanamayan prionlar kür neden olduğu spekülasyonpolyQ toksisite e spontan bastırıcılarının polyQ toksisite karakterize etmek ya da tanımlamak ve polyQ toksisite değiştiriciler karakterize amaçlayan bir deney başarılı sonuç tehlikeye sokabilecek. Bu spontan bastırıcılarının sık görülmesi önlemek için, çok etkili olduğu kanıtlanmış aşağıda belirtilen önlemler, izleyin: <ol><li> Toksisite denemeleri için taze maya hücreleri kullanın. Uzun süre için maya saklamayın. Sıkça dondurulmuş stokları taze maya kolonileri almak. </li><li> Sıkça floresan mikroskop ile ifade ve toksik polyQ-genişleme proteinlerin agregasyonu izlemek. </li><li> Herhangi bir zehirlenme ölçümler başlamadan önce her zaman toksik polyQ toksisite (tek karbon kaynağı olarak glukoz içeren seçici ortam yani) ifadesi bastırmak medyasında maya hücreleri tutun. </li><li> Her polyQ-toksisite deneyi için en az üç bağımsız transformantlar kullanın. &lt;/ Li&gt; </ol> 3. Büyüme Tahliller <ol><li> Her deneysel koşul için, tek karbon kaynağı olarak glukoz ile seçici maya medya 3 ml toksik polyQ-genişleme protein barındıran maya hücrelerinin üç bağımsız transformatlardan her biri koloni aşılamak. </li><li> 30 gece boyunca bu kültürlerin inkübe ° C Ortamda glukoz bunların ifadesi bastıran, çünkü bu koşullar altında, hücrelerin polyQ-genişletme proteini ifade değildir. Bu kültürlerin büyümek izin vermeyin; 1 Aşağıdaki gece kültürlerin OD 600 (600 nm dalga boyundaki ışık absorbans) tutmak. </li><li> Biz rutin olarak toksik polyQ-genişleme proteini ifade maya hücrelerinin büyümesini kusurları izlemek için üç farklı testler kullanabilirsiniz: </li></ol> Plakalar üzerine 3.1 Büyüme <ol><li> Bana seçici 0.0005 bir OD 600 (yani bir OD600 = 0.5 kültürün bir 1:1000 dilüsyon) için gece maya kültürleri (220 rpm çalkalama ile yetiştirilen) sulandırınızdia 7 içeren glikoz. </li><li> Eşit seçici tek karbon kaynağı olarak glukoz içeren orta ve tek karbon kaynağı olarak galaktoz içeren seçici besiyeri ile bir levha ile bir tabak (10 cm çapında) her seyreltilmiş kültürün 50 ul (ca sonuçlanan plaka başına. 700 koloni) yayıldı. </li><li> 30 ° C&#39;de 3-4 gün süreyle inkübe Inkübasyondan sonra, her plaka fotoğraflarını çekmek ve galaktoz tabaklarda glikoz ve kolonilerin sayısı kolonilerin sayısı. Çok zehirli bir polyQ-genişleme protein (103Q, Şekil 2a) ifade maya hücreleri kullanırken İdeal koşullar altında, galaktoz plaka üzerinde hiç ya da çok az koloniler olmalıdır. </li></ol> 3.2 Spotting deneyleri Bu test kaplama deneyi yukarıda açıklanan daha nicel ve böylece aynı plaka üzerinde aynı deneyi ile polyQ toksisite bile ince farklılıklar ortaya çıkarabilir. <ol><li> Overn sulandırınızight kültürlerin 0.1 bir OD 600 glukoz ile orta büyüdü. </li><li> Pipet 200 steril 96-tabak içine bu seyreltilmiş kültürlerin ul ve çok kanallı pipet ile steril su içinde beş beş kat seri dilüsyonlar hazırlamak. </li><li> Bir Frogger, (ayrıca hücre süspansiyonları transferi için 8 x 6 iğneli bir gözcü olarak adlandırılır) kullanarak tek karbon kaynağı ve tek karbon kaynağı olarak galaktoz seçici ortamı içeren plakalar olarak glukoz ile seçici ortam içeren plakalar üzerinde hücre süspansiyonları aktarın. </li><li> Plaka 30 bunları kuluçkaya ° C&#39;de üç dört gün için önce kurumasını bekleyin. </li><li> Inkübasyondan sonra, her plaka fotoğrafları (Şekil 2b) alır. </li></ol> 3.3. Sıvı kültürleri büyüme maya polyQ toksisite izlenmesi Bu protokol, burada açıklanan üç testlerinin (OD600 numaraları) en nicel ve hatta polyQ toksisite çok ince farkları tespit edebilir. Anılan ocspontan bastırıcılarının currence Ancak, potansiyel olarak yanıltıcı sonuçlar üretebilir. Bu nedenle yukarıda tarif edilen iki kaplama deneyleri en az biri ile bu tahlilde birleştirerek önermektedir. Biz bu deneyler için BioscreenC enstrüman kullanmayı öneriyoruz. BioscreenC tanımlanan ajitasyon ile tanımlanmış sıcaklıklarda inkübe ise otomatik olarak 100 de plaka maya kültürleri optik yoğunluğunu ölçen bir alettir. Maya hücreleri büyüyor ve optik yoğunluk ölçüm için başka yöntemler de uygulanabilir. <ol><li> 3 ml steril su içinde tek karbon kaynağı olarak üç kat glukoz ihtiva eden en az ortamından maya hücrelerinin yıkanır. </li><li> 0.1 bir OD 600 galaktoz orta yetiştirilen gecede kültürleri sulandırınız. </li><li> Maya kültürlerin 300 ul 100 kuyucuklu BioscreenC plakanın her doldurun. </li><li> Kolay Bioscreen Deney programını açın. Eğer (boşlukları ve orta dahil olmak üzere sadece izlemek isteyen örneklerin sayısını belirlemekontrol), 30 ° C sıcaklığı ayarlamak 3 gün deneyler uzunluğunu ayarlamak, 15 dakika ölçüm aralıkları ayarlamak, 600 nm filtre / Kahverengi ayarlayın ve her ölçümden önce 15 saniye sallayarak modunu ayarlamak Orta kuvvette. </li><li> BioscreenC alet ve ekli yaz deney sırasında alınan her bir veri noktasının Excel tabloları üretecek. </li><li> Her numune için Excel ile büyüme eğrileri hazırlayın ve farklı örnekleri (Şekil 2c) büyüme karşılaştırın. BioscreenC deneyler tarafından üretilen verinin analizi ayrıntılı bir açıklaması 10 daha önce sağlanmıştır. </li></ol> 4. Temsilcisi Sonuçlar <img alt="Şekil 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> Şekil 1.. Maya polyQ modeli. kısa, toksik olmayan polyQ ifade maya hücreleri gösteren zehirli polyQ-genişleme protein. b a) şematik) Floresans mikroskobugenişleme protein (25Q, sol panel) ve maya hücreleri uzun, toksik polyQ-genişleme protein (103Q, sağ panel) ifade. <img alt="Şekil 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> Şekil 2. PolyQ-genişleme proteinleri maya hücrelerinin büyümesini tahlillerin Temsilcisi sonuçları. a) Kaplama testi. yaklaşık 700 maya hücreleri 30 ° C&#39;de üç gün boyunca plakalar üzerine yayılmış ve inkübe edildi Üst panel glukoz ihtiva eden bir orta plaka gösterir, toksik polyQ-genişletme protein (103Q) ifadesi, yani uyarılan değildir. Alt panelini galaktoz içeren ortam, bir maya plakası gösterir, toksik polyQ-genişletme proteininin ifadesi yani indüklenir. Deneyde Burada gösterilen dikkat, herhangi bir spontan bastırıcı oluştu. B) assay Spotting. Maya hücrelerinin Beş beş kat seri seyreltiler ya bir toksik olmayan polyQ protein (25Q) veya bir toksik polyQ-genişletme protei barındırann (103Q) proteinleri (sol panel) veya onların ifadesi neden galaktoz tabaklarda ifade bastırmak glukoz plakalar üzerinde tespit edildi. Sonra plakalar ° C. C) BioscreenC deneyi. Ya bir toksik olmayan polyQ protein (25Q) veya bir toksik polyQ-genişletme protein (103Q) eksprese maya hücresi kültürlerinin büyümesi ile izlendi 30 az üç gün süreyle inkübe edildi BioscreenC alet. Deneysel koşullar ve BioscreenC deneylerin analizi temel Metin tarif edilmektedir.

<ol>
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Çıkar çatışması ilan etti.

Bu yazının toksik polyQ-genişleme proteinleri maya hücrelerinin azalması büyüme dayalı model organizma maya polyQ-toksisite ölçmek için üç tamamlayıcı deneysel yaklaşımlar özetliyor. Maya İş polyQ-genişleme proteinlerin 9,11,12 hatalı katlanması ve toksisite dahil proteinin hatalı katlanması temel hücresel ve moleküler mekanizmalar ve onun ardından gelen toksisitesi, içine derin kavrayışlar teklif etti. Burada sunulan protokolleri esas deneyler zaten polyQ toksisite 5-8,13,...

<table border="1"><tbody><tr><td> Cihaz Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Frogger (6x8 pin) </td><td> V &amp; P Bilimsel, San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves ABD </td><td> 5101370 </td></tr><tr><td> 100-iyi Petek plakaları </td><td> Growthcurves ABD </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Protein hatalı katlanması, Alzheimer hastalığı, Parkinson hastalığı ve Huntington hastalığı 1 olduğu kadar çok insan hastalığı, nörodejeneratif hastalıkların, özellikle, ile ilişkilidir. Huntington hastalığı (HD) protein Huntinğtin içinde bir poliglutamin (polyQ) bölgenin anormal genişlemesi nedeniyle oluşur. PolyQ-genişletilmiş Huntinğtin proteini Aberan konformasyonu (yani misfolds) ulaşır ve hücresel toksisite 2 neden olur. En az sekiz daha nörodejeneratif hastalıklar Spinoserebellar ataksiler ve Kennedy hastalığı da dahil olmak üzere 3 polyQ-açılımları, kaynaklanır. Model organizma maya polyQ-genişleme ifade polyQ-toksisite intra-ve inter-moleküler faktörlerin etkisi ve hücre bozulmaktadır hücresel yollarının belirlenmesi de dahil olmak üzere, polyQ-toksisite hücresel ve moleküler temeli içine önemli anlayışlar kolaylaştırdı proteinler 3-8. Önemlily, maya bulundu polyQ-toksisite birçok yönü dolayısıyla polyQ-toksisite yatan temel mekanizmaların keşfi için maya modelinin önemini gösteren diğer deney sistemleri ve HD hastalardan alınan örnekleri bir ölçüde yeniden üretildi. Maya polyQ-toksisite belirlemek için doğrudan ve nispeten basit bir şekilde polyQ-genişleme proteinleri maya hücrelerinin büyüme kusurları ölçmektir. Bu yazının polyQ-genişleme proteinleri maya hücrelerinin büyümesini ölçerek maya polyQ-toksisite belirlemek için üç tamamlayıcı deneysel yaklaşımlar anlatılmaktadır. İlk iki deneysel yaklaşımlar plakalar üzerinde maya büyüme izlemek, üçüncü bir yaklaşım BioscreenC aracı kullanarak likit maya kültürlerinin büyümesi izler. Ayrıca, bu yazının maya polyQ modelleri işlerken meydana gelebilir deneysel zorlukların açıklanır ve veya önlemek için yardımcı olacak stratejileri ana hatlarıylaBu zorlukların en aza indirmek. Burada anlatılan protokoller tespit etmek ve genetik yollar ve polyQ-toksisite modüle küçük moleküllerin karakterize etmek için kullanılabilir. Ayrıca, açıklanan deneyler maya modelleri diğer hastalığı ile ilişkili katlanan proteinleri neden olduğu toksisite doğru analizler için şablon olarak hizmet verebilir.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Bu yazıda maya poliglutamin toksisite (polyQ)-genişletme proteinlerin değerlendirilmesi için üç tamamlayıcı protokoller açıklar<em&gt; Saccharomyces cerevisiae</em&gt;. Bu protokoller kolayca maya diğer katlanan proteinleri toksisitesini izlemek için değiştirilebilir.

Büyüme Tahliller Mayalarda poliglutamin toksisite değerlendirilmesi için

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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13.5K Görüntüleme.  Boston Biomedical Research Institute. Bu yazıda maya poliglutamin toksisite (polyQ)-genişletme proteinlerin değerlendirilmesi için üç tamamlayıcı protokoller açıklar Saccharomyces cerevisiae. Bu protokoller kolayca maya diğer katlanan proteinleri toksisitesini izlemek için değiştirilebilir.

Video: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Büyüme inhibisyonu fenotipleri tanımlanması Mayalarda bakteriyel Tip III Efektör İfade İndüklenmiş

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Biyoloji

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Büyüme Faktörü Progranulin ile etkileşimde Proteinler tanımlamak Modifiye Maya-İki Hybrid Sistemi

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Kabuklu uzun vadeli Lethal Toksisite Testi Artemia Franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Yöntemler içinde Muscle hücrealtı Kompartmanları DeğerlendirecekÜSKÜP<em&gt; ° C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Spri, Nano-Spri ELISA Tahliller Kullanma Serum İnsan Büyüme Hormonu Karşılaştırmalı Analizi

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Maya Büyümesinin Çok Parametreli Niceleşmesi İçin Büyük Ölçekli Bir Yöntem

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Floresan proteinlerin etkisi Polyglutamine toksisite deneyleri Maya kullanılarak füzyon ortakları

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Orta-den geçerek deneyleri Ca2 +etkileri değerlendirilmesi için eleme-sinyal ve insan Sperm Acrosome reaksiyon

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Floresans canlı hücre mikroskobu ile mitotik ve meiotic Fission Maya nükleer dinamiklerinin incelenmesi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans RAN Peptid Toksisitesi Ölçme

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Methods Article

Büyüme Tahliller Mayalarda poliglutamin toksisite değerlendirilmesi için