Name: preparation of quality inositol pyrophosphates
Uploaded: 2011-09-03T14:00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

A. Riccio yararlı yorumlar için teşekkür ediyor ve el yazması okumak için. Bu çalışma Hücre Biyolojisi Birimi Tıbbi Araştırma Konseyi (MRC) finansman ve İnsan Frontier Bilim Programı Hibe (RGP0048/2009-C) tarafından desteklenmiştir.

1. Enzimatik Reaksiyon - 1. gün (öğleden sonra 1 saat) <ol><li> IP 6 K1 veya VIP1 pyrophosphorylated izoformları IP 6 dönüştürmek 10-20 bağımsız enzimatik reaksiyonları hazırlamak için ilk adım. </li><li> E. gelen arıtılmış Onun IP 6 K1 ve GST-Vip1 enzimler coli protokole göre, daha önce açıklanan 17,18. </li><li> 1X reaksiyon tamponu (pH 6,8 30 mM HEPES, 50 mM NaCl, 6 mM MgSO 4, 1 mM DTT), 6 mM phosphocreatine (PCr), 25 U / ml CreatinePhosphoKinase (CPK), 5 mM ATP (Mg içeren 50 mcL reaksiyonlar hazırlayın tuz), 0.3 mM IP6, 0.05-0.1 mg Onun IP 6 K1 veya GST-Vip1. MiliQ GKD 2 O. ile ses seviyesini ayarlayın </li><li> Kısaca 37 reaksiyon ve inkübe ° C gecede rotasyon ile dönerler. </li></ol> 2. Poliakrilamid jel döküm ve yükleme günlük 2 (öğleden sonra 4 saat) <ol><li> Poliakrilamid jel, 24 cm uzunluğunda, 18 cm genişliğinde cam plakalar ve 1.5 mm genişliğinde ayırıcılar kullanılarak hazırlanmıştır. Genellikle 16 şeritli veya preparatif tek şeritli tarak kullanılır. </li><li> % 35.5 (w / v) Akrilamid:: Bis-Akrilamid 19:01, 1X Tris / Borat / EDTA (TBE),% 0,05 (w / v) amonyum persülfat (APS aşağıdaki maddeleri içeren bir karışımı (50 ml / jel) hazırlayın ) 0.05% (w / v) Temed. Ön-döküm cam plakalar arasındaki karışımı dökün tarak yerleştirin ve 30-60 dakika oda sıcaklığında polimerize izin. </li><li> Jel polimerize sonra, soğuk oda cihazın transferi ve 200-300 Volt 30-60 dakika için 1X TBE önceden çalıştırın. </li><li> Her reaksiyon OrangeG boya 1X (10 mM Tris-HCl pH7.0, 1 mM EDTA,% 30 gliserol,% 0,1 OrangeG) ekleyin. Standart bir kontrol olarak yüklemek için IP 6 2 nmol içeren bir örnek hazırlayın. </li><li> Herhangi bir çökelti kaldırmak için bir şırınga ve bir 21G iğne kullanarak tampon çalışan her bir kuyunun iyice yıkayın, sonra jel yükleyin. Yan kuyu yükleme kaçının. </li><li> OrangeG boya grubu, son 10 cm içerisinde jel alttan kadar, 450-550 Volt (7 mAmp / jel) geceleme jel çalıştırın. </li></ol> 3. IP 7 İzolasyon - 3. gün (4 saat) ve günde 4 (6-7 saat SpeedVac kurutma işlemi) <ol><li> Jel aygıtı sökün ve bir cam levha diğeri jel bırakarak dikkatlice kaldırın. Şekil 1&#39;de gösterildiği gibi alt IP 6 standart ve bir örnek şerit içeren jel OrangeG boya bant üzerinde sadece küçük bir kısmı, kesin. </li><li> Lekelere kesme Toluidine Blue (% 0,1 (w / v)% 20 (w / v) metanol, toluidin mavi,% 2 (w / v) gliserol) ile bir kaç dakika jel bölümü (1-3 dk) veya inositol pirofosfat grubu görünene kadar. Daha önce boyanmamış jel kurumasını önlemek için jel üstünde kaldırıldı cam plaka koyun. </li></ol> IP 7 bant IP 6 standart biraz daha yavaş çalışır görünür olmalıdır. ATP, IP 6 daha hızlı çalışır, aynı zamanda görünür olmalıdır (Şekil 1). Transfer jel lekeli kısmı birkaç dakika için de boyama solüsyonu (% 20 (w / v) metanol), Toluidine Blue herhangi bir aşırı yıkayacak ve lekesiz bir jel ile jel yeniden konumlandırmak. Yüksek pyrophosphorylated inositol izoformları (IP 8 ve IP 9) görselleştirme gerekiyorsa, oda sıcaklığında 20 dakika Toluidine Blue boyama solüsyonu ile jel leke. Daha sonra, yaklaşık 15 dakika için de boyama çözüm Toluidine Blue yıkayın. <ol start="3"><li> Bir jilet boyanan jel (Şekil 1) ile belirlenen IP 7 göçmen konumu referans olarak kullanarak jel lekesiz bir kısmı IP 7 bant kesilmiş . </li><li> IP 7 band 15 ml tüp jel kesilen koyun ve MilliQ GKD 2 O. 10 ml Oda sıcaklığında 10 dakika süreyle dönüşümlü olarak tüpler koyun. TBE ve mikroskopik akrilamid parçacıkların aşırı kaldırmak için sıvı atın. </li><li> Daha sonra, iki dehidratasyon hidrasyon döngüleri gerçekleştirin. IP 7 içeren jel ile tüpe 5 ml% 50 (w / v) metanol ekleyin ve 2 saat boyunca oda sıcaklığında döndürmek. MilliQ GKD 2 O 5 ml içeren yeni bir 15 ml tüp jel dilim transferi ve 2 saat boyunca oda sıcaklığında döndürmek. Tüplerinden metanol ve MilliQ H 2 O atmayın. Önceden kullanılan 15 ml tüpler yeniden transfer poliakrilamid jel dehidratasyon hidrasyon döngüsü bir kez daha tekrarlayın. Yıkar biri gece boyunca yapılabilir. </li><li> Yıkandı, IP 7 konsantre için 60 ısıtılmış bir SpeedVac ° C ile birlikte 10 ml (5 ml MilliQddH 2 O ve 5 ml% 50 (w / v) metanol), kuru </li><li> Örneklerin neredeyse kuru sonra kalan sıvı (300-600 ul), ± 1.5 ml santrifüj tüp ve 5000 rpm&#39;de 2 dakika spin aktarın. </li><li> 1,5 mL taze bir santrifüj tüpü içine supernatant ve transfer toplayın; terkalt 20-30 mcL akrilamid parçacıklar içerebilir. </li><li> Gerekirse bir ısıtmasız SpeedVac kullanarak kurutma işlemi devam etmektedir. Örnekleri hacmi 100-300 mcL ulaştığınızda örnekleri tamamen kurumasını, IP 7 kurtarma önemli ölçüde azalır, bu nedenle kurutma işlemi sona erdirmek. </li></ol> 4. IP 7 konsantrasyonu ve saflık tayini. <ol><li> Bölüm 2.2-2.5 benzer, 2-5 PAGE jel üzerinde çalıştırmak için kurtarılan IP 7 örnek mcL kullanın. , Konsantrasyon standart ve 4 Poly-P marker nmol olarak 6 IP birkaç dilüsyonları (yani 0.5, 1, 2, 4 nmol) yükleyin . Jel çalıştırdıktan sonra, Bölüm 3.2 (Şekil 2A) açıklanan prosedürü izleyerek, boyama ve Toluidine Blue çözüm ile de boyama tüm jel inositol pirofosfat izoformları görselleştirmek. </li><li> Toluidine boyama sonra, konsantrasyonları Şekil 2B gösterildiği gibi, jel, tarama ve IP 6 ve 7 IP arasında yoğunluk farklılıkları karşılaştırarak, Resim-J gibi görüntüleme yazılımı kullanarak tespit edilebilir. </li></ol> 5. Temsilcisi Sonuçlar: IP 6 K1 ve VIP1 enzimler kullanarak IP 7 IP 6 hazırlık enzimatik dönüşüm PAGE analizi (Şekil 1) kullanarak kolayca çözülebilir. Inositol halka üzerinde bulunan fosfat grupları sayısına göre daha yavaş çalışabilir Toluidine Blue jel boyama ile birlikte bir boyut kontrolü gibi IP 6 yükleme, pyrophosphorylated türevleri belirlenmesini sağlar. Yukarıda açıklanan prosedür 7 IP kolay arıtma sağlar. PAGE ile saflaştırılmış inositol pirofosfat analizi bizim IP 7 (Şekil 2A) saflığını ortaya çıkardı. İlginçtir ki, VIP1 IP 7 ürün 1/3PP-IP 5 izomeri IP 6 K1 tarafından oluşturulan IP 7 5PP IP 5 izomeri biraz daha yavaş göç eder. Arındırılmış IP 7 (Şekil 2B) konsantrasyonu kolay bir kantifikasyon izin IP 6 standartlarının kullanın. Daha fazla deneyler için IP 7 kullanmadan önce, biyolojik aktivite tespit edilebilir (Şekil 3). 5PP IP 5 VIP1 ve IP 7 fosfataz DDP1 (diphosphoinositol polifosfat phosphohydrolase) ile inkübe edilir. Rutin, saflaştırılmış IP 7 DDP1 (Şekil 3) VIP1 tarafından 8 IP ve IP 6 dönüştürülür. <img alt="Şekil 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Şekil 1: IP 7 bant ve izolasyon SAYFA Toluidine boyama standart (IP 6) içeren jel kısmı kesilmiş ve Toluidine Blue çözüm kullanılarak boyandı. Üç grup (yukarıdan aşağıya) IP 7, IP 6 ve ATP temsil eder. Jel lekeli kısmı daha sonra kalan jeli ile uyumlu hale getirilmiştir. Bu IP 7, (kesikli kutu) kesilmiş ve temizlenmiş olabilir içeren jel kısmı lokalizasyonu sağlar. <img alt="Şekil 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Şekil 2: IP 6 K1 ve VIP1 tepkime ürünleri SAYFA analizi. A) Toluidine Blue boyama ile IP 6 Analiz (4, 2, 1, 0.5 nmol) IP 6 K1 (5PP IP 5) ve VIP1 (1/3PP-IP hem arınmış IP 7 konsantrasyonunu belirlemek için kullanılan 5) reaksiyonlar. B) Scatter plot analiz saflaştırılmış IP 7 konsantrasyonunu belirlemek için. Konsantrasyonları ImageJ yazılım kullanarak IP 6, önceden belirlenmiş miktarda göre hesaplanan bant yoğunluğu, göre belirlenmiştir. X-ekseni şiddetleri temsil eder; Y ekseni nmol ifade konsantrasyonları temsil eder. <img alt="Şekil 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Şekil 3: IP 7 biyolojik aktivite analizi saflaştırılmış IP 7 kalitesini belirlemek için DDP1 VIP1 veya IP 7 fosfataz 5PP IP 5 (IP 6 K1 IP 7) inkübe ve ardından SAYFA reaksiyon çözümlenir. DAPI ve Toluidine boyama VIP1 tarafından 8 IP beklenen üretim ve DDP1 IP 6 ila 7 IP dönüşüm ortaya çıkardı.

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Çıkar çatışması ilan etti.

Biyokimya inositol pirofosfat ciddi bu tür bileşiklerin ticari kullanılamaması ve mevcut algılama yöntemleri kötü hassasiyeti ile sınırlıdır. Farklı fosfat grupları sayısı ve Toluidine Blue (Şekil 1), fosfat grupları bağlanır metakromatik boya, sahip moleküllerin ayrılmasını sağlayan SAYFA kombinasyonu araştırma 20 yeni yollar açılması inositol pyrophoshate izoformları kolay tespit edilmesini sağlar. Outby yapılan enzimatik reaksiyon inositol pirofosfat ürünler arındırmak için SAYFA teknoloji açıklanan kullanım IP 6 K1 veya VIP1 ya da basit, ekonomik ve güvenilir, yüksek kaliteli IP 7 büyük miktarlarda üretim için izin veren bir yöntemdir . Yukarıda açıklanan yöntem 7 IP basit arıtma sınırlı değil ama açıklanan protokol küçük değişiklikler inositol pyrophosphates farklı bir dizi arıtma izin verebilir. Sekizden fazla fosfat grupları içeren Yüksek fosforile inositol pirofosfat izoformları, IP IP 6, 7 veya farklı miktarlarda bir substrat 20,6 olarak tespit edilebilir. Bu inositol pyrophosphates boyama prosedürünü uzunluğunun artırılması ve daha sonra saflaştırılmış (bölüm 3.2) tespit edilebilir. Ayrıca, IP 5 enzimatik reaksiyon için substrat olarak kullanımı 5 PP-IP arıtma ve inositol halka bir hidroksil grubu içeren diğer inositol pyrophosphates izin verecek. Sonuç olarak, bu basit yöntem, böylece, bu heyecan verici araştırma alanı için yeni yollar açılması, yaygın kullanılan enstrümanlar ile inositol pyrophosphates miligram miktarda güvenilir arıtma sağlar.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Fitik asit (IP 6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Poly-P (sodyum hexametaphosphate) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> ATP-Mg 2 + tuz </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> Phosphocreatine (PCr) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> CreatinePhospho Kinaz (CK) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> GST-Vip1 </td><td> 17 </td><td> 17 </td></tr><tr><td> His-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> His-Ddp1 </td><td> Laboratuarda </td><td> Laboratuarda </td></tr><tr><td> Akrilamid: Bis-Akrilamid 19:01 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Amonyum persulfate (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Tris / Borat / EDTA (TBE) </td><td> Sigma-Aldrich </td><td> T 9060 </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> Toluidine Blue </td><td> Sigma-Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> Mesih </td><td> 100218 </td></tr><tr><td> Jel aparat </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Vakum manifoldu </td><td> Mesih </td><td> Alpha 2-4 </td></tr><tr><td> Vakum pompası </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol, doğa ya da değiştirilmemiş veya daha karmaşık fosforile türevleri mevcuttur. Son ki, ökaryotik hücrelerde en bol iki inositol pentakisphosphate (IP 5) ve inositol hexakisphosphate (fitik asit ya da IP 6). IP 5, IP 6, bir ya da daha fazla pirofosfat bağlar 1 içeren inositol pirofosfat moleküllerin habercileri . IP 6 Fosforilasyon diphoshoinositolpentakisphosphate (IP 7 veya 5 PP-IP) ve bisdiphoshoinositoltetrakisphosphate (IP 8 veya (PP) 2-IP 4) üretir. İnositol pyrophosphates şimdiye kadar üzerinde çalışılan bütün ökaryotik organizmalardan izole edilmiştir. Buna ek olarak, iki ayrı sınıflar inositol pirofosfat sentezi için sorumlu enzimlerin yüksek 2-4 evrim boyunca korunmuş. IP 6 kinazlar (IP 6 Ks) sahip muazzam bir katalitik esneklik, 5 IP ve IP 6, PP-IP 4 ve sırasıyla IP 7 ve daha sonra, substrat olarak bu ürünleri kullanarak, daha karmaşık moleküller nesil 5,6 teşvik dönüştürme . Son zamanlarda, pirofosfat üreten enzimlerin ikinci sınıf bir IP 7 ve IP 8 7,8 6 IP dönüştürmek mümkün maya protein VIP 1 formu (PP-IP 5 K olarak anılacaktır), tespit edilmiştir. İnositol pyrophosphates insülin salgılanması 9, telomer uzunluğu 10,11, kemotaksi 12, veziküler kaçakçılığı 13, fosfat homeostazı 14 ve HIV-1 gag sürümü 15 gibi birçok farklı hücresel süreçleri düzenler. Moleküllerin bu sınıf için eylem iki mekanizma ileri sürülmüştür. Allosterically AKT 16 gibi spesifik proteinlerin etkileşim hücresel fonksiyonları etkileyebilir. Alternatif olarak, bir fosfat pirofosfat grubu ön fosforile proteinler 17 bağışta bulunabilirsiniz. Bu araştırma alanında büyük bir potansiyel inositol pyrophosphates, pek çok bilim adamı bu moleküller ve bu yeni post-translasyonel değişiklik eğitim engelleyen bir ticari kaynak yokluğunda tarafından engellenmektedir. Inositol pyrophosphates izole etmek için mevcut yöntemleri gelişmiş kromatografi 18,19 gerektirir. Bu prosedürler, inositol pirofosfat bozulması 20 ve böylece kötü kurtarma yol açabilir asidik koşullarda kullanın . Ayrıca, hantal bir post-kolon tuzsuzlaştırma prosedürleri uzman laboratuarlar kullanımını kısıtlamak. Bu çalışmada, IP, 6-kinaz ve PP-IP 5-kinazlar reaksiyonlar ürünleri üretimi, izolasyon ve saflaştırma için basit bir yöntem tarif . Bu yöntem, yüksek fosforile inositol polifosfatlar çözmek için 20 poliakrilamid jel elektroforezi (PAGE) yeteneği sayesinde mümkün oldu . IP 6 K1 ve yüzey gibi IP 6 kullanarak IP PP-5 K enzimatik reaksiyonların ardından, SAYFA, daha sonra suda yıkandı, oluşturulan inositol pyrophosphates ayırmak için kullanılır oldu.

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

İnositol pyrophosphates kanser, diyabet ve obezitenin insan patolojilerin önemli bir rol oynar; ancak, etki mekanizması tam bir anlaşmazlık meselesidir. Piyasada bulunan inositol pyrophosphates eksikliği detaylı çalışmalar sorunlu vermektedir. Burada inositol pyrophosphates miligram üretmek ve izole etmek için basit bir protokol.

Kalite İnositol Pyrophosphates hazırlanması

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Research

JoVE Journal

Biology

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA Mikroarray&#39;ler: Örnek Kalite Kontrol, Dizi Hibridizasyon ve Tarama

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Biyoloji

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Elusive Küçük Memeliler Yüksek Kalite DNA Edinme Noninvaziv Saç Örnekleme Tekniği

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

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 ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Makromoleküler 3D İmar için bir örnek Hazırlanması Astar ve Yüksek Kalite Veri Toplama: Cryo-elektron mikroskobu Yapılması ve Yapılmaması Gerekenler

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

Yaratma Yüksek Kalite Makine okunabilir Biyolojik Pathways için Web Aracı

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Sperm yoğunluğu degrade Santrifüjü kullanarak fark kalitesini topluluğu

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Taşınabilir QPCR Sistemi ile Matricaria chamomilla Alan Tanımlama

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Kök Eksüdalarında Rhizobacterial Kemoattractants'ın Hızlı Tanımlanması için Geliştirilmiş Bir Kemotaksi Tahlili

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

Laktik Asit Bakterilerinin Yetiştirilmesi, Büyümesi ve Yaşayabilirliği: Kalite Kontrol Perspektifi

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

Proses geliştirme yoluyla püskürtülerek kurutulmuş probiyotik ürün kalitesinin optimize edilmesi

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

Yazar Spotlight: Probiyotik Bakterilerin Püskürtülerek Kurutulması için Proses Geliştirme ve Ürün Kalitesinin Değerlendirilmesi  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Probiyotik bakterilerin sprey kurutulması için proses geliştirme ve ürün kalitesinin değerlendirilmesi

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...