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

私たちは、有益なコメントをA. Riccioのを感謝し、原稿を読み取るために。この作品は、細胞生物学ユニットの医学研究評議会（MRC）の資金によって、ヒューマンフロンティアサイエンスプログラムのグラント（RGP0048/2009-C）によってサポートされていました。

1。酵素反応 - 1日目（午後の1時間） <ol><li>最初のステップは、IP 6 K1またはVIP1がpyrophosphorylatedアイソフォームにIP 6に変換する10月20日独立した酵素反応を準備することです。 </li><li>我々はE.から精製された彼の- IP 6 K1とGST - VIP1酵素を使用以前に17,18を記載されたプロトコールに従って大腸菌 。 </li><li> 1X反応バッファー（30mMのHepes緩衝液pH6.8、50mMのNaCl、6 mMのMgSO 4を 、1mMのDTT）、6 mMのクレアチンリン酸（PCR）、25 U / mLのCreatinePhosphoKinase（CPK）、5mMのATP（Mgを含む50μL反応を準備する塩）、0.3mMのIP6、0.05から0.1μgの彼- IP 6 K1またはGST - VIP1。 MiliQのddH 2 Oで音量を調節</li><li>簡単に言うと37℃の反応とインキュベート℃で一晩回転でも回転させています。 </li></ol> 2。ポリアクリルアミドゲルキャスティングと荷重 - 2日目（午後の4時間） <ol><li>ポリアクリルアミドゲルは、24センチ、18センチ幅のガラス板と1.5 mm幅のスペーサを用いて調製される。通常16車線または取シングルレーンコームが使用されます。 </li><li> 35.5パーセント（w / v）のアクリルアミド：ビスアクリルアミド午前19時01分、1Xトリス/ホウ酸/ EDTA（TBE）、0.05％（w / v）の過硫酸アンモニウム（APS以下を含む混合（50mLの/ゲル）を準備する）、0.05％（w / v）をTEMED。 、事前に鋳造ガラスプレートの間にミックスを注ぎコームを挿入し、室温で30-60分間重合させる。 </li><li>ゲルが重合した後、寒い部屋に装置に転送し、200から300ボルトで約30〜60分間、1 × TBEで事前に実行してください。 </li><li>各反応にOrangeG染料（10mMトリス- HCl pH7.0、1mMのEDTA、30％グリセロール、0.1％OrangeG）の1Xを追加。標準コントロールとしてロードするためにIP 6の2 nmolのを含むサンプルを準備します。 </li><li>沈殿物を除去するためにシリンジと21G針を使用してバッファを実行しているとよく徹底的にそれぞれを洗い、その後、ゲルをロードします。側のウェルにローディングすることは避けてください。 </li><li> OrangeG染料のバンドがゲルの底から最後の10cm以内になるまで、450から550ボルト（7 MAMP /ゲル）で一晩ゲルを実行します。 </li></ol> 3。 IP 7の分離- 3日目（4時間）と4日目（6-7時間のSpeedVac乾燥工程） <ol><li>ゲル装置を分解し、慎重に他のいずれかにゲルを残して1枚のガラスプレートを取り外します。 図1に示すように、ちょうどOrangeG色素のバンドを上からIP 6の標準を含む底部と一つのサンプルのレーンにゲルのわずかな部分をカット。 </li><li>トルイジンブルー（0.1％（w / v）のトルイジンブルー、20％（w / v）のメタノール、2％（w / v）のグリセロール）数分間（1〜3分）とゲルの切断部を染色またはイノシトールピロリン酸のバンドが表示されるまで。以前に乾燥から未染色ゲルを防ぐためにゲルの上に戻って取り外したガラス板を置く。 </li></ol>それはIP 6標準よりも少し遅くなるので、IP 7バンドが見えるはずです。 IP 6よりも速く実行されるATPは、、また、（ 図1）可視でなければなりません。数分間脱染色液（20％（w / v）のメタノール）でゲルの染色された部分を転送する、トルイジンブルーの余った部分を洗い流すと染色ゲルでゲルを再配置。 高いpyrophosphorylatedイノシトールアイソフォーム（IP 8 IP 9）の可視化が必要な場合は、室温で20分間、トルイジンブルー染色液でゲルを染色。その後、約15分間脱染色液でトルイジンブルーを洗い流す。 <ol start="3"><li>かみそりの刃でリファレンスとして染色ゲル（ 図1）で決定したIP 7移行の位置を用いて、ゲルの染色部分にIP 7バンドをカット。 </li><li> 15 mLの試験管にゲルから切り出されたIP 7バンドを入れて、ミリQのddH 2 O 10mLを追加する室温で10分間回転でチューブを入れる。 TBEおよび顕微鏡アクリルアミド粒子の過剰を削除するには、液体を捨てる。 </li><li>その後、二つの脱水和のサイクルを実行します。 IPの7を含むゲルをチューブに50％（w / v）のメタノール5 mLを加え、2時間室温で回転する。ミリQのddH 2 O 5 mLを含む新しい15mLのチューブにゲルスライスを移し、2時間室温で回転する。チューブからメタノールとミリQ H 2 Oを破棄しないでください。以前に使用されて15 mLの試験管にポリアクリルアミドゲルを再転送することにより、再び脱水和のサイクルを繰り返します。洗浄の一つを一晩行うことができます。 </li><li>溶出されたIP 7を集中して、60℃に加熱SpeedVac℃を用いて（5 mLをMilliQddH 2 O及び5mLの50％（w / v）のメタノール）を一緒10mLを乾燥させる</li><li>サンプルはほぼ乾燥していると、± 1.5 mLの遠心分離管と5000 rpmで2分間スピンして、残りの液体（300〜600μl）を移す。 </li><li>新しい1.5 mLの遠心チューブに上清と転送を収集する。残すそれは、アクリルアミドの粒子を含んでいる可能性があるため、底部20〜30μL。 </li><li>必要な非加熱Speed​​Vacを使用して、乾燥プロセスを続行する場合。サンプルは100〜300μLの容積に達したときにサンプルが完全に乾く場合は、IP 7の回復が劇的に減少され、したがって、乾燥工程を終了する。 </li></ol> 4。 IP 7濃度及び純度の決定。 <ol><li>セクション2.2から2.5までと同様に、PAGEゲル上で実行するように回復したIP 7サンプル2〜5μLを使用してください。濃度の標準とポリ- Pマーカーの4 nmolのようにIP 6のいくつかの希釈（すなわち0.5、1、2、4ナノモル）を読み込みます。ゲルを実行した後、3.2節（ 図2A）で説明されている手順に従って、染色とトルイジンブルー液で脱染色ゲル全体によるイノシトールピロリン酸のアイソフォームを可視化する。 </li><li>トルイジン染色した後、濃度は、 図2Bに示すように、イメージ- J、などのイメージングソフトウェアを使用して、ゲルをスキャンし、IP 6とIP 7の間の強度の違いを比較することによって決定することができます。 </li></ol> 5。代表的な結果： IP 6 K1とVIP1酵素を使用してIP〜7 IP 6の調製用酵素的変換は、簡単にページの解析（ 図1）を使用して解決することができます。彼らはイノシトール環上に存在するリン酸基の数に応じて実行速度が遅くなるので、トルイジンブルーのゲルの染色と一緒にサイズのコントロールとしてIP 6のローディングは、pyrophosphorylated誘導体の同定が可能になります。上記の手順では、IP 7の容易に精製することができます。 PAGEで精製したイノシトールピロリン酸の分析は、当社のIP 7（ 図2A）の純度を明らかにした。興味深いことに、VIP1の1/3PP-IP 5 IPの異性体7の製品は、IP 6 K1によって生成されるIP 7 5PP - IP 5異性体より僅かに遅い移行されます。精製したIP 7（ 図2B）の濃度を容易に定量化を可能にするIP 6の基準を使用してください。さらなる実験のためにIP 7を使用する前に、その生物活性を評価することができます。 （ 図3）。 5PP - IP 5は VIP1でとIP 7ホスファターゼDDP1（diphosphoinositolポリリン酸のリン酸ヒドロラーゼ）でインキュベートする。日常的に、精製されたIP 7 DDP1（ 図3）によってVIP1でIPを8にし、IP 6に変換されます。 <img alt="図1" src="/files/ftp_upload/3027/3027fig1.jpg" /> 図1：IP 7バンドのページと分離のトルイジン染色は、標準（IP 6）を含むゲルの部分を切断し、トルイジンブルー液を用いて染色した。 3つのバンド（上から下へ）IP 7、IP 6とATPを表しています。ゲルの染色された部分は、ゲルの残りに整列した。これは、（点線で囲まれた部分）切断して精製することができるIPの7を含むゲルの部分のローカライズが可能になります。 <img alt="図2" src="/files/ftp_upload/3027/3027fig2.jpg" /> 図2：IP 6 K1とVIP1反応生成物のPAGE分析。 A）トルイジンブルー染色によるIP 6の解析（4、2、1、0.5 nmolのは）IP 6 K1（5PP - IP 5）とVIP1（1/3PP-IP両方から精製したIP 7濃度を決定するために使用されていました5）精製されたIP 7の濃度を決定するために反応が。B）散布図の分析。濃度は、IP 6の所定の量と比較するとImageJのソフトウェアを用いて計算したバンド強度、、にしたがって決定した。 X -軸は強度を表し、Y軸は、ナノモルで表した濃度を表します。 <img alt="図3" src="/files/ftp_upload/3027/3027fig3.jpg" /> 図3：IP 7生物学的活性の解析精製されたIP 7の品質を判断するには、我々は、DDP1 VIP1、またはIP 7ホスファターゼと5PP - IP 5（IP 6 K1生成されたIP 7）インキュベートし、ページ上で反応して解決。 DAPIとトルイジン染色はVIP1でIP 8の予想される生産とDDP1でIP 6〜IP 7の変換を明らかにした。

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利害の衝突は宣言されません。

生化学におけるイノシトールピロリン酸の使用は厳しく、このような化合物の商業利用できないと、既存の検出方法の乏しい感度によって制限されます。異なるリン酸基の数、およびトルイジンブルー（ 図1）、リン酸基に結合する異染染料を、有する分子の分離を可能にするページ、の組み合わせは、研究20の新たな道を開くイノシトールpyrophoshateのアイソフォームの容易な検出を行うことができます。 酵素反応のイノシトールピロリン酸の製品を精製するためにこのページの技術を説明する使用はoutbyどちらIP 6 K1またはVIP1を行い、高品質のIP 7の大量の生産を可能にするシンプルで、経済的かつ信頼性の高い方法です。上記の方法は、7 IPの簡易精製法に限定されるものではなく、記述されたプロトコルのマイナーな修正は、イノシトールピロリン酸の異なる範囲の精製を可能にすることができる。個以上のリン酸基を含む高リン酸化イノシトールピロリン酸のアイソフォームは、基板20,6として、IP 7またはIP 6の異なる量を用いて検出することができる。これらのイノシトールピロリン酸は、染色手順の長さを増加させ、続いて（セクション3.2）、精製によって検出することができます。また、酵素反応の基質としてIP 5の使用は、PP - IP 5の精製とイノシトール環に ​​ヒドロキシル基を含む他のイノシトールピロリン酸塩をできるようになります。 結論として、この多くを求めない方法は、このように、このエキサイティングな研究分野のための新しい道を開いて、広く利用可能な楽器とイノシトールピロリン酸のミリグラム単位の信頼性の高い精製が可能になります。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>フィチン酸（IP 6） </td><td>シグマアルドリッチ</td><td> P8810 </td></tr><tr><td>ポリ- P（ヘキサメタリン酸ナトリウム） </td><td>シグマアルドリッチ</td><td> P8510 </td></tr><tr><td> ATPとMg 2 +塩</td><td>シグマアルドリッチ</td><td> A9187 </td></tr><tr><td> OrangeG </td><td>シグマアルドリッチ</td><td> O3756 </td></tr><tr><td>クレアチンリン酸（PCR） </td><td>シグマアルドリッチ</td><td> P7936 </td></tr><tr><td> CreatinePhosphoキナーゼ（CPK） </td><td>シグマアルドリッチ</td><td> C3755 </td></tr><tr><td> GST - VIP1 </td><td> 17 </td><td> 17 </td></tr><tr><td>彼の- IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td>彼の- Ddp1 </td><td>研究室で利用可能</td><td>研究室で利用可能</td></tr><tr><td>アクリルアミド：ビスアクリルアミド19時01分（40％） </td><td> Flowgen </td><td> H16972 </td></tr><tr><td>過硫酸アンモニウム（APS） </td><td>シグマアルドリッチ</td><td> A9164 </td></tr><tr><td>トリス/ホウ酸/ EDTA（TBE） </td><td>シグマアルドリッチ</td><td> T 9060 </td></tr><tr><td> TEMED </td><td> BDH </td><td> 43083G </td></tr><tr><td>トルイジンブルー</td><td>シグマアルドリッチ</td><td> 198161 </td></tr><tr><td> Speed​​Vac </td><td>キリスト</td><td> 100218 </td></tr><tr><td>ゲル装置</td><td> Hoefer </td><td> SE600 </td></tr><tr><td>真空マニホールド</td><td>キリスト</td><td>アルファ2月4日</td></tr><tr><td>真空ポンプ</td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

ミオイノシトールは、修正されていないか、より複雑なリン酸化誘導体のいずれかに自然に存在しています。最新の、真核細胞の中で最も豊富な二人はイノシトールpentakisphosphate（IP 3）とイノシトール六リン酸（フィチン酸またはIP 6）です。 IP 5とIP 6は、1つまたは複数のピロリン酸結合1を含むイノシトールピロリン酸の分子の前駆体である。 IP 6のリン酸化がdiphoshoinositolpentakisphosphate（IP 7またはPP - IP 5）及びbisdiphoshoinositoltetrakisphosphate（IP 8または（PP）2 - IP 4）を生成します。イノシトールピロリン酸塩は、これまでに学んだすべての真核生物から単離されている。さらに、イノシトールピロリン酸の合成を担う酵素の2つの別個のクラスは、高度に進化2月4日にわたって保存されている。 IP 6キナーゼ（IP 6 KS）保有基質として、これらの製品を使用することにより、それぞれ、その後IP 5、PP - IP 4、IP〜7 IP 6を変換する巨大な触媒柔軟性は、より複雑な分子5,6の生成を促進する。最近、酵素を生成するピロリン酸第二のクラスは、IP 7やIP 8 7,8にIP 6を変換することができる酵母タンパク質VIP 1（また、PP - IP 5 Kと呼ぶ）、の形で同定された。 イノシトールピロリン酸は、インスリンの分泌9、テロメアの長さ10,11、走化性12、小胞輸送13、リン酸の恒常性14およびHIV - 1のgagリリース15として、多くの異なる細胞プロセスを調節する。アクションの2つのメカニズムが分子のこのクラスのために提案されている。彼らは、アロステリックAKT 16のような特定の蛋白質と相互作用することにより細胞機能に影響を与える可能性があります。また、ピロリン酸基は、事前にリン酸化タンパク質17にリン酸を寄付することができます。この研究分野の大きな潜在力は、これらの分子の研究から多くの科学者を妨げているイノシトールピロリン酸の商業的供給源、およびこの新しい翻訳後修飾の欠如によって妨げられている。イノシトールピロリン酸塩を分離するために現在利用可能なメソッドは、洗練されたクロマトグラフ装置18,19が必要です。これらの手順は、イノシトールピロリン酸の分解20とこうして貧しい回復につながる可能性のある酸性条件を使用してください。さらに、面倒なポストカラムの脱塩の手続きは専門の研究所にその使用を制限する。 本研究では、IP 6 -キナーゼとPP - IP 5 -キナーゼ反応の生成物の生成、単離および精製の ​​ための多くを求めない方法を説明します。このメソッドは、高度にリン酸化されたイノシトールポリリン酸20を解決するためにポリアクリルアミドゲル電気泳動（PAGE）の能力によって可能であった。 IP 6 K1と基質としてのIP 6を使用してPP - IP 5 Kの酵素反応に続いて、PAGE、続いて水で溶出し、生成されたイノシトールピロリン酸塩を分離するために使用されていました。

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

Preparation of Quality Inositol Pyrophosphates

イノシトールピロリン酸は人間の病態、例えば癌、糖尿病や肥満の重要な役割を果たしているが、作用の正確なメカニズムは、紛争の問題です。市販のイノシトールピロリン酸の不足は、詳細な研究が問題とレンダリング。ここでは、イノシトールピロリン酸のミリグラムを生成し、分離するために単純なプロトコルを記述する。

品質イノシトールピロリン酸塩の調製

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マイクロアレイ：サンプルの品質管理、アレイハイブリダイゼーションとスキャニング

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

生物学

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.

見つけにくい小型哺乳類から高品質のDNAを得るために非侵襲ヘアサンプリング法

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.

甲殻類との長期致死毒性試験アルテミア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.

高分子の3次元再構成のための試料調製のプライマーおよび高品質データ収集：クライオ電子顕微鏡ののいいことといけないことをやる

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.

生成高品質の機械可読の生物学的経路のためのWebツール

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.

密度勾配遠心法を用いた差動品質の精液採取

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.

ポータブルqPCRシステムを用いた マトリカリアシャモミラ のフィールド同定

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.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

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.

乳酸菌の培養・増殖・生存率:品質管理の視点から

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

プロセス開発による噴霧乾燥プロバイオティクス製品の品質の最適化

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.

著者スポットライト:プロバイオティクス細菌の噴霧乾燥のためのプロセス開発と製品品質の評価  

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

プロバイオティクス菌の噴霧乾燥プロセス開発と製品品質評価

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