データのグラフィカルな表現にアンドリューラングの援助は大歓迎です。実験的な業務を遂行する装置や材料を提供するためのメトラートレドのおかげ。

はじめ Ugi反応は迅速に化合物（1-3）の様々なライブラリを作成する便利な方法であることが証明されている。それは、アミンとの反応、アルデヒド、カルボン酸と室温でメタノールで一般的にイソニトリルを伴います。 Ugi反応はしばしば薬理学的に活性な分子の合成のツールと​​して使用されており、我々はそれがすぐに新しい抗マラリア剤（4）の検索では化合物へのアクセスに利用してきた。それは、UGI製品が時々反応混合物（1,5）から純粋な形で沈殿することが観察されている。これは、次いで、反応物を容易に、クロマトグラフィーなどの高価な精製工程を必要とせずにスケールアップできるので、非常に幸運な結果です。それは、さらなる治療なしで反応混合物をフィルタリングから直接取得したUGIの製品の歩留まりを最適化するために最も有益である。この目的のために我々は、ろ過チューブを搭載した48スロットメトラートレドMiniBlock（6）利用。メトラートレドMiniMapper（7）自動化された液体のハンドラは、試薬および溶媒を提供する目的で使用されました。興味のあるパラメータは、濃度、溶媒組成や試薬のいくつかの過剰であった。 実験追加の溶剤、フルフリルアミン（メタノール中2M）、ベンズアルデヒド（メタノール中2M）、BOC -グリシン（メタノール中2M）：MiniMapper自動化された液体ハンドラは、48ポジションMiniBlockの空のフィルターチューブに以下の順序で液体を提供するようにプログラムされたとt - butylisocyanide（メタノール中2M）。デフォルトの添加量は100マイクロリットルであった。試薬が過剰に添加されるものとして記載されている場合は、120マイクロリットルが配信されました。 MiniBlockは、家の掃除を使用してフィルタ処理される前に16時間シェーカー上に置いた。 2回の洗浄は濾過する前に振っ15分間に続いて各試験管にメタノール（1 mL）を追加することによって実行されます。チューブは、少なくとも30分間デシケーター中で高真空下で乾燥させた。収量はフィルターチューブの重量増加から計算した。純度は、それぞれの溶媒系と濃度から一つのサンプルのためにH NMRにより評価した。反応は三連で実行された（8-10）、平均収率を表1に報告されています。 UGIの製品の特性評価 <a href="http://www.chemspider.com/Chemical-Structure.21106059.html">tert -ブチル（2 - {[2 - （tert -ブチルアミノ）-2 -オキソ-1 -フェニルエチル]（フラン-2 -イルメチル）アミノ} -2 -オキソエチル）カーバメート</a> ：白色固体、融点（11）202から204 C、1H NMR（12 <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp203A11-HNMR.html">スペクトル</a> ）（500MHzの、δppm、CDCl3）1.33（S、9H）、1.45（S、9H）、4.21（m、2H）【、4.49（D、J = 18Hz、1H）、4.50（ D、J = 18Hz、1H）、5.47（s、1H）【、5.60（s、1H）【、5.62（s、1H）【、5.89（s、1H）【、6.10（s、1H）【、7.19（s、1H）【 、フェニル7.21から7.37（M、5H）、13C NMR（13、 <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/Exp02060822080933CNMR.html">スペクトラム</a> ）（500MHzの、δppm、CDCl3）28.3、28.6、42.3、42.8、51.7、62.9、79.5、107.7、110.4、128.5、128.7、129.6、134.7 、141.9、149.8、155.7、168.4 170.2、IR（14、 <a href="http://showme.physics.drexel.edu/mirza/Software/JCAMP/ugiproductpaper03IR.html">スペクトラム</a> ）（Ʋmax cm - 1のATR）：1645,1673,1699、3331、FAB - HRMS（15、 <a href="http://showme.physics.drexel.edu/mirza/Exp099Chighres.JPG">スペクトラム</a> ）（C24H33N3O5のために計算されたm / zの444.2498の[M + H]、444.2517を得た。） データ解析沈殿物の収量データは、単一要素または2つの因子（複製付）Microsoft Excelで利用可能な分散（ANOVA）のツールの分析を用いて解析した。 （16）分散分析で統計的に有意であることが判明、これらの変数は、Fisherの最小有意差（LSD）のテストは、UGIの沈殿物（17）の収率で有意差を示す特定の実験的な設定を決定するために使用された。すべての有意差検定は、95％の信頼レベルで実行されました。 結果と考察 図1は、UGIの沈殿物の収量に及ぼす溶媒組成物の効果をまとめたものです。単因子ANOVAは、0.2Mの試薬の濃度で学んだfour溶剤の平均利回りに大きな差があることを示します。フィッシャーのLSD検定は、メタノールとエタノールで得られた沈殿物の収率が95％の信頼水準（図中の溶剤の名前以上のバーで示さ）で統計的に異なっていないことを示します。UGIの沈殿物の収率は、エタノールとメタノールで有意に大きいアセトニトリルよりも、どの順番でTHFのそれより有意に大きい。 <img src="/files/ftp_upload/old/942_2008_9_26_0/942_Figure-01.png" alt="図1" /> 図1：レアージュにおけるUGIの沈殿物の収量に及ぼす溶媒組成物の効果のまとめ0.2MのNTの濃度。記載されている溶剤が、この濃度で40％のメタノールが含まれています。 試薬濃度の影響を考慮すると、表1のデータは、0.2Mから溶媒としてメタノール、エタノールおよびアセトニトリルを使用して0.07Mに行く際に収率の急落が見つかった示し、収量は0.2Mで、メタノールで同等であったと0.4 Mの濃度。 UGIの反応は一般に0.5 - 2Mの範囲（2）で実行されていることに注意してください。しかし、自動化された液体ハンドラを使用するとき、それはこのように、約0.5Mでの最終濃度に実用的な上限を設定すること、ずっとより濃縮された200万以上の試薬溶液で開始することが困難な場合があります。非線形効果が遊んで明らかなので、この最適化の研究の結果は、0.2M以下UGI反応の成功または失敗の解釈には注意を示唆している。 図2は、0.2Mの試薬の濃度でUGIの沈殿物の収率に対する溶媒組成や試薬の過剰の影響をまとめたものです。二因子分散分析の結果は統計的に有意な溶剤と試薬の過剰な効果を示している。さらに、2つの変数間の統計的に有意な相互作用があります。相互作用の詳細は、沈殿物の収率は使用する溶媒によって影響されていることを示している図2に明らかにされています。再び図中の要因以上のバーは収量が95％の信頼水準で統計的に異なっていないことを示すことに注意してください。アセトニトリルのために、大幅に改善された結果は、過剰のアミン、アルデヒドまたはイソニトリルを得ている間に、エタノールとメタノールの両方のために最高の収量は、過剰のイミンまたはイソニトリルで得られる。表1の結果の議論では上述したように、沈殿物の全収率は、溶媒としてTHFのために低くなり、イミンの唯一の過剰は、沈殿物の収率で統計的に有意な改善につながる。 four溶剤のための改善された収率のこれらの異なるパターンは統計的に有意な相互作用の効果につながるものであり、異なる溶媒によるUGIの製品のさまざまな相互作用の指標となる可能性があります。表1の数値結果は、最高の収率は過剰イミンの1.2当量、0.2Mアセトニトリル/メタノールとも大幅な利回りを提供0.2Mエタノール/メタノール混合物を0.4Mメタノールで得られたを示しているが。 0.2MのTHF /メタノール混合物のすべてが非常に悪い結果を与えたことに特に注意してください。 Ugi反応が正常にTHF（2）を含む溶剤の様々な、で実施されています。ここで、低収量はUgi反応は、沈殿物が形成されていないことだけが、THFの混合物で行われなかったことを意味しな​​いことに注意してください。 <img src="/files/ftp_upload/old/942_2008_10_13_5/942_Figure-02.png" alt="図2" width="335" height="325" /> 図2：0.2Mの試薬 ​​の濃度でUGIの沈殿物の収率に対する溶媒組成や試薬の過剰の影響の概要。記載されている溶剤が、この濃度で40％のメタノールが含まれています。 図3は、溶媒としてメタノールを使用して沈殿物UGIの収量に試薬の濃度と試薬の過剰の影響をまとめたものです。二因子分散分析の結果は統計的に有意な濃度と試薬の過剰な効果を示している。前述したように、0.2Mと0.4Mのの試薬の濃度で沈殿物の収率は（が互いに異なっていない）0.07Mよりもかなり大きいです。さらに、研究の下に二つの変数の間に統計的に有意な相互作用があります。過剰試薬のアイデンティティと沈殿物の収率改善のパターンは、より高い試薬の濃度で、一般的に同じであることに注意してください。 <img src="/files/ftp_upload/old/942_2008_9_26_2/942_Figure-03.png" alt="図3" width="384" height="255" /> 図3：UGIの収率に対する濃度と試薬の過剰の影響の概要は、メタノール溶媒で沈殿する。 全体的に、図2と図3で説明した結果、溶媒の選択、試薬の濃度と過剰の試薬のアイデンティティの間の相互作用があったことを示しているが、全体的にこれらはストレート溶剤と濃縮効果より微妙だった。 Ugi反応（18）最適化する際に他の人が同様の結果を発見した。 結論最適化の実験は48回の実行の最高収率は1.2当量と0.4 Mのメタノールから66％であることがわかった。イミンの。これは大幅にメタノールで0.4 Mでの等モル濃度の下での最初の反応から得られた49％の利回りを超えています。沈殿物の収率の良好な再現性を積極的に識別される微妙な相互作用の影響を考慮し、これらの反復実験で得られた。

<ol>
	<li>Marcaccini, S., Torroba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Ugi+four-component+condensation.">The use of the Ugi four-component condensation.</a> Nature Protocols. 2, 632-63 (2007).</li><li>Domling, A., Ugi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+reactions+with+isocyanides+Angew.">Multicomponent reactions with isocyanides Angew.</a> Chem. Int. Eng. Ed. 39, 3168-31 (2000).</li><li>Domling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Developments+in+Isocyanide+Based+Multi-Component+Reactions+in.">Recent Developments in Isocyanide Based Multi-Component Reactions in.</a> Applied Chemistry, Chem. Rev.. 106, (2006).</li><li>Bradley, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> http://usefulchem.blogspot.com/2008/01/we-have-anti-malarial-activity.html. , .</li><li>Sanudo, M., Marcaccini, M., Basurto, S., Torroba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+3-Hydroxy-6-oxo[1,2,4]triazin-1-yl+Alaninamides,+a+New+Class+of+Cyclic+Dipeptidyl.">Synthesis of 3-Hydroxy-6-oxo[1,2,4]triazin-1-yl Alaninamides, a New Class of Cyclic Dipeptidyl.</a> Ureas J. Org. Chem.. 71, 4578-45 (2006).</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> UsefulChem EXP201 http://usefulchem.wikispaces.com/EXP201. , .</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EXP202 http://usefulchem.wikispaces.com/EXP202. , .</li><li>Bradley, J. -. C., Mirza, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> EXP203 http://usefulchem.wikispaces.com/EXP203. , .</li><li>p, m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>Bradley, J. -. C., NMR, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> spectrum 203A11 from UsefulChem EXP203 http://usefulchem.wikispaces.com/EXP203. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C NMR spectrum 206A from UsefulChem EXP206 http://usefulchem.wikispaces.com/EXP206. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> IR spectrum from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FAB-MS from compound UC-099C from EXP099 http://usefulchem.wikispaces.com/EXP099. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis details available at http://usefulchem.wikispaces.com/space/showimage/EXP201-203_KO-A.xls. , .</li><li>Montgomery, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Analysis+of+Experiments.">Design and Analysis of Experiments.</a> Nature Protocols. , (2005).</li><li>Tye, H., Whittaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+Design+of+Experiments+approach+for+the+optimisation+of+a+microwave+assisted+Ugi+reaction.">Use of a Design of Experiments approach for the optimisation of a microwave assisted Ugi reaction.</a> Org. Biomol. Chem.. 2, 813-813 (2004).</li></ol>

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Furfurylamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F20009</td>
<td>&nbsp;</td>
<tr>
<td>Benzaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>12010</td>
<td>&nbsp;</td>
<tr>
<td>Boc-glycine</td>
<td>Reagent</td>
<td>TCI America</td>
<td>B1185</td>
<td>&nbsp;</td>
<tr>
<td>t-butylisocyanide</td>
<td>Reagent</td>
<td>TCI America</td>
<td>B1274</td>
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optimization of the ugi reaction using parallel synthesis and automated liquid handling

フルフリルアミン、ベンズアルデヒド、BOC -グリシンおよびt - butylisocyanideの混合を含むUgi反応の最適化が記述されています。三連が48並列の実験の実行濃度、溶剤と試薬のいくつかの余分を変え、報告されている。生成物の単離は、単純なろ過と洗浄の手順によって達成された。得られた最高収率は1.2当量と0.4 Mのメタノールから66％であった。イミンの。これは大幅にメタノールで0.4 Mでの等モル濃度の下での最初の反応から得られた49％の利回りを超えています。 0.07Mですべての溶媒系は不振ながら、0.4Mまたは0.2Mの試薬の濃度のメタノール溶液には優れた収量を与えた。 THF /メタノール（60/40）が悪いし、アセトニトリル/メタノール（60/40）は、中間いた間0.2M、メタノールおよびエタノール/メタノール（60/40）混合物で統計学的に同等に良好であった。沈殿物の収率の良好な再現性を積極的に識別される微妙な相互作用の影響を考慮し、これらの反復実験で得られた。

Watch this Scientific Journal Video about Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling at JoVE.com

Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Ugi反応は迅速に化合物の多様なライブラリを作成する便利な方法であることが証明されている。それは、アミンとの反応、アルデヒド、カルボン酸と室温でメタノールで一般的にイソニトリルを伴います。このビデオでは、我々は、48スロットのメトラートレドMiniBlockろ過チューブを装備し、メトラートレドMiniMapper自動化された液体ハンドラが試薬と溶媒を配信するために使用されたことを利用しています。興味のあるパラメータは、濃度、溶媒組成や試薬のいくつかの過剰であった。

パラレル合成と自動液体ハンドリングを使用してUgi反応の最適化

The optimization of a Ugi reaction involving the mixing of furfurylamine, benzaldehyde, boc-glycine and t-butylisocyanide is described.  Triplicate runs ...

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Biology

16.2K ビュー.  Drexel University. Ugi反応は迅速に化合物の多様なライブラリを作成する便利な方法であることが証明されている。それは、アミンとの反応、アルデヒド、カルボン酸と室温でメタノールで一般的にイソニトリルを伴います。このビデオでは、我々は、48スロットのメトラートレドMiniBlockろ過チューブを装備し、メトラートレドMiniMapper自動化された液体ハンドラが試薬と溶?...

ビデオ: Optimization of the Ugi Reaction Using Parallel Synthesis and Automated Liquid Handling

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

スモールハーツの半自動光ビート解析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek&#39;s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.
PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: 
 
&#9679; Set up reactions and thermal cycling conditions for a conventional PCR experiment 
&#9679; Understand the function of various reaction components and their overall effect on a PCR experiment 
&#9679; Design and optimize a PCR experiment for any DNA template 
&#9679; Troubleshoot failed PCR experiments

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

ポリメラーゼ連鎖反応：基本的なプロトコルプラスのトラブルシューティングと最適化戦略

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of ...

Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals across the landscape is of primary importance in watershed management. Traditional economics methods of finding the lowest-cost solution in the watershed context (e.g.,5,12,20) assume that off-site impacts can be accurately described as a proportion of on-site pollution generated. Such approaches are unlikely to be representative of the actual pollution process in a watershed, where the impacts of polluting sources are often determined by complex biophysical processes. The use of modern physically-based, spatially distributed hydrologic simulation models allows for a greater degree of realism in terms of process representation but requires a development of a simulation-optimization framework where the model becomes an integral part of optimization.
Evolutionary algorithms appear to be a particularly useful optimization tool, able to deal with the combinatorial nature of a watershed simulation-optimization problem and allowing the use of the full water quality model. Evolutionary algorithms treat a particular spatial allocation of conservation practices in a watershed as a candidate solution and utilize sets (populations) of candidate solutions iteratively applying stochastic operators of selection, recombination, and mutation to find improvements with respect to the optimization objectives. The optimization objectives in this case are to minimize nonpoint-source pollution in the watershed, simultaneously minimizing the cost of conservation practices. A recent and expanding set of research is attempting to use similar methods and integrates water quality models with broadly defined evolutionary optimization methods3,4,9,10,13-15,17-19,22,23,25. In this application, we demonstrate a program which follows Rabotyagov et al.'s approach and integrates a modern and commonly used SWAT water quality model7 with a multiobjective evolutionary algorithm SPEA226, and user-specified set of conservation practices and their costs to search for the complete tradeoff frontiers between costs of conservation practices and user-specified water quality objectives. The frontiers quantify the tradeoffs faced by the watershed managers by presenting the full range of costs associated with various water quality improvement goals. The program allows for a selection of watershed configurations achieving specified water quality improvement goals and a production of maps of optimized placement of conservation practices.

This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs.

SWATモデルを用いた農業保全の実践の空間的な多目的最適化と進化的アルゴリズム

Finding the cost-efficient (i.e., lowest-cost) ways of targeting conservation practice investments for the achievement of specific water quality goals ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

エア·液界面での人間の鼻上皮細胞の培養

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

プロシージャおよび自動キャピラリー電気泳動を用いた免疫測定法のためのキーの最適化戦略

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

蛍光灯、摂取昆虫における流体吸収能力を決定するための磁気ナノ粒子

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

自動イメージングシステムを用いたiPS細胞の合流点の測定

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.