著者らは、バージニア工科大学カリリオン研究所所長のマイケル・J・フリードランダー博士が研究努力を奨励することを認めている。このプロジェクトは、S.M.MとD.F.K.への開発資金と、バージニア工科大学の重要技術応用科学研究所のナノバイオイニシアチブによって支えられました。

1. アフィニティ キャプチャ デバイスの準備16
<ol><li>窒化ケイ素Eチップを15mlのアセトンで2分間インキュベートし、2分間15mlのメタノールを2分間洗浄します(図1A)。薄層の空気流の下でチップを乾燥させます。</li>
	<li>乾燥したチップを加熱した攪拌板に150°Cで1.5時間インキュベートし、使用前に室温まで冷却します。</li>
	<li>ハミルトン注射器を使用して、小さなガラス管の脂質混合物を構成して25%クロロホルムを含み、 55%DLPC(1,2-ジロイル・ホスホコリン)クロロホルム(1mg/ml)および20%Ni-NTA脂質(1,2-ジオレオロイルイミノ酢酸-サクシニルニッケル塩)をクロロホルム(1mg/ml)で合計体積40μlとした。</li>
	<li>湿度の高いペトリ皿のパラフィルムに、15μlのミリ-Q水の各15μl滴の混合物のアリコートを適用する(図1B)。氷上でサンプルを少なくとも60分間インキュベートします。</li>
</ol>2. 高分子の捕獲17
<ol><li>150 nm の統合スペーサを備えた E チップを単層サンプルの上に置き、1 分間インキュベートします (図 1C)。</li>
	<li>サンプルからチップをそっと持ち上げ、His タグ付きプロテインA.インキュベートの3 μl アリコートを室温で 1 分間インキュベートします (図 1D)。</li>
	<li>Whatman#1ろ紙を使用して余分な滴を消し去り、すぐに抗体溶液の3μlアリコートを加えます。室温で1分間インキュベートします。</li>
	<li>ハミルトンシリンジを使用して余分な溶液を除去し、50 mM HEPES(pH 7.5)、150 mM NaCl、10 mM MgCl2および10 mM CaCl2を含む緩衝溶液に1μlのロタウイルスDLP(0.1mg/ml)のアリコートをすぐに加える。室温で少なくとも2分間インキュベートする。DMP の準備については、前に18を説明しました。
</li>
</ol>3. マイクロ流体室を組み立て、イン ・ザ・イン ・シーク・フィエサー
<ol><li>ウイルスサンプルを含むウェットEチップをマイクロ流体試料ホルダーの先端にロードします。2番目のフラットEチップを1分間グロー放電し、スペーサーチップの上に積み込みます(図2、 パネル1-5)。</li>
	<li>あるいは、生体高分子のコントラストを高めるために、重金属染色(例えば0.2%ウラニルシフィジ)を、湿った試料に第2のEチップを置く前に湿った標本に加えることができる。しかし、試料を含むEチップは、造影試薬を添加する前にミリQ水で洗浄する必要があります。</li>
	<li>アセンブリ全体を挟み合わせて密閉されたエンクロージャを形成し、3本の真鍮ネジでホルダー内に機械的に保持します(図2、パネル6-8)。</li>
	<li>アセンブリに続いて、ホルダーの先端は、TEMの内側にホルダーを配置する前に、ターボポンプドライポンプ場を使用して10-6 Torrにポンプで送られます。</li>
</ol>4. 透過型電子顕微鏡を用いた液体中のイメージング
<ol><li>LaB6フィラメントを搭載し、120kVで動作する透過型電子顕微鏡(FEI社)にインザッチの標本ホルダーをロードします。</li>
	<li>TEMフィラメントをオンにし、ウォブラー機能を使用して、カラム内のサンプルを前後に-15°から+15°まで傾けることで、標本に対して顕微鏡ステージのユーセントリックな高さを調整します。この手順は、液体チャンバの適切な厚さを考慮するために、Z方向のステージを調整します。このステップはまた、正確な倍率が画像を記録するときに使用されることを保証します。</li>
	<li>最初に、エッジに沿って、マイクロ流体室の隅の領域に画像を記録します。通常、これらの領域には最も薄いソリューションが含まれます。CCDカメラを用いて低線量条件(1-3電子/Å2)の下での検体の画像を記録します。6,000X ~ 30,000X の公称倍率を使用します。</li>
	<li>流体チャンバの端に焦点を合わせ、適切な焦点脱焦点値を決定します。30,000X倍率で画像を記録するには、-1.5 μmの値を使用します。厚い溶液が発生した場合、または造影剤が試料調製に使用されていない場合は、-2〜-4 μmの範囲でより高い脱焦点値を使用してください。</li>
	<li>実験中にマイクロ流体室に溶液が含まれていることを確認するには、デバイス内の液体に気泡が形成されるまで電子線を焦点を合わせます。</li>
</ol>

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著者のマデリーン・J・デュークスは、株式会社プロトチップスの従業員です。

今回の研究では、テザーロタウイルスDMPに対するアフィニティキャプチャアプローチをマイクロ流体プラットフォームに採用しました。これは、液体微小環境における高分子複合体の画像化をその 場 で可能にした。キャプチャアプローチは、生物学的標本をイメージングウィンドウに局地化して、液体中の画像を記録している間に発生する大きな拡散性の問題を否定するため、 ...

<a href="https://app.jove.com/t/6605">This corrects</a> the article <a href="https://dx.doi.org/10.3791/50936">10.3791/50936</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/50936">In situ TEM of Biological Assemblies in Liquid</a>. The Representative Results and References sections were&#160;updated.

Erratum: In situ TEM of Biological Assemblies in Liquid

生物学者や技術者の共通の目標は、分子機械の内部の仕組みを理解することです。透過型電子顕微鏡(TEM)は、原子に近い分解能1~2でこれらの複雑な細部を可視化するのに理想的な装置です。TEMの高真空システムを維持するために、生体試料は、典型的には、ビトレウス氷3の薄膜に埋め込まれ、糖4、重金属塩5、またはそれらの組み合わせ6が挙げられる。その結果、埋め込み標本の画像は、動的プロセスの限られたスナップショットのみを明らかにすることができる。
環境液体室で水和した生物学的標本を維持する初期の試みは、パーソンズと同僚が差動ポンプステージを使用して行った。未染色カタラーゼ結晶の電子回折パターンは、水和状態で3Åの分解能に記録された7-8.さらに、相分離脂質ドメインは、ヒト赤血球9〜10の水和膜で調べることができた。しかし、液体の拡散や電子線との干渉による運動は、重度の分解能損失をもたらし、生物学的標本を用いたさらなる実験は最近まで試みられなかった。
半導体マイクロチップを利用してマイクロスケール環境室を形成する新開発のマイクロ流体試料ホルダーが導入されました。これらのデバイスは、TEM 列11 から 12に配置されている間、サンプルを液体に保持できます。TEMイメージングにおけるこの技術的ブレークスルーにより、研究者は分子レベル13で初めて進行性のイベントを見ることを可能にしました。この新しいモダリティを「その時点での 分子顕微鏡」と呼び、EMカラム14-15の「内部」で実験を行うことができるようになりました。この方法の全体的な目的は、ナノメートル解像度でのそれらの動的挙動を観察するために液体中の生物学的アセンブリを画像化することです。開発された技術の背後にある根拠は、リアルタイムの観測を記録し、溶液中の生物学的機械の新しい特性を調べることです。この方法論は、細胞生物学および分子生物学におけるより広範な目的のためのTEMの使用を拡大する 12-16.
現在のビデオ記事では、市販のマイクロ流体標本ホルダーを組み立て、使用するための包括的なプロトコルを紹介しています。これらの専門のホルダーは、溶液の微細なボリュームを囲む液体チャンバーを形成するために、統合されたスペーサーで製造されたシリコン窒化マイクロチップを利用します。薄く透明な窓は、撮像目的のためにマイクロチップにエッチングされる12。TEMを用いて、マイクロ流体ホルダーを用いて、液体中のシミアンロタウイルス二重層粒子(DRP)を調べるのが適切な方法を示す。DMPなどの生物学的アセンブリがイメージング中に遠距離で急速に拡散しないようにするために、我々は、それらをマイクロ流体室16の表面にテザーするためにアフィニティキャプチャアプローチを採用する。この分子捕獲ステップは下流の処理ルーチンに使用されるイメージの獲得を可能にするので液体の生物学的標本をイメージするための 代替技術より大きな利点 がある。マイクロ流体イメージングと組み合わせて使用されるこのキャプチャステップは、当社の手順17に固有です。TEMまたはマイクロ流体イメージングチャンバを使用して構造生物学アプリケーションを採用している読者は、分子レベルでの動的観測が最終目標である場合、アフィニティーキャプチャ技術の使用を検討することができます。

<table border="0" cellpadding="0" cellspacing="0" width="832">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">E-chips, spacer chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPB-52TBD</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">E-chip, top chip</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;">EPT-45W</td>
			<td style="width:167px;">400 &#956;m x 50 &#956;m window</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ni-NTA lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">790404P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">DLPC (12:0) lipid</td>
			<td style="width:183px;">Avanti Polar Lipids</td>
			<td style="width:245px;">850335P</td>
			<td style="width:167px;">Powder form</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Volumetric flasks&#160;</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">20-812A; 20-812C</td>
			<td style="width:167px;">1 ml; 5 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Hamilton Syringes</td>
			<td style="width:183px;">Hamilton Co.</td>
			<td style="width:245px;">80300, 80400</td>
			<td style="width:167px;">1-10 &#956;l; 1-25 &#956;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Whatman #1 filter paper</td>
			<td style="width:183px;">Whatman</td>
			<td style="width:245px;">1001 090</td>
			<td style="width:167px;">100 pieces, 90 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Petri dishes</td>
			<td style="width:183px;">Corning&#160;</td>
			<td style="width:245px;">70165-101</td>
			<td style="width:167px;">100 mm x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass Pasteur pipettes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">14673-010</td>
			<td style="width:167px;">14673-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glass culture tubes</td>
			<td style="width:183px;">VWR</td>
			<td style="width:245px;">47729-566</td>
			<td style="width:167px;">6 mm x 50 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Acetone</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A11-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Methanol</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">&#160;A412-1</td>
			<td style="width:167px;">1 L</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Chloroform&#160;</td>
			<td style="width:183px;">Electron Microcopy Sciences</td>
			<td style="width:245px;">12550</td>
			<td style="width:167px;">100 ml&#160;</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">His-tagged Protein A</td>
			<td style="width:183px;">Abcam, Inc.</td>
			<td style="width:245px;">ab52953</td>
			<td style="width:167px;">10 mg</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">Milli-Q water system</td>
			<td style="width:183px;">EMD Millipore Corp.</td>
			<td style="width:245px;">Z00QSV001</td>
			<td style="width:167px;">Ultrapure Water</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:237px;">HEPES</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;">BP310-500</td>
			<td style="width:167px;">500 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Equipment&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:245px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Poseidon In situ specimen holder</td>
			<td style="width:183px;">Protochips, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;FEI compatible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">FEI Spirit BioTwin TEM</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>120 kV</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:237px;">Eagle 2k HS CCD camera</td>
			<td style="width:183px;">FEI Co.</td>
			<td style="width:245px;"></td>
			<td>10&#160;&#197;/pixel sampling at 30,000X</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:237px;">Gatan 655 Dry pump station</td>
			<td style="width:183px;">Gatan, Inc.</td>
			<td style="width:245px;"></td>
			<td>Pump holder tip to 10-6&#160;range</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:237px;">PELCO easiGlow, glow discharge unit</td>
			<td style="width:183px;">Ted Pella, Inc.</td>
			<td style="width:245px;"></td>
			<td>&#160;Negative polarity mode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Isotemp heated stir plate</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:245px;"></td>
			<td>Heat to 150 &#186;C for 1.5 hr</td>
		</tr>
	</tbody>
</table>

in situ tem of biological assemblies in liquid

研究者は定期的に透過電子顕微鏡(TEM)を使用して生物学的実体を調べ、新しい材料を評価します。ここでは、液体環境でのウイルスアセンブリの表示- これらの機器のための追加のアプリケーションを説明します。この新しい生物学的構造の可視化手法は、最近開発されたマイクロ流体ベースの検体ホルダーを利用しています。ビデオ記事では、TEM内の液体標本を画像化するためにマイクロ流体ホルダーを組み立てて使用する方法を示しています。特に、モデルシステムとしては、シミアンロタウイルスの二重層粒子(DMP)を用います。また、DDLPsを視受窓につなげたアフィニティバイオフィルムで液体チャンバーの表面をコーティングする手順についても説明する。これにより、3D構造の決定に適した方法でアセンブリをイメージすることができます。このように、我々は、天然の液体環境における亜ウイルス粒子の最初の一見を提示する。

グロー放電されたEチップを用いた液体中のDMPの代表的な画像(図3A)は、アフィニティキャプチャデバイスで富化されたDMPと比較して、拡散によるものと考えられる特定の視聴領域におけるDMPの数が少なくなっています(図3B)。イメージングチャンバにウラニルのフォーマットを加えて、試料のコントラストが高まり、溶液中の個々のDMPの視認性が高まります(図3C?...

Watch this Scientific Journal Video about In situ TEM of Biological Assemblies in Liquid at JoVE.com

In situ TEM of Biological Assemblies in Liquid

ここでは、透過型電子顕微鏡を用いて、ナノメートル分解能でウイルス複合体を画像化する手順について説明する。

その時 液体中の生物学的集合体のTEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

in-situ-tem-of-biological-assemblies-in-liquid

Research

JoVE Journal

Bioengineering

In situ TEM of Biological Assemblies in Liquid

9.9K ビュー.  Protochips, Inc.. ここでは、透過型電子顕微鏡を用いて、ナノメートル分解能でウイルス複合体を画像化する手順について説明する。

ビデオ: In situ TEM of Biological Assemblies in Liquid

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工学

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

パラメータ化拡張期充満フォーマリズムを経由Transmitral流れの運動学的モデルベースの​​解析によるグローバル拡張期機能の定量化

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

生物学的液体培地中でスケーラブルな、メタリック高アスペクト比のナノコンポジットの生成

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

粒子追跡マイクロレオロジーによりバイオフィルムの機械的特性のその場でのマッピング

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

制御された薬物放出のための磁場応答性ハイブリッドゼラチンマイクロゲルを交互に

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

生物学的設定における生成と観測化学発光のための新しい技術

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

モジュラー DNA デバイスの液体ハンドリング アセンブリを自動化

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D空間の細胞培養のための細胞骨格などの生体適合性液晶エラストマー発泡体の合成

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

生体組織の刺激のための電界・磁場装置

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

その時 液体中の生物学的集合体のTEM

その時液体中の生物学的集合体のTEM