The authors would like to acknowledge their gratitude to the U. S. Army Environmental Quality 6.1 Basic Research program, the US Army Institute of Surgical Research, and NSRDEC&#8217;s Continuous Product Improvement program and Expeditionary Base Camp TecD for funding this work. We are grateful to Adam Driks (Loyola University Medical Center) for the micrograph shown in Figure 6B.

1。ポータブル化学殺菌（PCS） <ol><li>装備。 PCSは、ポータブル、エネルギーに依存しない、使用場所の医療殺菌のための革新的なデバイスです。これらの目的のために、商業PELICAN硬質プラスチックスーツケースを殺菌します（ 図1）を収容するための特別な設計上の特徴で装飾した。 </li><li>機器設計。 A）広口反応容器を乾燥化学試薬と水を受け取る。 b）は壁に設置つのチェックバルブが1 psiで圧力を緩和する; C）フィルタ入口弁は空気が室内を洗浄するためのポンプで注入することができます。 D）循環チューブは、チャンバ滅菌後に空気を配布する。 E）使い捨て乾燥スクラバー（活性炭）出口逆止弁を介して移植されたデバイスは、残差を削除し、ユーザーと環境の健康と安全を確保すること。そしてf）ケースの基部に竹馬は、外科用器具トレイまたは他の多孔板に対応し、中にガス流量を最大紅潮。 </li><li>操作。レベルの表面上のPCを置き、ふたを開ける。きれいな、非滅菌の楽器を含む外科トレイを置き、ケース内支柱の上に青色のオートクレーブ紙に包ま。閉じてロック次いで、化学反応を開始するために広口反応容器中-乾燥化学薬品および水（他の組み合わせが可能であり、例えば 、93グラム亜塩素酸ナトリウム63グラム亜硫酸ナトリウム、25gの炭酸水素ナトリウム、アスコルビン酸および300mlの水）を混合する蓋。 2分の下では、反応は、多量の二酸化塩素の殺菌剤、熱、湿気を生成する。 25分の時点で、入口弁へのバッテリ駆動または手動駆動のエアポンプを接続し、約ためにチャンバ内に空気を流す。 5分（ 図2）。 </li><li>サイクルの完了および再利用。ケースを開けて、滅菌器の外科トレイを取り外します。反応槽（水と良性化学塩）を処分。 PCSは、手術器具の別のトレイにすぐに再使用のために利用可能で、ドライ化学物質と水の新鮮なセットです。 </li><li>バイオインディケータによる殺菌の検証。 G.いずれかの紙（〜10 5胞子/ユニット）に含浸させた胞子を含む市販のB / T確かに生物学的指標を配置（湿熱に使用） ステアロサーモフィルスまたはB滅菌サイクルの場合の内部アトロフェウス （エチレンオキサイドガス滅菌のために使用される）。のClO 2滅菌サイクルの完了時に、殺菌を検証するために24〜48時間の指標をインキュベートして、指標を削除し、アクティブにします。 </li><li>胞子懸濁液を用いて滅菌を検証。 Gの水性懸濁液を配置のClO 2滅菌サイクルへの暴露のため、PCS内部ステアロサーモフィルスの胞子（〜10 5 CFU / mL）を加えた。 G.を回復ステアロサーモフィルスの胞子を1％可溶性デンプン8（回復なしには無菌性がないことを示す）と抗生物質アッセイ媒体に二酸化塩素処理に暴露した。 treateの屈折性を検討する位相差顕微鏡とD胞子（のClO 2により不活性化胞子が相明るいキャラクター14を保持）。 </li><li>硬い表面の滅菌を検証。 G。ステアロサーモフィルスの胞子の水性懸濁液（5〜10）のガラス又は金属製の硬質の、非多孔性表面を接種する。のClO 2滅菌サイクルを用いた治療のために、PCSに接種材料を配置します。市販のディフコHY-Aチェック綿棒で処理された表面をサンプリングしない成長を得ないことは、無菌性を確認する。 </li></ol> 2。「D-FENS」 <ol><li>装備。 「D-FENSは「手動スプレーのトリガー装置を備え、折り畳み携帯用ボトルです。柔軟性のあるプラスチックボトルは、スタンドアップをするための完全なマチ付き底部を有し、かつ化学的性プラスチックは、噴霧器ごとに複数の再利用する（ 図3）が得られる。 </li><li>水溶液のClO 2のS olutionを生成します。 D-FENS、ドライ再1-10グラム、合計量を使用しています薬剤50を800mlまで生成する- 500 ppmの二酸化塩素溶液（ 例えば 、4.7グラムの亜塩素酸ナトリウム1.6グラムの亜硫酸ナトリウム、および可能な順列を有する1.3グラムのアスコルビン酸ナトリウム）。水に塩素を生成するには、その最終的な作業容量に溶液を希釈-小さなボリューム内のすべての試薬 ​​を溶解し、かつII反応後の希釈 - 私は前濃縮：「動態管理」を含む新規2段階の混合プロセスを使用2-9分でスプレーボトルの中の二酸化ソリューションを提供しています。消毒液は、意図された表面を消毒や除染する微細な霧またはエアロゾルとして噴霧される。 D-FENSにおける二酸化塩素溶液は、最小で8時間シフトのための安定したままで残っている溶液がバイオフィルムをパージするシフトの終了時に、シンク、床排水溝を下に空にすることができる。 </li><li>微生物学的検証 - 多孔質表面に接種する。卵黄を含むベアード·パーカー寒天（BPA）のペトリ皿を準備します亜テルル（EYT）および酵母抽出物（YE）とエンテロトキシンA、 黄色ブドウ球菌を生成し、黄色ブドウ球菌の3 -ひずみカクテル（ 黄色ブドウ球菌 、A-100の〜10 6 CFU / mlの懸濁液0.1mlを寒天表面に接種エンテロトキシンDを生産するエンテロトキシンBを生成し、ATCC 14458、および黄色ブドウ球菌993）7。S.それがはっきりと明らかに黒色コロニーを生成するので、不活性化されていない場合は、 黄色ブドウ球菌は 、標的生物として選定した。 </li><li>微生物学的検証 - テスト多孔性の表面。 D-FENSを使用すると、多孔寒天表面上に消毒液を噴霧する、二酸化塩素溶液を含むスプレーボトル。スプレートリガパルスあたりの溶液約等容量を分配するために一貫性のある、安定した力を使用しています。寒天表面の均一な被覆を適用するためにプレートを連続するパルス間の90度回転します。また、ガラスホッケースティックでこのテクニックを使用し、塩素系の普及に軽い圧力を適用する機械的研磨用の寒天表面上のNE二酸化液（拭くか、スクラブと同等- 図4を参照）。 </li><li>微生物学的検証 - 硬い表面。細菌細胞の水性懸濁液の0.2ミリリットル容量（ 例えば 、 黄色ブドウ球菌、大腸菌、またはリステリア菌 ）で滅菌したカスタムステンレス鋼（総面積10.16センチメートル2と4 &quot;×4&quot;、）（タイプ304）クーポンを接種し、クーポン表面全体に均一に接種材料に広がる。層流フード内で室温で30分間空気乾燥クーポン。滅菌ピンセットを接種したクーポンをピックアップし、100ミリリットルストマッカー袋に二酸化塩素を含んだ20ミリリットル試験溶液に浸す。 0.5〜5分間の接触時間の後、固体の亜硫酸ナトリウムを少量添加することにより、殺菌剤溶液をクエンチし、次いで、ストマッカー中で2分間溶液を練り。袋を取り外し、層流フード内の上澄み液を撤回し、シリアルにsolutioを希釈N既製の寒天プレートには、生存者を列挙するため24時間インキュベートする。 </li></ol>新鮮な果物や野菜のための3。PCS 新鮮な農産物に有害な食品媒介病原（ 大腸菌及びL.モノサイトゲネス ） を死滅させる還元PCS処理の能力は、病原体の高レベルのトマト楔の外面上にスポットしたスポット接種法を用いて試験した。 <ol><li>接種。 10 5個/ g L·どちらかとトマトのくさびの25グラムのサンプルの外表面に接種リステリア OSY-8578または10 6個/ g Eで大腸菌 ATCC 11229は、空気乾燥、滅菌バイオフード中15分間。 </li><li> PCS処理。接種物が乾燥した後、二酸化塩素濃度と露光時間の様々な条件下でPCSおよび試験（滅菌手袋を着用）トマトウェッジを配置。いくつかの例では、G.の胞子生体指標を配置するステアロサーモフィルス AND B.トマトウェッジに同行し、滅菌処理します（ 図5）を検証するため、PCS内部アトロフェウス 。 </li><li>微生物の回復。 PCS処理後、ストマッカーブレンダーで2分間練り次いで、水性リン酸緩衝液（pH7）50mlでストマッカー袋にトマトウェッジを配置する。 </li><li>列挙。としてシリアルに寒天プレートに10倍希釈液を練りソリューションを分配トリプシン大豆寒天-酵母エキス（TSAYE）及びL.のための栄養寒天（NA） monocyotgenesおよびEそれぞれ大腸菌 、および、ガラスホッケースティックでカバーを広げ、24〜48時間のために、T = 35℃で培養する。微生物の不活性化を確認するために、コロニーカウンターを使用して生存者を列挙します。 </li><li>ポリフェノールオキシダーゼ（「褐変」）酵素を不活性化する。 PCS内部の別々のペトリ皿に未接種リンゴのスライスを配置し、二酸化塩素（ 図5）に公開します。処理後、ペトリ皿を取り外して、目を公開周囲環境におけるEリンゴのスライス。目視観察は、最大1週間処置後には褐変を示さなかった。 </li></ol> 4。「D-FEND ALL」 <ol><li>水溶液のClO 2のS olutionを生成します。 ALLは、0.5〜3.0分で、二酸化塩素溶液を生成するために、水中に乾燥化学物質（亜塩素酸塩及びサミア）の少量を使用するD-FEND。例えば、二酸化塩素溶液を生成するために水溶液15-1,200 mlの試薬の0.8〜3.3グラムを混合する。 </li><li>微生物学的検証 - テキスタイル。 炭疽菌スターン又はバチルスが胞子アミロリケファシエンスの水性懸濁液を用いて織物サンプルの無菌ストリップに接種し、層流フードの繊維ストリップ空気乾燥してみましょう。滅菌ピンセットでストリップをピックアップし、ストマッカー袋100ミリリットル中の二酸化塩素溶液20ml中に浸す。 10分後、固体の亜硫酸ナトリウムを少量添加することで胞子に影響を与えずにプロセスをクエンチし、次いで織物片、溶液を素練りストマッカーでの2分間。袋を取り外し、層流フード内で連続的に、あらかじめ用意された寒天プレート上に溶液を希釈し、24時間インキュベートし、除染を検証するために列挙する。 </li></ol>

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This foundational R&#38;D has set new research and technical directions through collaborations with academia, other Government agencies, and industry that have led to the commercialization of novel, environmentally-friendly (&#8220;green&#8221;) technologies. Chlorine dioxide is the first method approved by the National Sanitation Foundation in 20 years for safer, faster, and more environmentally-friendly sterilization than conventional treatments. The PCS, D-FENS, and D-FEND ALL prototypes have been validated as bench-s...

商用デバイスは、以前に存在しなかった、新鮮な食材を3,6,9-13,15に栄養病原体を殺すために、または細菌胞子を除染するために実証済みの能力を持っている消毒剤の二酸化塩素（のClO 2）を生成し、そこからのPCS技術が進む。6、 14,15,17 PCSは研究室では、 ゲオバチルス·ステアロサーモフィルス （GS）の胞子（関連文献8を参照）、Gの胞子バイオ指標の生きた培養に対する殺菌を実現するために特別に検証されていますステアロサーモフィルス及びバチルスアトロフェウス （BA）6,15,16。 PCSはまた、例えば、ホールトマトなどの生鮮食品にリステリア菌、大腸菌を 、病原体、新鮮なカット農産物の貯蔵寿命を延ばすために栄養を不活性化することによって、食品の安全性を確保するために、より低いストリンジェントな条件で動作するように適合されている、INACによるスライスされたりんご6,15におけるポリフェノールの褐変酵素をtivating。二酸化塩素を生成するために、PCSは、従って、保管、取り扱い、および遠前方軍事酸性廃棄物を配置し、酸及び配送の固有の難しさの使用を排除し、ほぼ中性のpHでの酸化還元反応を介して進行する新規なエフェクター化学を使用して展開1,2,4,17。軍事に加えて、PCSは、国土安全保障/防衛でも使用することができます。電力、飲料水、および廃棄物の除去へのアクセスを無力自然災害（Superstormサンディ、津波、ハリケーン·カトリーナ）の間;オンサイト緊急第一応答者による;そして停電（計画停電とブラウンアウト）の間に地域の病院や学校で。 開発 isinfectant -のF oodsとEN vironmentally優しいのS anitation（D-FENS）用の噴霧器もエフェクター化学（3化学成分）と2段階の混合プロセスを（使用しています<em&gt; I。予備濃縮IIが続く。反応後希釈）の水性二酸化塩素を生成するには、主に陸軍フィールドキッチンと衛生センターや海軍の軍物資の表面の汚染除去、食品取り扱い機器とフィールド給電機器用折りたたみ式スプレーボトル中ガレー船は、医療装置、シャワー、トイレ配備要員の任意の場所に多数の、近接5,6に共存できます。検証テストは、D-FENSは多孔性の表面14に、病原体黄色ブドウ球菌 、一般的な食中毒病原体を排除することを示した。 「D-FEND ALL」（JA vironmentallyに優しい開発 econtamination用のD isinfectant、 万能 ）細菌を除染するために、水性二酸化塩素を製造するための比類のない汎用性（2化学成分）に簡単、より便利（1段階混合）代替手段を提供サニを促進するための表面消毒用テキスタイル上の胞子、テーション衛生及び清潔な飲料水のために生成するように設計された新規雑排水のリサイクル技術の用途のための出発物質の少量を用いて希薄二酸化塩素溶液の大量の迅速な産生を必要とするアプリケーションに特定の利点を有する水の品質と安全性を向上させる遠征ベースキャンプ2。 様々なメカニズムは、国家の材料利益のために技術の開発と商業化を促進する方法として、非連邦エンティティへの連邦政府の技術の移転を促進するために、連邦技術移転法に基づいて存在しています。したがって、特許ライセンス契約および商業評価ライセンスを経由して多くの軍と民間の使用のための彼らの急成長の可能性と、PCS、D-FENSおよびD-FENDすべてのテクノロジは、特許を取得されており、実用化のために、業界に移す。 ＆＃いわゆるD FENS（のゆっくりとした、制御放出バージョン8220、D-FENSライト」）技術は、フレッシュベリーの貯蔵寿命を延長する包装材料への組み込みのための商業業界に移し、PCSは、技術は、他の技術との比較テストのために大学とその他の政府機関に転送された生鮮食品の商品と食品安全の研究のため、学部理科教育を強化するため。 PCSおよびその化学の技術移転は、従来のホルムアルデヒドの治療法に比べて、時間、コスト、および環境保護の改善とバイオフード滅菌のために承認された商用製品につながった。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chlorite</td>
			<td>Sigma-Aldrich</td>
			<td>244155</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>239312</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D-16</td>
			<td></td>
		</tr>
		<tr>
			<td>BT Sure biological indicator (steam)</td>
			<td>Thermo Fisher Sci</td>
			<td>AY759X3</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ Test (EtO)</td>
			<td>SGM Biotech Inc</td>
			<td>EZG/6</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Hy-check</td>
			<td>Becton-Dickinson/ Difco</td>
			<td>290002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Difco</td>
			<td>236950</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Agar</td>
			<td>Difco</td>
			<td>213000</td>
			<td></td>
		</tr>
		<tr>
			<td>Baird-Parker Agar</td>
			<td>Difco</td>
			<td>276840</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Yolk-Tellurite</td>
			<td>Difco</td>
			<td>277910</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Yeast extract</td>
			<td>Difco</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Peptone</td>
			<td>Difco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Tryptone</td>
			<td>Difco</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Soluble starch</td>
			<td>Difco</td>
			<td>0178-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Lemco Beef Extract</td>
			<td>Oxoid</td>
			<td>L29</td>
			<td></td>
		</tr>
		<tr>
			<td>Masticator - Classic</td>
			<td>IUL Instruments</td>
			<td>Cat. No. 400</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher bags</td>
			<td>Seward</td>
			<td>Stomacher &#8216;400&#8217; bags</td>
			<td></td>
		</tr>
	</tbody>
</table>

the portable chemical sterilizer (pcs), d-fens, and d-fend all: novel chlorine dioxide decontamination technologies for the military

明記軍は前方外科チームが使用できるように、フィールドのポータブル、非蒸気滅菌技術が必要とされている、歯科企業、獣医サービスサポート分遣隊、戦闘支援病院、地域医療研究所手術器具を滅菌すると、病理学的滅菌する手術室、緊急治療領域、および集中治療室で廃棄前に標本。小説の次のアンサンブル、「クリーンでグリーン」二酸化塩素技術は除染6,15のための重要な軍事的必要性の数を満たすために適応する汎用性と柔軟性があります。具体的には、p ortable のC hemical 用S terilizer（PCS）は、緊急の戦場のニーズを満たすため、高強度のエネルギー独立性、軽量移植性、迅速な機動性、そして堅牢な耐久前進配備3のための重要な機能のギャップを埋めるために発明されました。外科sterilizatの革命的な技術革新などイオン技術、PCSは、オンサイトで依存している現代のフィールドオートクレーブで、ユースポイント、AT-意志の二酸化塩素の代わりに蒸気を発生。二つの（2）のPCS単位は（なぜなら、その面倒なサイズ、かさばる寸法、重量の展開での愛称「ベルタ」）1大型蒸気オートクレーブの同等のスループットで、1時間に4外科トレイを滅菌する。しかし、PCSが100％少ない電力（0対9キロワット）を使用して、98％少ない水で動作（10対640オンス）、大幅に95％重量を削減（20対450ポンド、4人のリフト）事実上96パーセントキューブ（2.1対60.2フィート3）、その修理や、信頼性の高い動作を維持持ち上げ、輸送、スチームオートクレーブに必要な電力の前方展開で困難な課題を解消。

The easy-to-operate PCS was designed to achieve sterility by inactivating bacterial spore suspensions or bacterial spore bio-indicators in 30-minute treatments involving the controlled production of chlorine dioxide by unique effector chemistry. Specifically, microbiological validation studies verified that the PCS achieved sterility by inactivating bio-indicators containing spores (105 spores/ml) of either G. stearothermophilus or B. atrophaeus, that are intended to indicate sterilization by...

Watch this Scientific Journal Video about The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military at JoVE.com

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

ポータブル化学殺菌（PCS）、D-FENSおよびD-FEND ALL：小説二酸化塩素除染技術の軍事用

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

the-portable-chemical-sterilizer-pcs-d-fens-d-fend-all-novel-chlorine

Research

JoVE Journal

Bioengineering

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

14.4K ビュー.  United States Army-Natick Soldier RD&E Center, Warfighter Directorate. The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally...

ビデオ: The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

単一細胞の多彩な化学分析マイクロ流体チップ

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

生物工学

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

のリグニンダウンレギュレーション<em&gt;トウモロコシ</em&gt; dsRNAiとクラソンリグニン分析によって

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

多様な微生物群集の高密度栽培のための新しいバイオリアクター

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水質浄化のための膜のような防汚特性を持つハイドロゲルの合成

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

組織特異的細胞培養および配信プラットフォームとして、細胞外マトリックス由来のフォームおよびマイクロキャリアの作製

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

トリマーエピトーププレゼンテーションを必要とするワクチンに対する大腸菌の自己組織化タンパク質ナノ粒子の産生

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

ヒト好中球群れの化学化学および分子解析のための生体微粒子マイクロアレイ

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

細胞を含まない細菌抽出物、リポソーム、エマルジョン移動を用いた合成細胞の調製

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

細菌スクリーニング、選択、および分離のための光分解性ヒドロゲルインターフェース

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.