リスト謝辞と資金源。 NIH-ES015022およびES018274（TRN） NSF-協力協定1003907（VCM）

全身ナノ粒子吸入暴露ステップごとの操作手順は、次のように記載されている。 注 ：1）手順1と3はドラフト内で行うべきである、2）演算子は（呼吸器、ゴーグル、ゴム手袋）適切な個人保護具を着用しなければならない。 1。エアコンの TiO 2ナノ粒子乾燥粉末<ol><li>不透明な容器にナノTiO 2の粉末を置きます。 </li><li>容器の蓋を開いたままにします。 </li><li>コンディショニングのために少なくとも24時間乾燥し、デシケーター中で容器を置きます。 </li></ol> 2。データ収集と制御システム、SMPSとELPIとすべてトランスデューサウォーミングアップ<ol><li>空気の監視やデータ収集システムとエアロゾルのモニタリングSMPS（TSI社、ショアビュー、ミネソタ州）とELPI（Dekati、タンペレ、フィンランド）のための電源スイッチをオンにして、少なくとも1時間のシステムをウォームアップ。 </li><li>電源を入れるすべてのトランスデューサ内のスイッチは、少なくとも1時間のためにそれらをウォームアップする。 </li></ol> 3。エアロゾル発生器への TiO 2ナノ粒子の乾燥粉末のロード<ol><li>エアロゾル発生器のシリンダーキャップを開き、エアゾール発生器のフィルタを交換してください。注：Oneエーロゾル発生器が一つの気筒を有する。使用するエアロゾル発生器の数は、露光チャンバ内の粒子の所望の質量濃度に依存する。 </li><li> 〜4グラムのナノTiO 2の粉末を秤量し、各気筒でそれらをロードします。 </li><li>シリンダーキャップを交換してください。 </li><li>すべてのエリアでは、TiO 2の汚染の疑いが拭い濡らさなければならない。 </li></ol> 4。吸入暴露チャンバーにエアロゾル発生器を接続する<ol><li>吸入暴露室（TSEシステムズ社、バートホンブルク、ドイツ）の入口であるサイクロンセパレータマニホールドを経由してエアロゾル発生器のすべてのコンセントに接続してください。 </li><li>圧縮空気配管に接続しエアロゾル発生器におけるベンチュリ分散機。 </li></ol> 5。吸入暴露室に大気モニタリングやエアロゾルサンプリングインレットを接続<ol><li>温度と相対湿度（RH）、圧力、吸入暴露室で大気モニタリングポートをテストし東証システムから供給されるO 2＆CO 2センサーを接続します。 </li><li>吸入暴露チャンバーでエアロゾルのサンプリングポートの1つにエアロゾル希釈器の入口を接続し、ELPIの入口にその出口を接続します。 </li><li>吸入暴露チャンバーでエアロゾルのサンプリングポートのいずれかにSMPSを接続します。 </li><li>曝露チャンバーにエアロゾルのサンプリングポートのいずれかに粒子濃度モニタ（東証システム）の入口に接続します。 </li><li> PTFEメンブレンフィルター（P / N 66149、ポール·コーポレーション、アナーバー、ミシガン州）を計量し、ステンレス製フィルターホルダー（インToxの製品、モリアーティNM）にフィルタをロードします。 </li><li>の入口を接続吸入暴露チャンバーでエアロゾルのサンプリングポートのいずれかにフィルタ予め秤量、サンプリングポンプにその出口を接続してください。ステンレス製フィルターホルダー</li></ol> 6。データ収集システムをアクティブに<ol><li>アクティブELPIデータ収集ソフト、ELPIVIは 、設定パラメータをチェックして、約5分間水洗ポンプをオンにしてから、ELPIをゼロ。録音前暴露濃度。 </li><li> SMPSデータ集録ソフトウェアをアクティブにします。録音前暴露濃度。 </li><li> O 2、CO 2、空気流量、温度およびRHチャンバ圧力、温度およびRHの監視および制御するためのソフトウェア、DACO（TSEシステム）を活性化する。 </li></ol> 7。吸入暴露チャンバー内に実験動物のロード<ol><li>実験動物を計量。 </li><li>動物が暴露した後、同じケージ内に戻すことができるように、実験動物とケージをマークする旧姓なded。 </li><li>吸入暴露室の扉を開き、有線ケージに実験動物をロードします。 </li><li>水は、動物のために提供されてもよい。 </li><li>吸入暴露室のドアを閉じ、固定します。 </li><li>頻繁に苦痛の兆候曝露室観察窓から動物を観察する。動物は、リラックスして正常に動作しなければならない。急速に/呼吸こじつけ場合露出を停止、外観異常、姿勢異常や不動が観察される。動物を外し、元のケージに戻し、出席獣医師に連絡し、/または適切な動物実験を開始し、委員会の手順を使用します。 </li></ol> 注：ステップ8.7、8.8と8.17を実行する際にオペレータは個人用保護具を着用しなければならない。 8。ナノ粒子エアロゾルに小動物を公開する<ol><li>吸入暴露室の排気真空ポンプの電源をオンにします。 </li><li>にデータ収集ソフトウェア、DACOを実行して、a）b）は、露光チャンバ内の圧力を制御し、及びc）は圧力、温度、RH、O、露光環境のデータを収集し、露光室に濾過乾燥空気を供給する2、CO 2。 </li><li>チャンバ圧力でわずかに負圧を（セットポイント= -0.2ミリバール）を確立。 </li><li>エアロゾル発生器の電源をオンにします。 </li><li>連続的に吸入暴露チャンバ内の粒子サイズおよび相対質量濃度を監視するためにELPIとSMPSデータ収集ソフトウェアを実行する。 </li><li>エアロゾル濃度が安定しているとき、ELPIモニタ上の濃度プロファイルは、（通常：エアゾール発生器が動作中になった後、これは20分かかります）プラトーに達した、つまり （例えば、1時間）サンプリング時間を設定し、エアロゾルのサンプリングをオンにするフィルタを使用してナノ粒子の代表的なサンプルを収集するためにポンプ。 </li><li>サンプリング時間に達すると、フィルタを削除し、saのを差し込む暴露室から逃げるの試験材料を防ぐためにゴム製のプラグでポートをmpling。 </li><li>フィルタを秤量し、上記のように露光チャンバ内の平均質量濃度を算出する。 </li><li>平均濃度が目標濃度オフになっている場合は、手動でターゲット濃度が達成されていることを確認するために発電機で空気の流れを調整します。 </li><li> 時刻t =露光時間D = C×垂直メートル XTX F rは 、試験材料のD =用量、C =平均質量濃度、V mは分時拍出量、などの動物の肺における粒子堆積を計算し、F はr =画分の材料それは、堆積又は吸収される。 </li><li>クリーン、プレ加重フ​​ィルタをフィルターホルダーにフィルターを交換し、ステップ8.6と8.8を繰り返します。 </li><li>動物の肺の曝露室と標的粒子沈着の本当の質量濃度に基づき、残りのEXPを見積もる、tは残るとしてosure時間=（D ターゲット -D）/（C X V M X F R）tは残る 、=暴露期間まま、D ターゲット =ターゲット用量、C =試験材料の質量濃度、V Mを意味する =分容積、F R材料=分数堆積または吸収される。 </li><li> 残るトンに達したときにエアロゾル発生器の電源をオフにします。 </li><li>モニターに示されている粒子濃度がチャンバー内の暴露前粒子濃度に近くなるまで、曝露室から動物を削除する前に、濾過された空気を吸入暴露室をフラッシュします。 </li><li>チャンバー排気真空ポンプの電源をオフにします。 </li><li>データ収集ソフト、DACOを停止します。 </li><li>暴露後、正常な呼吸と動作を確認するために動物を観察し、文書のない他の研究合併症EXそのイスト。鼻汁、呼吸困難または他の動物愛護合併症が認められた場合には、主治医の獣医師および/に連絡するか、適切な動物実験を開始し、委員会の手順を使用します。 </li><li> ELPIとSMPSデータ収集ソフトウェアを停止します。 </li></ol> 9。テストレポートの作成 9.1試験条件は、 <ol><li>このテストで使用エーロゾル発生システム及びその動作パラメータの説明。 </li><li>露光中に使用される設計、種類、寸法およびその動作パラメータを含む露光装置の説明。 </li><li>測定温度、湿度、粒子サイズ、および実際の濃度ための機器。 </li><li>排気と使用した場合の試験室に動物を収容する方法の治療。 </li></ol> 9.2露出の雰囲気のデータが含まれる<ol><li>吸入装置のエアフロー率。 </li><li>の温度と湿度空気。 </li><li>動物ケージの近くにあるエアロゾルのサンプリングゾーンの実際（分析または重量）の濃度。 </li><li>粒度分布、および計算されたカウントメディアン空気力学的直径および幾何標準偏差。 </li><li>なぜ希望チャンバー濃度および/または粒子サイズなどの説明は（該当する場合）を達成し、そして努力がガイドラインのこれらの側面に準拠するために取ることができませんでした。 </li></ol>その他9.3 <ol><li>吸入設備を含む部屋の中でわずかに負圧が吸入暴露室から逃げるの試験材料を防ぐために維持されるべきである。 </li><li>動物性廃棄物の影響を排除するために、毎日暴露室を清掃してください。 </li><li> ELPI、SMPSや他の楽器を洗浄し、ユーザマニュアルに基づいて校正する必要があります。 </li></ol>

<ol>
	<li>Bide, R. W., Armour, S. J., Yee, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allometric+respiration/body+mass+data+for+animals+to+be+used+for+estimates+of+inhalation+toxicity+to+young+adult+humans.">Allometric respiration/body mass data for animals to be used for estimates of inhalation toxicity to young adult humans.</a> J. Appl. Toxicol. 20 (4), 273-290 (2000).</li><li>Guyton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+respiratory+patterns+in+laboratory+animals.">Analysis of respiratory patterns in laboratory animals.</a> Am. J. Physiol. 150, 70-77 (1947).</li><li>Knuckles, T. L., Yi, J., Frazer, D. G., Leonard, H. D., Chen, B. T., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+alters+systemic+arteriolar+vasoreactivity+through+sympathetic+and+cyclooxygenase-mediated+pathways.">Nanoparticle inhalation alters systemic arteriolar vasoreactivity through sympathetic and cyclooxygenase-mediated pathways.</a> Nanotoxicology. , 1-12 (2011).</li><li>Pauluhn, J., Mohr, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+4-week+inhalation+exposure+of+rats:+effect+of+low-,+intermediate,+and+high-humidity+chamber+atmosphere.">Repeated 4-week inhalation exposure of rats: effect of low-, intermediate, and high-humidity chamber atmosphere.</a> Exp. Toxic Pathol. , 178-187 (1999).</li><li>Schmoll, L. H., Elzey, S., Grassian, V. H., O'Shaughnessy, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+aerosol+generation+methods+from+bulk+powders+for+inhalation+exposure+studies.">Nanoparticle aerosol generation methods from bulk powders for inhalation exposure studies.</a> Nanotoxicology. 3, 265-275 (2009).</li><li>To, D., Yin, X., Sundaresan, S., Dave, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deagglomeration+of+nano-particle+aggregates+via+rapid+expansion+of+high+pressure+suspensions.">Deagglomeration of nano-particle aggregates via rapid expansion of high pressure suspensions.</a> AIChE J. 55 (11), 2756-3032 (2009).</li><li>U.S. Environmental Protection Agency (US EPA). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+effects+test+guidelines:+OPPTS.,+870.1300.+Acute+inhalation+toxicity.">Health effects test guidelines: OPPTS., 870.1300. Acute inhalation toxicity.</a> EPA. , 712-C-98-193 (1998).</li><li>Wong, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+feedback+control+of+an+inhalation+exposure+system+with+discrete+sampling+intervals:+testing,+performance,+and+modeling.">Automated feedback control of an inhalation exposure system with discrete sampling intervals: testing, performance, and modeling.</a> Inhal. Toxicol. 15, 729-743 (2003).</li><li>Wong, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhalation+Exposure+Systems:+Design,+Methods+and+Operation.">Inhalation Exposure Systems: Design, Methods and Operation.</a> Toxicologic Pathology. 35, 3-14 (2007).</li><li>Yi, J., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+Aerosol+Generator.">Nanoparticle Aerosol Generator.</a> US patent. , (2011).</li></ol>

本報告書における調査結果と結論は著者のものであり、必ずしも国立労働安全衛生研究所の見解を表すものではありません。任意の会社名または製品の言及はNIOSHによる支持を意味するものではありません。また、代替製品が使用不能、または適切な評価の後に置換することができないことを意味するものではありません。

私たちは、組み立てと全身ナノ粒子エアロゾル吸入暴露系で、ここで説明している。システム機能は、最先端のナノ粒子のエアロゾルの特性評価技術で検証した。新規なナノ粒子のエーロゾル発生システムと、この吸入露光システムは、比較的一貫した温度、湿度、空気の流れ、および実験動物のための酸素含有量を十分に特徴づけられ、制御された均一なナノ粒子のエアロゾル試験雰囲気?...

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Inhalation exposure system</td>
			<td>TSE Systems GmbH, Bad Homburg, Germany</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Air monitoring system</td>
			<td>TSE Systems GmbH, Bad Homburg, Germany</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Titanium dioxide Aeroxide P25</td>
			<td>Evonik, Germany</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning mobility particle sizer-3936L75</td>
			<td>TSI Inc., Shoreview, MN</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Electric low pressure impactor, Standard 10 LPM</td>
			<td>Dekati, Tampere, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ultra Micro Balance, XP2U</td>
			<td>METTLER TOLEDO, Switzerland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Field Emission Scanning Electron Microscope-S-4800</td>
			<td>Hitachi, Japan</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Energy dispersive X-ray analysis </td>
			<td>Princeton Gamma-Tech, Rocky Hill, N.J.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nuclepore polycarbonate filters </td>
			<td>Whatman, Clinton, PA</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PTFE membrane filters </td>
			<td>Pall corporation, Ann Arbor, Michigan </td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

whole-body nanoparticle aerosol inhalation exposures

吸入すると、設計エアロゾル化ナノ材料（ENM）を扱う個人のための最も可能性の高い暴露経路である。適切にナノ粒子の吸入毒性試験を実行するには、チャンバハウジングにおけるエアロゾルは、実験動物が持っている必要があります：1）安定した濃度は全体の暴露期間のために必要なレベルに維持、2）均質な汚染物質の構成、3）安定した&lt;200 nmおよび幾何標準偏差はG &lt;2.5 5σ幾何平均径のサイズ分布。簡単に凝集粒子ので、ナノ粒子を含有するエアロゾルの発生が非常に困難である。これは非常に強力な粒子間力と数十または分割することが困難なサイズ6ミクロン、数百人の中で大規模なフラクタル構造の形成によるところが大きい。ネブライザー、流動床、ベンチュリアスピレーターとライトダストフィードを含むいくつかの一般的なエアロゾル発生器は、我々再テストしたが、いずれも、すべての基準5を満たしているナノ粒子エアロゾルを生成することができませんでした。 全身ナノ粒子エアロゾル吸入暴露システムは、製造され検証され、ナノTiO 2の吸入毒性試験に利用されました。重要なコンポーネント：1）小説ナノTiO 2のエアロゾル発生、2）0.5メートル3全身吸入暴露室、3）監視および制御システム。バルク乾燥ナノTiO 2の粉末（21nmで3.8グラム/ cm 3の嵩密度の一次径）から生成されたナノTiO 2のエアロゾルは90 LPM（10.8換気/時間）の流量で露光チャンバ内に配信された。粒度分布と質量濃度プロファイルを走査型モビリティ粒サイザー（SMPS）、電気低圧インパクタ（ELPI）を用いて連続的に測定した。エアロゾル質量濃度（C）は （ ミリグラム/ m 3）で重量測定法で確認した。質量（M）集めた粒子はM 前とM ポストサンプリング（mg）を前後のフィルタの質量であるM =（M ポスト M 前 ）として決定した。質量濃度は次のように計算されたC = M Qは流量（3メートル/分 ） をサンプリングしている/（Q * t）は、tはサンプリング時間（ 分 ）である。チャンバ圧力、温度、相対湿度（RH）、O 2及びCO 2濃度を連続的に監視し、制御した。 Nucleporeフィルター上に集め、ナノTiO 2のエアロゾルは、走査型電子顕微鏡（SEM）およびエネルギー分散型X線（EDX）分析で分析した。 1）安定した質量濃度を、汚染物質のフリー2）均一な組成物、3）カウント中位空力安定した粒度分布を要約すると、我々は我々の曝露チャンバーに生成され、配信ナノ粒子エアロゾルが持っていることを報告エアロゾル生成時に157ナノメートルの直径namic。このシステムは、確実に、繰り返し、労働環境や国内ENMエアロゾル曝露を模擬試験雰囲気を作成します。

吸入暴露試験では、通常、試験材料8,9の定義された濃度に実験動物を露出させ、既知および一定テスト環境で実験動物を維持することが含まれます。全身吸入ナノ粒子露光装置は、 図1に示されている。チャンバを通る空気の連続流90 LPMがあった場合に全身チャンバは、動的フローに基づいて運転した。この空気の流れは、急性吸入暴露7のために米国環境保護...

Watch this Scientific Journal Video about Whole-Body Nanoparticle Aerosol Inhalation Exposures at JoVE.com

Whole-Body Nanoparticle Aerosol Inhalation Exposures

全身ナノ粒子エアロゾル吸入暴露施設は、ナノサイズの二酸化チタン（酸化チタンのために構築した<sub&gt; 2</sub&gt;）の吸入毒性試験。このシステムは、ナノ酸化チタンを提供する<sub&gt; 2</sub持っている&gt;エアゾール試験雰囲気：1）安定した質量濃度、汚染のない2）均一な組成物、及びエアロゾル生成中3）安定した粒度分布。

全身ナノ粒子エアロゾル吸入暴露

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

whole-body-nanoparticle-aerosol-inhalation-exposures

Research

JoVE Journal

Biology

15.7K ビュー.  West Virginia University. 全身ナノ粒子エアロゾル吸入暴露施設は、ナノサイズの二酸化チタン（酸化チタンのために構築した 2）の吸入毒性試験。このシステムは、ナノ酸化チタンを提供する 2エアゾール試験雰囲気：1）安定した質量濃度、汚染のない2）均一な組成物、及びエアロゾル生成中3）安定した粒度分布。

ビデオ: Whole-Body Nanoparticle Aerosol Inhalation Exposures

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

拘束されていない全身プレチスモグラフィを使用したマウスにおける呼吸機能の測定

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

生物学

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

病気のベクトル蚊幼虫におけるキトサン/干渉RNAナノ粒子媒介遺伝子サイレンシング

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

比色金ナノ粒子アッセイに適用される構造スイッチングアプタマーを選択するための方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

インフィールドアプリケーションの設計およびアプタマー金ナノ粒子基づく比色アッセイの開発

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

毒性試験のための空気中の粒子への現実的な反復吸入暴露のための気管支上皮モデル

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

オルソ電圧(40-300 kV)X線設備を用いた細胞照射用線量測定

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

Construction of a Realistic, Whole-Body, Three-Dimensional Equine Skeletal Model using Computed Tomography Data

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches have rarely been used to study equine athletic injury. Degenerative osteoarthritis caused by mechanical stress can originate from chronic postural dysfunction, which, because the primary dysfunction is often distant from the site of tissue injury, is best identified through modeling whole-body biomechanics. To characterize whole-body equine kinematics, a realistic skeletal model of a horse was created from equine computed tomography (CT) data that can be used for functional anatomical and biomechanical modeling. Equine CT data were reconstructed into individual three-dimensional (3D) data sets (i.e., bones) using 3D visualization software and assembled into a complete 3D skeletal model. The model was then rigged and animated using 3D animation and modeling software. The resulting 3D skeletal model can be used to characterize equine postures associated with degenerative tissue changes as well as to identify postures that reduce mechanical stress at the sites of tissue injury. In addition, when animated into 4D, the model can be used to demonstrate unhealthy and healthy skeletal movements and can be used to develop preventative and rehabilitative individualized therapies for horses with degenerative lamenesses. Although the model will soon be available for download, it is currently in a format that requires access to the 3D animation and modeling software, which has quite a learning curve for new users. This protocol will guide users in (1) developing such a model for any organism of interest and (2) using this specific equine model for their own research questions.

The purpose of this protocol is to describe the method of creation of a realistic, whole-body, skeletal model of a horse that can be used for functional anatomical and biomechanical modeling to characterize whole-body mechanics.

コンピュータ断層撮影データを用いたリアルな全身3次元馬骨格モデルの構築

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

細胞外小胞の定量とサイズ決定のためのナノ粒子追跡解析

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

ナノハーブコーティング生地の抗菌効果

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

著者スポットライト:エッセンシャルオイルのナノハーブカプセル化を使用した抗菌生地  

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

エッセンシャルオイルのナノハーブカプセル化を使用した抗菌ファブリック

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...