1。細菌培養物<ol><li>グラム陰性P.緑膿菌株PAO1は、18時間37℃でルリアブロス（LB）中で増殖させた。 </li><li>細胞の培養物を5分間、7,500 rpmで（毎分ラウンド）で遠心分離し、上清を除去した。 </li><li>細菌を、10％LBに再懸濁し、培養が定常期を再入力することを可能にする別の2時間のために再成長させた。 </li><li>細菌懸濁液は、2つのグループに分けた：最初のグループ（2本）のない抗生物質我々はゲンタマイシン抗生物質（50μgの/ ml）を添加番目のグループに加えた。 </li></ol> 2。コロニー形成単位の判定（CFU） <ol><li>細胞生存率を決定するために、20μlのサンプルを、24時間の時間枠内でおよそ2時間の実験から採取した。サンプルの連続希釈が行われ、めっきさLB寒天プレート上、37℃で一晩インキュベートした。 </li><li>プラットフォームごとのCFUを各治療のため電子を測定し、比較する期間、様々な処置の間で行われた。式で説明したようCFUにおける対数減少を算出した。 （1）： ログイン削減= LOGU-LogC [CFU / mlの] ここで、Uは、各時点で単位値をコロニー形成であり、CFUに等しいCFU / mlの単位中にコロニー形成単位である。 CFU / mlの=（コロニーのx希釈係数の数）/（体積接種） 及びCは、開始時にコントロールサンプルにおいて見出さCFUである。 Uは、測定時点で要因を形成するコロニーを指定することに注意してください。 </li><li>それらのそれぞれの中の細菌の濃度が10分の1に減少しながら希釈係数は、希釈液の数である。接種量は常に200マイクロリットルであり、それは、我々の試験管のサイズに関係している。 </li></ol>したがって、ビューの濃度点を要約する、ゲンタマイシン抗生物質、50μgの/ mlの濃度であった。の終わりまで、細菌に関してプロセスは、我々は全体の8希釈していた。各希釈は10倍であり、それは、200μlにチューブ内で行われていた。出発点は、200μlのチューブに入れたサンプル20μlのだった（その結果、初期濃度は20/200C 0だったC 0と= 0.1C0サンプルを20μlの初期濃度である）、最終濃度が8減少した8希釈に起因桁違い。 3。照明<ol><li> CWのNd：YAGレーザー（532 nmおよび200 mWの平均光パワーの波長）の光、50％/ 50％ビームスプリッタを使用して二つの光路に分割された。ビーム径は約10mmであった。露出時間は24時間であった。 </li><li> QスイッチパルスNd：YAGレーザー（波長532nm、300mWの2.5 MWの光ピークパワーの平均電力）が、光の50％/ 50％ビームスプリッタを用いて2つの経路に分割された。スポット径は6であった。 Qスイッチレーザのパルス幅は6ナノ秒であり、繰り返しレートが15Hzのであった。ザ平均電力密度は106 MW / cm 2であった、ピークパワー密度は8.83キロワット/ mm 2であった。露出時間は24時間であった。 </li></ol>細菌懸濁液は、照射中に攪拌し、それが細菌の増殖（全てのチューブに細菌が成長できるようにするルリアのブロス培地があった）のために適切な培養条件下で維持されたことに注意してください。

<ol>
	<li>Feuerstein, O., Persman, N., Weiss, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxic+Effect+of+Visible+Light+on+Porphyromonas+gingivalis+and+Fusobacterium+nucleatum:+An+In+Vitro+Study.">Phototoxic Effect of Visible Light on Porphyromonas gingivalis and Fusobacterium nucleatum: An In Vitro Study.</a> Photochemistry and Photobiology. 80, 412-415 (2004).</li><li>Enwemeka, C. S., Williams, D., Enwemeka, S. K., Hollosi, S., Yens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+470-nm+light+kills+methicillin-resistant+Staphylococcus+aureus+(MRSA)+in+vitro.">Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro.</a> Photomed. Laser Surg. 27, 221-226 (2009).</li><li>Guffey, J. S., Wilborn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+bactericidal+effects+of+405-nm+and+470-nm.">In vitro bactericidal effects of 405-nm and 470-nm.</a> Photomed. Laser Surg. 24, 684-688 (2006).</li><li>Lipovsky, A., Nitzan, Y., Friedman, H., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Staphylococcus+aureus+strains+to+broadband+visible+light.">Sensitivity of Staphylococcus aureus strains to broadband visible light.</a> Photochem. Photobiol. 85, 255-260 (2008).</li><li>Lipovsky, A., Nitzan, Y., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+Mechanism+for+visible+light+induced+wound+healing.">A possible Mechanism for visible light induced wound healing.</a> Lasers in Surgery and Medicine. 40, 509-514 (2008).</li><li>Lipovsky, A., Nitzan, Y., Gedanken, A., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visible+light-induced+killing+of+bacteria+as+a+function+of+wavelength:+Implication+for+wound+healing.">Visible light-induced killing of bacteria as a function of wavelength: Implication for wound healing.</a> Lasers in Surgery and Medicine. 42, 467-472 (2010).</li><li>Feuerstein, O., Ginsburg, I., Dayan, E., Veler, D., Weiss, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Visible+Light+Phototoxicity+on+Porphyromonas+gingiwalis+and+Fusobacferium+nucleaturn.">Mechanism of Visible Light Phototoxicity on Porphyromonas gingiwalis and Fusobacferium nucleaturn.</a> Photochemistry and Photobiology. 81, 1186-1189 (2005).</li><li>Nussbaum, E. L., Lilge, L., Mazzulli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+630-,+660-,+810-,+and+905-nm+laser+irradiation+delivering+radiant+exposure+of+1-50+J/cm2+on+three+species+of+bacteria+in+vitro.">Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm2 on three species of bacteria in vitro.</a> J. Clin. Laser Med. Surg. 20, 325-333 (2002).</li><li>Dadras, S., Mohajerani, E., Eftekhar, F., Hosseini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+Photoresponses+of+Staphylococcus+aureus+and+Pseudomonas+aeruginosa+to+514,+532,+and+633+nm+Low+Level+Lasers+In+Vitro.">Different Photoresponses of Staphylococcus aureus and Pseudomonas aeruginosa to 514, 532, and 633 nm Low Level Lasers In Vitro.</a> Current Microbiology. 53, 282-286 (2006).</li><li>Stover, C. K., Pham, X. Q., Erwin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+Pseudomonas+aeruginosa+PAO1,+an+opportunistic+pathogen.">Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.</a> Nature. 406, 952-964 (2000).</li><li>Hamblin, M. R., Demidova, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+low+level+light+therapy.">Mechanisms of low level light therapy.</a> Proc. SPIE. 6140, 1-12 (2006).</li><li>Krespi, Y. P., Stoodley, P., Hall-Stoodley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+disruption+of+biofilm.">Laser disruption of biofilm.</a> Laryngoscope. 118, 1168-1173 (2008).</li><li>Reznick, Y., Banin, E., Lipovsky, A., Lubart, R., Zalevsky, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+laser+light+enhancement+of+susceptibility+of+bacteria+to+gentamycin+antibiotic.">Direct laser light enhancement of susceptibility of bacteria to gentamycin antibiotic.</a> Opt. Commun. 284, 5501-5507 (2011).</li></ol>

利害の衝突は宣言されていない。

光線療法は、治療多くの疾患のための有望なアプローチとして浮上し、近年では先進的な学際的研究分野となっています。この文脈において、可視領域の光の使用が広く研究されている。例えば、感染した創傷を殺菌のために、強い可視光にさらすことによって、より効果的に治癒することができることが見出された。このアプローチのための作用機構は、細菌を殺す11光誘?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Lauria Broth</td>
 <td>Difco</td>
 <td>241420</td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamycin</td>
 <td>Sigma </td>
 <td>G1914</td>
 <td></td>
 </tr>
 <tr>
 <td>Bacto Agar</td>
 <td>Difco </td>
 <td>231710 </td>
 <td></td>
 </tr>
 </tbody>
</table>

the synergistic effect of visible light and gentamycin on pseudomona aeruginosa microorganisms

最近では、可視光の殺菌効果に関するいくつかの出版物、それらのほとんどは、スペクトルのその青の部分を主張があった（400 NM-500 nm）の様々な病原体1-5を殺すために責任があります。青色光の光毒性効果は、光誘起反応性酸素種（ROS）主に青色領域4,6,7の光を吸収する内因性光感受性物質、細菌による形成の結果であることが示唆された。赤と近赤外8と同様に緑色の光9の殺菌効果の報告もあります。 本研究では、我々は我々が緑膿菌での高出力緑色（波長532nm）の連続（CW）とパルスQスイッチ（QS）光の影響を特徴づけるために許可された方法を開発した。この方法を用いて、我々はまた、細菌の生存に抗生物質治療（ゲンタマイシン）と組み合わせて緑色の光の効果を検討した。P.緑膿菌は、ACですommon noscomial日和見病原体は、様々な疾患を引き起こす。株は、様々な抗生物質に対してかなり耐性があり、多くの予測AcrB /メックス型RND排出システム10を多剤が含まれています。 この方法は、抗生物質、ゲンタマイシンを加えてとせずにQスイッチおよび/ ​​またはCWレーザに露出ルリアブロス（LB）培地中で増殖させ、（ 緑膿菌株 PAO1）自由生活固定相グラム陰性菌を利用した。細胞生存率は、異なる時点で測定した。得られた結果は、それだけでレーザー治療が唯一のPの生菌数は0.5 log減少をもたらした未処理の制御とそれだけでゲンタマイシン治療に比べて細胞の生存率を減少させなかった示した緑膿菌 。合成レーザおよびゲンタマイシン処理は、しかしながら、P.の相乗効果及び生存率をもたらし緑膿菌は、8ログの減少した。 提案手法では、さらに実装することができます同時に光で面積を照射しながら感染臓器に抗生物質溶液を注入することができる装置のようなカテーテルの開発を介しmented。

レーザーベースのセットアップを図1に概略的に示されている。第1実験条件は、CWのNd利用：とは200mWの平均光パワー：YAGレーザは、波長532nmの（YAGのNdの第2高調波）を有する。このビームは、各分割ビームが100mWでのパワーを持っていたこのような光の50％/ 50％ビームスプリッタを使用して二つの光路に分割された。ビーム径は約10mmであったため、電力密度は、約100mWの/ cm 2</s...

Watch this Scientific Journal Video about The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms at JoVE.com

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

我々は、抗生物質治療（ゲンタマイシン）と組み合わされて、連続またはパルス可視レーザーベースの治療を含むその発達生物医学装置の生存率の減少につながる統計的に有意な相乗効果で結果を表示する<em&gt; P.緑膿菌</em8ログが単独の抗生物質による治療と比較していることで&gt; PAO1、。

上の可視光およびゲンタマイシンの相乗効果<em&gt; Pseudomona緑膿菌</em&gt;微生物

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

the-synergistic-effect-visible-light-gentamycin-on-pseudomona

Research

JoVE Journal

Immunology and Infection

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

13.3K ビュー.  Bar-Ilan University. 我々は、抗生物質治療（ゲンタマイシン）と組み合わされて、連続またはパルス可視レーザーベースの治療を含むその発達生物医学装置の生存率の減少につながる統計的に有意な相乗効果で結果を表示する P.緑膿菌 PAO1、。

ビデオ: The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培養モデルの緑膿菌</emライブヒト気道細胞上に成長した>バイオフィルム

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

免疫・感染学

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

のRNAの単離緑膿菌マウス消化管に定着

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

生物発光細菌を使用してリアルタイムに急性肺炎のマウスモデルにおける肺炎球菌の毒性因子の影響を以下の

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

緑膿菌誘発肺傷害モデル

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

マクロファージコレステロール枯渇との食作用への影響<em&gt;クリプトコックス·ネオフォルマンス</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles (MPs) are one such type of vesicles, ranging in diameter from 0.1 &#956;m to 1 &#956;m, and typically shed from the plasma membrane of eukaryotic cells undergoing activation or apoptosis. Here we describe the generation of T lymphocyte&#8211;derived microparticles (LMPs) from apoptotic CEM T cells stimulated with actinomycin D. LMPs are isolated through a multistep differential centrifugation process and characterized using flow cytometry. This protocol also presents an in situ cell death detection method for demonstrating the proapoptotic effect of LMPs on bronchial epithelial cells derived from mouse primary respiratory bronchial tissue explants. Methods described herein provide a reproducible procedure for isolating abundant quantities of LMPs from apoptotic lymphocytes in vitro. LMPs derived in this manner can be used to evaluate the characteristics of various disease models, and for pharmacology and toxicology testing. Given that the airway epithelium offers a protective physical and functional barrier between the external environment and underlying tissue, use of bronchial tissue explants rather than immortalized epithelial cell lines provides an effective model for investigations requiring airway tract tissue.

Cell membrane&#8211;shed microparticles (MPs) are active biological vesicles that can be isolated and their pathophysiological effects investigated in various models. Here we describe a method for generating MPs derived from T lymphocytes (LMPs) and for demonstrating their proapoptotic effect on airway epithelial cells.

気道上皮細胞上でのアポトーシス促進効果のリンパ球微粒子および検出の生成

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

コットンボールワームでデンソウイルスのホストフィットネス上のホスト組織分布、伝送モード、および効果を調査するためのプロトコル

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

光側に来る: In Vivo糖尿病ヘアレス マウス モデルにおける慢性創傷における緑膿菌バイオ フィルム感染症の監視

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

発注済み、緑膿菌ひずみ PA14 トランスポゾン挿入変異体の非冗長ライブラリのレプリケーション

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...