The authors thank Therwa Hamza, John E. Tidwell, Nina Clovis, and Suzanne Smith for experimental assistance and Suzanne Smith for proofreading. The authors also thank John Thomas, PhD for providing the bacterial clinical isolates and John B. Barnett, PhD for his support and the use of the biological safety lab at the Department of Microbiology, Immunology and Cell Biology at West Virginia University. The authors acknowledge financial support from the Osteosynthesis and Trauma Care Foundation and National Science Foundation (#1003907). Microscope experiments and image analysis were also performed in the West Virginia University Imaging Facility, which is supported in part by the Mary Babb Randolph Cancer Center and NIH grant P20 RR016440.
Animal use for blood draws were approved by the West Virginia University Institutional Animal Care and Use Committee. All experiments were executed in compliance with all relevant guidelines, regulations, and regulatory agencies.

1。 PRPの作製とその活性化 1.1採血<ol><li>イソフルランの吸入（誘導のためのO 2中2％、保守のための1％）でウサギを麻酔。 </li><li> 20mlのシリンジに2ミリリットル0.129 Mのクエン酸三ナトリウムを（anticoagulentソリューション）を描画します。クエン酸三ナトリウム溶液を50 mlの蒸留H 2 O中の1.897グラムクエン酸三ナトリウムを溶解し、0.22μmの滅菌フィルターで濾過することによって調製される。 </li><li> 70％エタノールを用いてウサギの耳を殺菌。 </li><li>翼状針（25g）をシリンジに接続を介して、ウサギの耳静脈から（ 例えば 5ミリリットル）の血を引く。 </li><li>穏やかな攪拌によってクエン酸三ナトリウム溶液を用いて血液を混ぜる。血液とクエン酸三ナトリウム水溶液の体積比は9:1である。 </li></ol> 1.2 PRPの調製（ 図1） <ol><li> 50mlプラスチック遠心管に抗凝固処理血液を転送します。 10μlのアリコートを取る血球計算法を用いてベースライン血小板数を決定するために、血液。 </li><li>スイングアウトローター付き遠心機で室温（RT）で10分間、300×gで血液を遠心分離します。低い（ 図1）への加速とブレーキの速度を設定します。 </li><li>遠心後、血液は3層に分かれています。底層は、中間層（一般的に &quot;バフィーコート&quot;と呼ばれる）は、主に赤血球で濃縮血小板や白血球から構成され、最上位層は、主に血液の液体成分である血漿、血小板である（ 図1）。細胞培養用フードに遠心管を慎重に輸送;層を乱すことがありません。 1ミリリットルプラスチックピペットを用いて、15​​ mlプラスチックチューブに、血漿、バフィーコート、および2-3 mm厚赤血球層のすべてを転送します。 </li><li>室温で15分間3000 xgで転送サンプルをもう一度遠心します。上層（上清）を乏血小板血漿（PPP）と見なされますdは、新しいチューブに移す。 </li><li> 2.0×10 6血小板/μlを（血球計算法によって決定される）を得るために、PPPを使用して、残りの血液サンプル中の血小板濃度を調整することによって、PRPを得る。 </li></ol> 1.3 PRPの活性化<ol><li> 1,000 IU / mlの濃度になるように取り組んで5ミリリットル10％塩化カルシウムで5,000 IUウシトロンビンを溶解させることによって、PRPの活性化溶液を調製します。 </li><li> PRPとPPPにライセンス認証ソリューションを追加して、繰り返して、PRP-およびPPP-ゲルを形成するために、ピペッティングにより溶液を混ぜて使用する。 PRPまたはPPPへの活性化溶液の体積比は1:4です。 </li></ol> 2キルカーブアッセイを使用して、PRP の in vitro抗菌試験（ 図2） では、 <ol><li>無菌接種ループを使用して、Sのいくつかのコロニーを追加プラスチックチューブでミューラーヒントンブロス（MHB）5mlにその晩平板培養から黄色ブドウ球菌 。 Vortexは簡潔にしてから、37℃で2時間サンプルをインキュベート次の、細菌のメディアの光学濃度を分光光度計を用いて決定し、予め決められた標準曲線に基づいて〜1×10 8 cfu / mlに等しい光学密度に調整した。 </li><li> 1×10 6 CFU / mlを取得し、氷の上で接種を配置するPBSを用いて100倍希釈を作る。 </li><li>セットアップおよび滅菌、使い捨て5ミリリットル丸底ポリスチレンチューブにラベルなど、各チューブに2 mlの最終容積については、 表1に示されている以下のサンプル·グループを準備。 </li><li>活性化のためのトロンビン溶液（ゲル形成）が続いポリスチレンチューブにPRP、PPP、またはPBSを最初に追加します。次に、MHBを追加してから、S.球菌接種（1×10 6 CFU / ml）を1×10 5 CFU / mlの最終濃度を得ることができる。 </li><li> ℃で150rpmで攪拌​​しながら軌道37でチューブをインキュベートする。 </li><li>予め決められた時点（ 例えば 0,1、および2時間）で、繰り返しピペッティング（これを経由して各チューブ内の溶液を混合細菌は、PRPゲルの内部に閉じ込められるかもしれないので、ステップ）が重要である。 、サンプルの10μlを取り滅菌0.9％生理食塩水で順次希釈し、トリプシン大豆寒天（TSA、5％羊血液付き）CFUカウントのためのプレート上に各希釈液の100μlアリコートをピペット。 </li><li> 37℃で一晩培養寒天プレート°Cは、その後プレートのコロニーをカウントし、記録します。 y軸上のx軸およびCFU / mlの上の時間（hr）とlogarithimicスケールでデータをプロットします。 </li></ol>

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著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

多血小板血漿は、ますますその治癒促進特性15から17に起因する臨床応用に使用されている。本研究では、PRPは、感染予防のための新しいアプローチとして提示された。 PRPは、MRSA、MSSA、A群連鎖球菌 、および淋菌に対しても強い抗菌性を有することが見出された。感染予防のための従来の抗生物質治療と比較して、PRPの主な利点は、以下を含む：（1）現在の抗生物質療?...

インプラント関連感染は重要な臨床合併症である。 黄色ブドウ球菌 （ 黄色ブドウ球菌 ） は、インプラント関連感染症から分離された最も一般的な微生物の一つである。これは、インプラントの表面をカバーし、抗生物質耐性感染1,2につながる可能性がバイオフィルムを製造することができる。インプラント関連感染症の治療は、頻繁に繰り返さdebridementsかつ長期の非経口抗生物質治療のために長期入院を必要とします。抗生物質耐性例では、インプラントの除去が必要になることがあります。抗生物質に対する細菌の耐性が上昇するとしても、疾病管理予防センター（CDC）によって参照されている &quot;世界で最も緊急の健康問題の一つ。&quot;時間では、新しい効果的な抗菌治療法の開発がなければ、多剤耐性病原体は、従来の抗生物質による治療不可能となる可能性があります。インプラント関連の防止感染症は、ことが重要であると新規予防剤またはアプローチはこのような感染症を予防するために必要とされる。 多血小板血漿（PRP）は、骨と骨移植の治癒3から5を助けることができる30以上の成長因子が含まれている自己血の濃度である。骨再生や軟組織の成熟を高めるために、PRPの応用はますますため血小板がリリースし、様々な成長因子のその高濃度の診療所で報告されています。 PRPのいくつかの特徴では、PRPも抗菌特性6-9を持っているかもしれないことを示します。 PRPは、血小板の数が多い、白血球の高濃度（細菌および真菌に対する宿主防御アクションを持っているかもしれない）、複数の抗菌ペプチド7,8,10が含まれています。心臓外科患者の大規模コホートの最近の研究では、創傷閉鎖signif時PRPゲルの術中使用することが明らかになったicantly浅と深い胸骨感染11の発生率を減少させた。これらの理由及び観察のために、私たちは、PRPは、そのよく研究された治癒促進特性以外にも、抗菌特性を持っているという仮説を立てた。感染を防ぐためにPRPを使用しての潜在的な利点があり得る：（i）PRPが従来の抗生物質治療に比べて抵抗性を誘発する可能性が低くなります。 （ⅱ）PRPがまた感染予防に相乗効果があるかもしれ治癒を促進する性質をもっています。PRPの治癒促進特性は、病原体とヒトの細胞がインプラント表面12にはレースをしていると感染の確率を減らすことにより、細菌の付着を防止するためにシールを提供することができます、13。 （ⅲ）PRPは本質的に、生体適合性、安全かつ伝染性疾患のリスクがない。 私たちの長期的な目標は、インプラント関連infectiを防ぐための新たなアプローチとして、PRPを使用することですアドオン。本研究の目的は、PRP は 、in vitro抗菌特性の PRPのを調べるために、二回遠心分離のアプローチを使用して、そのような抗菌性を評価するためのプロトコルを記述するために準備することでした。

<table><tbody><tr><td>Bovine thrombin</td><td>King Pharmaceuticals, Inc</td><td>60793-215-05</td><td>Thrombin (bovine origin)</td></tr><tr><td>Calcium chloride</td><td>King Pharmaceuticals, Inc</td><td>60793-215-05</td><td>10% calcium chloride</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>1001936060</td><td></td></tr><tr><td>Mueller Hinton broth</td><td>Becton, Dickinson and Company</td><td>275710</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma-Aldrich</td><td>D8662</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Sigma-Aldrich</td><td>W302600</td><td></td></tr><tr><td>Tryptic soy agar</td><td>Fisher Scientific</td><td>R01202</td><td></td></tr><tr><td>Centrifuge</td><td>Kendro Laboratory Products</td><td>750043077</td><td></td></tr><tr><td>Syringe filter</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr></tbody></table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

インプラント関連感染が増加して抗生物質耐性、動物と人間の間に抗生物質耐性菌の伝送、および感染症の治療コストの高さに起因する世界的なヘルスケア産業にますます困難になってきています。 そこで本研究では、多血小板血漿（PRP）の潜在的な抗菌特性に基づいてインプラント関連感染の予防に有効である可能性がある新たな戦略を開示している。治癒を促進するためのよく研究された特性のために、PRP（生物学的製剤）はますます整形外科手術、歯と口腔の手術、顎顔面手術、整形手術、スポーツ医学などを含む臨床応用に使用されている PRPはインプラント関連感染の防止に従来の抗生物質治療に高度な代替手段である可能性があります。 PRPを使用することにより、従来の抗生物質治療Siに比べて有利であるかもしれないPRPのNCE抗生物質耐性を誘導する可能性が低いとPRPの抗菌と癒しの促進特性は感染予防上の相乗効果があるかもしれません。それも病原体とヒトの細胞がインプラント表面のためにレースをしていることが知られており、治癒を促進するのPRPのプロパティには、それによって感染の確率を減らす人間の細胞接着を向上させることができます。また、PRPは本質的に、生体適合性、安全かつ伝染性疾患の危険性から自由である。 我々の研究のために、私たちは一般的に整形外科の感染症で発見され、PRPは、in vitroでこれらの細菌に対する抗菌性を持っているかどうかを検討されているいくつかの臨床菌株を選択しました。我々は、同じ血小板濃度がすべてのサンプルを得ることができるようになり二回遠心分離のアプローチを使用してPRPを用意しました。我々は、一貫性のある抗菌性の調査結果を達成し、PRPは、in vitroでメティのような細菌に対する抗菌性を強いていることを発見したウイルスバスター感受性およびメチシリン耐性黄色ブドウ球菌、A群連鎖球菌 、および淋菌 。したがって、PRPの使用は、感染を防ぐために、インプラント関連感染の高価な術後治療の必要性を低減する可能性を秘めているかもしれません。

PRPは、再現性回遠心分離法（ 図1）を用いて調製される。 PRPは、メチシリン耐性S.に対する in vitro抗菌特性で （CFUsで最大100倍の減少）は、強力な存在が判明した一般的に世界中の病院14に見られる黄色ブドウ球菌 （MRSA）（ 図3）。同様に、PRPはメチシリン感受性球菌に対して強い抗菌性を持って黄色ブドウ球菌 （MSSA）、A?...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

インプラント関連感染は重要な臨床合併症である。この研究は、インプラント関連感染を防ぐために、多血小板血漿（PRP）を用いたアプローチを説明し、一定の血小板濃度でPRPを調製するためのプロトコルを提示して、PRPの新たに同定された抗菌性や抗菌性を調べるための関連プロトコルを報告<emin vitroで&gt;。</em

感染を防ぐための新しいアプローチとして、PRP：準備と<em&gt;インビトロ</emPRPの&gt;抗菌特性

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

prp-as-new-approach-to-prevent-infection-preparation-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K ビュー.  West Virginia University. インプラント関連感染は重要な臨床合併症である。この研究は、インプラント関連感染を防ぐために、多血小板血漿（PRP）を用いたアプローチを説明し、一定の血小板濃度でPRPを調製するためのプロトコルを提示して、PRPの新たに同定された抗菌性や抗菌性を調べるための関連プロトコルを報告。

ビデオ: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Treatment of Platelet Products with Riboflavin and UV Light: Effectiveness Against High Titer Bacterial Contamination

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. Products are routinely stored at 22 &#176;C on an agitating shaker, a condition that can promote bacterial growth. Although the total number of bacteria believed to be introduced into a platelet product is extremely low, these bacteria can multiply to a very high titer prior to transfusion, potentially resulting in serious adverse events. The aim of this study was to evaluate a riboflavin based pathogen reduction process against a panel of bacteria that have been identified as common contaminants of platelet products. This panel included the following organisms: S. epidermidis, S. aureus, S. mitis, S. pyogenes, S. marcescens, Y. enterocolitica, B. neotomae, B. cereus, E. coli, P. aeruginosa and K. pneumoniae. Each platelet unit was inoculated with a high bacterial load and samples were removed both before and after treatment. A colony forming assay, using an end point dilution scheme, was used to determine the pre-treatment and post-treatment bacterial titers. Log reduction was calculated by subtracting the post-treatment titer from the pre-treatment titer. The following log reductions were observed: S. epidermidis 4.7 log (99.998%), S. aureus 4.8 log (99.998%), S. mitis 3.7 log (99.98%), S. pyogenes 2.6 log (99.7%), S. marcescens 4.0 log (99.99%), Y. enterocolitica 3.3 log (99.95%), B. neotomae 5.4 log (99.9996%), B. cereus 2.6 log (99.7%), E. coli &#8805;5.4 log (99.9996%), P. aeruginosa 4.7 log (99.998%) and K. pneumoniae 2.8 log (99.8%). The results from this study suggest the process could help to lower the risk of severe adverse transfusion events associated with bacterial contamination.

The storage conditions of platelet products create an ideal environment for bacterial contaminants to multiply to dangerous levels. The goal of this study was to use a colony forming assay to evaluate a riboflavin based pathogen reduction process against high titer bacterial contamination in human platelet products.

リボフラビンとUVライトと血小板製品の治療：高力価の細菌汚染に対する有効

Contamination of platelet units by bacteria has long been acknowledged as a significant transfusion risk due to their post-donation storage conditions. ...

免疫・感染学

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医療機器のための細菌撥ポリマーのハイスループット同定

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

生物工学

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非正規のアミノ酸類の選択的な圧力の取り込みによって生成される抗菌ペプチド

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

バイオ エンジニア リング アプリケーション用先端材料の抗菌特性

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

細菌の光力学的不活性化における光活性分子を評価するためのLEDベース In Vitro スクリーニング

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

生化学

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

浄化、体外(p) ppGppクロストリジウム ・ ディフィシルから合成酵素の活性

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

抗生物質耐性プラットフォームを用いた抗生物質脱複製

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

in vitroでのナノ粒子およびナノ構造表面の抗菌活性の評価

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

<meta charset="utf8"></head><body>ポイントオブケア使用のための最適化されたCRISPRベースの分子診断プロトコル

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

<meta charset="utf8"></head><body>著者スポットライト:感染症の迅速な検出のための簡易CRISPRベースの検査の開発

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

プレミックス試薬とフリーズドライ試薬によるポイントオブケアCRISPRベースの診断

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...

感染を防ぐための新しいアプローチとして、PRP：準備と

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リボフラビンとUVライトと血小板製品の治療：高力価の細菌汚染に対する有効

医療機器のための細菌撥ポリマーのハイスループット同定

非正規のアミノ酸類の選択的な圧力の取り込みによって生成される抗菌ペプチド

バイオエンジニアリングアプリケーション用先端材料の抗菌特性

細菌の光力学的不活性化における光活性分子を評価するためのLEDベース In Vitro スクリーニング

浄化、体外(p) ppGppクロストリジウム・ディフィシルから合成酵素の活性

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プレミックス試薬とフリーズドライ試薬によるポイントオブケアCRISPRベースの診断

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