博士。コペンハーゲン大学（デンマーク）からCJP Grimmelikhuijzenとマイケルウィリアムソンは、イクオリンはプラスミドの提供のために高く評価されています。私達の協力者、ARS - USDA（米国テキサス州）からのロナルドJ. Nachmanは、ペプチド合成のためとNOVOstarプレートリーダーを提供するための知られています。

<ol>
	<li>Kozak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+5'-noncoding+sequences+from+699+vertebrate+messenger+RNAs.">An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.</a> Nucleic Acids Research. 15, 8125-8148 (1987).</li><li>Nachman, R., Pietrantonio, P. V., Geary, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+mimetic+analogs+of+insect+kinin+neuropeptides+with+arthropod+receptors.">Interaction of mimetic analogs of insect kinin neuropeptides with arthropod receptors.</a> Neuropeptide systems as Targets for Parasite and Pest Control. , (2010).</li><li>Nachman, R. J., Pietrantonio, P. V., Coast, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+the+development+of+novel+pest+management+agents+based+upon+insect+kinin+neuropeptide+analogs.">Towards the development of novel pest management agents based upon insect kinin neuropeptide analogs.</a> Annals of the New York Academy of Sciences. , 1163-11251 (2009).</li><li>Taneja-Bageshwar, S., Strey, A., Isaac, R. E., Coast, G. M., Zubrzak, P., Pietrantonio, P. V., Nachman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biostable+agonists+that+match+and/or+exceed+the+activity+of+insect+kinins+on+recombinant+arthropod+GPCRs.">Biostable agonists that match and/or exceed the activity of insect kinins on recombinant arthropod GPCRs.</a> General and Comparative Endocrinology. 162, 122-128 (2009).</li><li>Taneja-Bageshwar, S., Strey, A., Kaczmarek, K., Zabrocki, J., Pietrantonio, P. V., Nachman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+insect+kinin+analogs+with+cis-peptide+bond,+type+VI-turn+motifs+identifies+optimal+stereochemistry+for+interaction+with+a+recombinant+arthropod+kinin+receptor+from+the+southern+cattle+tick+Boophilus+microplus.">Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus.</a> Peptides. 29, 295-301 .</li><li>Taneja-Bageshwar, S., Strey, A., Zubrzak, P., Williams, H., Reyes-Rangel, G., Juaristi, E., Pietrantonio, P. V., Nachman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+selective+and+non-selective,+biostable+beta-amino+acid+agonists+of+recombinant+insect+kinin+receptors+from+the+southern+cattle+tick+Boophilus+microplus+and+mosquito+Aedes+aegypti.">Identification of selective and non-selective, biostable beta-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti.</a> Peptides. 29, 302-309 .</li><li>Taneja-Bageshwar, S., Strey, A., Zubrzak, P., Pietrantonio, P. V., Nachman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+structure-activity+analysis+of+insect+kinin+core+analogs+on+recombinant+kinin+receptors+drom+southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+and+mosquito+Aedes+aegypti+(Diptera:+Culicidae).">Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors drom southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae).</a> Archives of Insect Biochemistry and Physiology. 62, 128-140 (2006).</li><li>Pietrantonio, P. V., Jagge, C., Taneja-Bageshwar, S., Nachman, R. J., Barhoumi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mosquito+Aedes+aegypti+(L.)+leucokinin+receptor+is+a+multiligand+receptor+for+the+three+Aedes+kinins.">The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins.</a> Insect Molecular Biology. 14, 55-67 (2005).</li><li>Holmes, S. P., Barhoumi, R., Nachman, R. J., Pietrantonio, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+a+G+protein-coupled+receptor+from+the+Southern+cattle+tick+Boophilus+microplus+(Acari:+Ixodidae)+identifies+it+as+the+first+arthropod+myokinin+receptor.">Functional analysis of a G protein-coupled receptor from the Southern cattle tick Boophilus microplus (Acari: Ixodidae) identifies it as the first arthropod myokinin receptor.</a> Insect Molecular Biology. 12, 27-38 (2003).</li><li>Staubli, F., Jorgensen, T. J. D., Cazzamali, G., Williamson, M., Lenz, C., Sondergaard, L., Roepstorff, P., Grimmelikhuijzen, C. J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+the+insect+adipokinetic+hormone+receptors.">Molecular identification of the insect adipokinetic hormone receptors.</a> Proceedings of the National Academy of Sciences USA. 99, 3446-3451 (2002).</li><li>Stables, J., Green, A., Marshall, F., Fraser, N., Knight, E., Sautel, M., Milligan, G., Lee, M., Rees, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioluminescent+assay+for+agonist+activity+at+potentially+any+G-protein+coupled+receptor.">A bioluminescent assay for agonist activity at potentially any G-protein coupled receptor.</a> Analytical Biochemistry. 252, 115-126 (1997).</li></ol>

利害の衝突は宣言されません。

我々は、このプロトコルを使用して、クモ（ダニ、ダニやクモ）、ダニのキニンの受容体から最初に検出された神経ペプチド受容体の機能解析を行うことができた。このメソッドは、主に次の3つのアプリケーションを持っています。最初に、技術はリガンド活性の測定によって受容体のdeorphanizationに適用することができます。第二に、アッセイは、リガンド - 受容体の構造活性相関（SAR）を解決することができます。第三に、メソッドは、創薬に使用することができます。さらに、このプロトコルは、ほとんどすべてのGPCRに関するアゴニストまたはアンタゴニストの活性を研究するために使用することができます。私たちは、小さなライブラリーのスクリーニングのためにこのプロトコルを適応し始めている。我々が利用細胞株では、ユビキタスGタンパク質G 16を発現しない。我々は、Gqをタンパク質と細胞内カルシウムのカスケードを通して節足動物キニン受容体のシグナルので、それを必要としなかったし、ここに示すように、彼らは、哺乳動物細胞でこのシグナル伝達特性を節約。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> DMEM </td><td>インビトロジェン</td><td> ABCD1234 </td><td></td></tr><tr><td> CHO - K1細胞</td><td> ATCC </td><td> CCL - 61 </td><td>マナッサス、VA、アメリカ合衆国</td></tr><tr><td> F12K培地</td><td>インビトロジェン</td><td> 21127 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>シグマアルドリッチ</td><td> F0643 </td><td></td></tr><tr><td>トリプシン- EDTA（10倍） </td><td>インビトロジェン</td><td> 15400 </td><td></td></tr><tr><td>抗生物質、抗真菌</td><td>インビトロジェン</td><td> 15240 </td><td></td></tr><tr><td>のOpti - MEM Iは、血清培地を削減</td><td>インビトロジェン</td><td> 31985 </td><td></td></tr><tr><td>リポフェクチン試薬</td><td>インビトロジェン</td><td> 18292-011 </td><td></td></tr><tr><td> GENETICIN </td><td>インビトロジェン</td><td> 10131035 </td><td></td></tr><tr><td> MC1061/P3 Ultracomp </td><td>インビトロジェン</td><td> C663 - 03 </td><td></td></tr><tr><td> QIAprep Spin Miniprepキット</td><td>キアゲン社</td><td> 19064 </td><td></td></tr><tr><td> FuGENE 6トランスフェクション試薬</td><td>ロッシュ</td><td> 11 814 443 001 </td><td></td></tr><tr><td> 96ウェル白色の薄い底のmicrotitereプレート</td><td>共演スター</td><td> 3610 </td><td></td></tr><tr><td>カルシウム不含DMEM培地</td><td>インビトロジェン</td><td> 21068 </td><td></td></tr><tr><td>セレンテラジン</td><td>インビトロジェン</td><td> C - 2944 </td><td></td></tr><tr><td>ブライトライン血球計</td><td> Hausserサイエンティフィック</td><td></td><td>ホーシャム、ペンシルバニア州</td></tr><tr><td> NOVOstar </td><td> BMGのLabtechnologies </td><td></td><td></td></tr><tr><td>プリズムソフトウェア4.0 </td><td>グラフパッドソフトウェア株式会社</td><td></td><td>サンディエゴ、カリフォルニア州、米国</td></tr><tr><td> T - 25およびT - 75フラスコ</td><td> BDファルコン</td><td> 353014と353135 </td><td></td></tr></tbody></table>

a calcium bioluminescence assay for functional analysis of mosquito (aedes aegypti) and tick (rhipicephalus microplus) g protein-coupled receptors

彼らは多くの重要な生理学的および行動のプロセスを調節すると節足動物のホルモン受容体は、新規農薬の潜在的なターゲットです。それらの大半は、Gタンパク質共役受容体（GPCR）のスーパーファミリーに属する。我々は、ダニや蚊から節足動物キニン受容体を特徴付けるに焦点を当てている。節足動物のキニンはmyotropic、利尿、および神経伝達物質の機能を備えた多機能神経ペプチドである。ここでは、二つの異種キニン受容体を発現しているシステム上の昆虫キニンの構造活性相関の系統的分析のための方法が記載されている。我々は、利尿、myotropic、および/またはダニや蚊の消化過程を妨害する可能性のある生体安定キニン類似の開発に関連する重要な情報を提供しています。 南部牛のダニ、 ウシマダニ属microplus（Canestrini）、及び蚊ネッタイシマカ （リンネ）、からキニン受容体を安定に哺乳類細胞株CHO - K1で発現させた。これらの受容体の機能解析は、これらの組換え体細胞にペプチドアプリケーションによって細胞質カルシウム濃度を決定するために細胞内の生物発光を測定するカルシウムの生物発光プレートアッセイを用いて完成した。このメソッドは、イクオリンの蛋白質、発光クラゲから単離された発光タンパク質を利用しています。我々は、一時的に安定してキニン受容体を発現する細胞株で（mtAEQ/pcDNA1）エクオリンプラスミドをトランスフェクション。これらの細胞は、どの細胞イクオリンと複合体因子のセレンテラジン、で処理した。この結合はカルシウム濃度を示す発光レベルを放出、カルシウムの存在下で分解。キニン受容体が細胞内カルシウムの放出を介して信号として、信号の強度は、ペプチドの効力に関連しています。 このプロトコルは、変更といくつかの以前に記載されているプロトコルの合成であり、。。、それは機能的なプレートアッセイ（Staubly ら 、2002年と厩舎ら 、1997年まで哺乳類細胞株におけるGPCRの安定した発現のためのステップバイステップの手順を説明）。この手法を用いて、我々は蚊やダニのキニン受容体を発現する安定細胞株を樹立することができた、三蚊のkininsの効力を比較し、リガンドとレセプターの相互作用に重要なアミノ酸位置を特定し、ペプチドの半スループットスクリーニングを行うライブラリ。昆虫キニンは内因性ペプチダーゼによる高速酵素分解を受けやすいので、それらは深刻な害虫駆除や内分泌学的研究のためのツールと​​して使用が制限されています。したがって、我々はまた彼らの効力とbiostabilityを強化するアミノ酸イソ酪酸（AIB）を含むキニン類似をテストした。このペプチダーゼ抵抗性アナログは、生体安定昆虫キニン類似体の開発において重要なリードを表し、神経ペプチドベースの節足動物の制御戦略の開発を支援することがあります。

Watch this Scientific Journal Video about A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors at JoVE.com

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

このプロトコルは、クローン細胞ラインの選択とその同族のGPCRで合成された節足動物の神経ペプチドの構造活性相関を分析するためにカルシウムの生物発光アッセイの手順について説明します。このアッセイは、受容体のdeorphanizationと合成アナログ設計およびペプチド/薬物リード発見のための構造活性相関の研究にも使用できます。

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

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Research

JoVE Journal

Immunology and Infection

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

15.5K ビュー.  Texas A&M University (TAMU). このプロトコルは、クローン細胞ラインの選択とその同族のGPCRで合成された節足動物の神経ペプチドの構造活性相関を分析するためにカルシウムの生物発光アッセイの手順について説明します。このアッセイは、受容体のdeorphanizationと合成アナログ設計およびペプチド/薬物リード発見のための構造活性相関の研究にも使用できます。

ビデオ: A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

黄熱病の蚊から血球を収集し、染色のためのプロトコルネッタイシマカ</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

免疫・感染学

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

の遺伝的操作<em&gt;Δku80</emの機能ゲノム解析のための&gt;株<em&gt;トキソプラズマ</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

飼育<em&gt; Ixodes肩甲骨、</em&gt;ブラック・ティック・ティック：未熟期をマウスに与える

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

炎症性腸疾患における癒着防止治療の機能分析のための動的接着アッセイ

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

受容体と酵素間の機能的相互作用を研究するため Co 免疫沈降分析

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

無脊椎動物の病理学研究に向けた Hemolymph のMetarhizium Blastospores の収集とRhipicephalus Microplusダニへの接種の開示

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

ジカウイルスを用いた Aedes aegypti蚊の ベクターコンピテンス分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

アエデスアエジプティ蚊における微小胞体寄生虫・エダリア・アダイスの伝播

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

アエデスアエジプティ蚊の動物由来血液と人工食事の摂食と定量化

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

ハマダラカ媒介動物における熱帯熱マラリア原虫感染の検出のための標準的な膜摂食アッセイ

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...