Drs. PJC Grimmelikhuijzen et Michael Williamson de l&#39;Université de Copenhague (Danemark) sont appréciés pour fournir l&#39;aequorine plasmide. Notre collaborateur, le Dr Ronald J. Nachman de ARS-USDA (TX, USA), est reconnu pour la synthèse peptidique et de fournir au lecteur de plaque Novostar.

<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>

Nous avons été en mesure d&#39;effectuer la caractérisation fonctionnelle du récepteur neuropeptide découvert des arachnides (tiques, acariens et araignées), le récepteur kinine tique, en utilisant ce protocole. Cette méthode a trois applications principales. Tout d&#39;abord, la technique peut être appliquée pour deorphanization récepteurs grâce à des mesures d&#39;activité ligand. Deuxièmement, le dosage peut résoudre ligand-récepteur relations structure-activité (SAR). Troisièmement, les méthodes peuvent être utilisées dans la découverte de médicaments. Par ailleurs, ce protocole peut être utilisé pour étudier l&#39;activité des agonistes ou antagonistes sur presque n&#39;importe quelle RCPG. Nous commençons à adapter ce protocole pour le dépistage des petites bibliothèques. La lignée cellulaire nous avons utilisé n&#39;exprime pas la protéine ubiquitaire G G 16. Nous n&#39;avons pas besoin, car arthropodes récepteurs des kinines signal par la protéine Gq et la cascade intracellulaire de calcium et ils conserver cette propriétés de signalisation dans les cellules de mammifères, comme indiqué ici.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> DMEM </td><td> Invitrogen </td><td> ABCD1234 </td><td></td></tr><tr><td> Cellules CHO-K1 </td><td> ATCC </td><td> CCL-61 </td><td> Manassas, VA, USA </td></tr><tr><td> F12K moyennes </td><td> Invitrogen </td><td> 21127 </td><td></td></tr><tr><td> Sérum de veau fœtal </td><td> Sigma-Aldrich </td><td> F0643 </td><td></td></tr><tr><td> Trypsine-EDTA (10x) </td><td> Invitrogen </td><td> 15400 </td><td></td></tr><tr><td> Antibiotiques Antimycosique </td><td> Invitrogen </td><td> 15240 </td><td></td></tr><tr><td> Opti-MEM I Réduction milieu sans sérum </td><td> Invitrogen </td><td> 31985 </td><td></td></tr><tr><td> Réactif Lipofectin </td><td> Invitrogen </td><td> 18292-011 </td><td></td></tr><tr><td> Généticine </td><td> Invitrogen </td><td> 10131035 </td><td></td></tr><tr><td> MC1061/P3 Ultracomp </td><td> Invitrogen </td><td> C663-03 </td><td></td></tr><tr><td> QIAprep Spin Miniprep Kit </td><td> Qiagen Inc </td><td> 19064 </td><td></td></tr><tr><td> Réactifs de transfection FuGENE 6 </td><td> Roche </td><td> 11 814 443 001 </td><td></td></tr><tr><td> 96 puits blanche fine plaque inférieure microtitere </td><td> Costar </td><td> 3610 </td><td></td></tr><tr><td> sans calcium milieu DMEM </td><td> Invitrogen </td><td> 21068 </td><td></td></tr><tr><td> Coelentérazine </td><td> Invitrogen </td><td> C-2944 </td><td></td></tr><tr><td> Bright-Ligne hémacytomètre </td><td> Hausser scientifique </td><td></td><td> Horsham, Pennsylvanie </td></tr><tr><td> Novostar </td><td> Labtechnologies BMG </td><td></td><td></td></tr><tr><td> Prism Software 4.0 </td><td> GraphPad Software Inc </td><td></td><td> San Diego, Californie, USA </td></tr><tr><td> T-25 et T-75 Flacons </td><td> BD Falcon </td><td> 353014 353135 et le </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

Récepteurs hormonaux arthropodes sont des cibles potentielles pour les pesticides roman comme ils régulent de nombreux processus essentiels physiologiques et comportementaux. La majorité d&#39;entre eux appartiennent à la superfamille des G récepteurs couplés aux protéines (RCPG). Nous nous sommes concentrés sur la caractérisation des récepteurs des kinines arthropodes de la tique et de moustiques. Kinines arthropodes sont des neuropeptides multifonctionnel avec myotropes, diurétique, et la fonction des neurotransmetteurs. Ici, une méthode pour l&#39;analyse systématique des relations structure-activité des kinines insectes sur deux hétérologue kinine exprimant le récepteur des systèmes est décrite. Nous fournissons des informations importantes relatives au développement des analogues des kinines biostable avec le potentiel de perturber le diurétique, myotropes, et / ou processus digestifs chez les tiques et les moustiques. Les récepteurs des kinines par la tique du bétail sud, Boophilus microplus (Canestrini), et le moustique Aedes aegypti (Linné), ont été exprimés de manière stable dans la lignée cellulaire de mammifère CHO-K1. Les analyses fonctionnelles de ces récepteurs ont été réalisées en utilisant un dosage du calcium plaque de bioluminescence que les mesures de bioluminescence intracellulaire afin de déterminer les niveaux de calcium cytoplasmique lors de l&#39;application de peptides à ces cellules recombinantes. Cette méthode tire parti de la protéine aequorine, une photoprotéine isolé luminescente méduses. Nous transfectées transitoirement l&#39;aequorine plasmide (mtAEQ/pcDNA1) dans des lignées cellulaires qui ont exprimé de façon stable les récepteurs des kinines. Ces cellules ont ensuite été traitées avec le cofacteur coelentérazine, qui se complexe avec l&#39;aequorine intracellulaire. Ce lien ruptures dans la présence de calcium, en émettant des niveaux de luminescence indique la concentration de calcium. Comme le récepteur de signaux kinine par la libération de calcium intracellulaire, l&#39;intensité du signal est liée à la puissance du peptide. Ce protocole est une synthèse de plusieurs protocoles décrits précédemment avec des modifications, il présente, étape par étape les instructions pour l&#39;expression stable des RCPG dans une lignée cellulaire de mammifère par le biais des tests fonctionnels plaque (Staubly et al, 2002 et Stables et al, 1997.. ). En utilisant cette méthodologie, nous avons été en mesure d&#39;établir des lignées cellulaires stables exprimant les moustiques et les récepteurs des kinines tique, comparer la puissance de trois kinines moustiques, d&#39;identifier les positions des acides aminés essentiels pour l&#39;interaction ligand-récepteur, et d&#39;effectuer de semi-criblage d&#39;un peptide bibliothèque. Parce que les kinines insectes sont sensibles à la dégradation enzymatique rapide par les peptidases endogènes, ils sont sévèrement limités dans l&#39;utilisation comme outils de lutte contre les ravageurs ou les études endocrinologiques. Par conséquent, nous avons également testé les analogues des kinines contenant de l&#39;acide aminé isobutyrique (AIB) afin d&#39;améliorer leur efficacité et biostabilité. Cet analogue peptidase-résistante représente une avance importante dans le développement de biostable analogues des kinines insectes et peut aider au développement du neuropeptide basé stratégies de contrôle des arthropodes.

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

Ce protocole fournit des instructions pour la sélection de la lignée clonale de cellules et un dosage du calcium bioluminescence d&#39;analyser les relations structure-activité des neuropeptides synthétisés arthropodes sur leur apparenté RCPG. Ce test peut être utilisé pour deorphanization récepteurs et des études de relation structure-activité pour la conception et le peptide analogue synthétique / drogue mènent la découverte.

Un test de bioluminescence calcium pour l&#39;analyse fonctionnelle des moustiques ( Aedes aegypti) Et cochez (<emRhipicephalus> microplus) G récepteurs couplés aux protéines

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

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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 vues.  Texas A&M University (TAMU). Ce protocole fournit des instructions pour la sélection de la lignée clonale de cellules et un dosage du calcium bioluminescence d'analyser les relations structure-activité des neuropeptides synthétisés arthropodes sur leur apparenté RCPG. Ce test peut être utilisé pour deorphanization récepteurs et des études de relation structure-activité pour la conception et le peptide analogue synthétique / drogue mènent la découverte.

Vidéo: 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.

Un protocole pour le recueil et la coloration hémocytes du moustique Fièvre jaune Aedes aegypti</em

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

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Immunologie et infection

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.

Manipulation génétique<em&gt; Δku80</em&gt; Les souches pour l&#39;analyse génomique fonctionnelle de<em&gt; Toxoplasma gondii</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.

Élevage<em&gt; Ixodes scapularis,</em&gt; La tique à pattes noires: alimentation sur des souris étapes Immature

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.

Dosage d’adhérence dynamiques pour l’analyse fonctionnelle des thérapies contre les adhérences dans la maladie intestinale inflammatoire

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.

Co-Immunoprécipitation dosage pour l’étude des Interactions fonctionnelles entre les récepteurs et les Enzymes

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.

Divulgation de la collection d’hémolymphe et inoculation des blastospores de Métarhizium dans les tiques de Rhipicephalus MICROPLUS vers les études de pathologie des invertébrés

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.

Analyses de compétence vectorielle sur les moustiques Aedes aegypti utilisant le virus Zika

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.

Propagation du parasite microsporidien Edhazardia aedis chez les moustiques Aedes aegypti

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.

Alimentation et quantification du sang d’origine animale et des repas artificiels chez les moustiques Aedes aegypti

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.

Dosage standard d’alimentation par membrane pour la détection de l’infection à Plasmodium falciparum chez les moustiques vecteurs anophèles

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 ...

Un test de bioluminescence calcium pour l'analyse fonctionnelle des moustiques ( Aedes aegypti) Et cochez ( microplus) G récepteurs couplés aux protéines