<p class="jove_content"&gt; ونحن نشكر Nadeshda وولف، Eschen نادين وديلونغ سوزان لتربية الحيوانات ممتازة. كان مدعوما من قبل CW زمالة الدكتوراه من &quot;برنامج هيلمولز<em&gt; برنامج BioInterfaces يقوم</em&gt; الدولي خريج المدرسة (BIFIGS). وقدمت مزيدا من الدعم من قبل منحة KIT-RISC وكذلك من خلال منح ماري كوري لإعادة الإدماج في إطار البرنامج الأوروبي 7 إطار المجتمع (PIRG07-GA-2010-267552) إلى الفريق الاستشاري. وأيد عبد الحميد شومان من قبل جريئة (علم الأحياء من الكبد والبنكرياس والتنمية مرض) - شبكة التكوين الأولي ماري كوري (238821). كان مدعوما من قبل UL Dopaminet FP7 (سابعا اطار برنامج) (223744).</p&gt;

<ol>
	<li>Peterson, R. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+use+of+small+molecules+for+probing+biological+processes+in+zebrafish.">Discovery and use of small molecules for probing biological processes in zebrafish.</a> Methods Cell Biol. 76, (2004).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nat. Rev. Drug. Discov. 4, 35-44 (2005).</li><li>Loynes, C. A., Martin, J. S., Robertson, A., Trushell, D. M., Ingham, P. W., Whyte, M. K., Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+advance:+pharmacological+manipulation+of+inflammation+resolution+during+spontaneously+resolving+tissue+neutrophilia+in+the+zebrafish.">Pivotal advance: pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish.</a> J. Leukoc. Biol. 87, 203-212 (2009).</li><li>Martin, J. S., Renshaw, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+in+vivo+zebrafish+models+to+understand+the+biochemical+basis+of+neutrophilic+respiratory+disease.">Using in vivo zebrafish models to understand the biochemical basis of neutrophilic respiratory disease.</a> Biochem. Soc. Trans. 37, 830-837 (2009).</li><li>Renshaw, S. A., Loynes, C. A., Trushell, D. M., Elworthy, S., Ingham, P. W., Whyte, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108, 3976-3978 (2006).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Dev. Biol. 7, 42 (2007).</li><li>Redd, M. J., Kelly, G., Dunn, G., Way, M., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+macrophage+chemotaxis+in+vivo:+studies+of+microtubule+function+in+zebrafish+wound+inflammation.">Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrafish wound inflammation.</a> Cell. Motil. Cytoskeleton. 63, 415-422 (2006).</li><li>D'Alençon, C. A., Peña, O. A., Wittmann, C., Gallardo, V. E., Jones, R. A., Loosli, F., Liebel, U., Grabher, C., Allende, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+chemically+induced+inflammation+assay+in+zebrafish.">A high-throughput chemically induced inflammation assay in zebrafish.</a> BMC Biol. 8, 151 (2010).</li><li>Haas, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+signaling+mediates+self-organizing+tissue+migration+in+the+zebrafish+lateral+line.">Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line.</a> Dev Cell. 10, 673-680 (2006).</li><li>Luetjohann, D. S., Shah, A. H., Christen, M. P., Richter, F., Knese, K., Liebel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sciencenet'+-+towards+a+global+search+and+share+engine+for+all+scientific+knowledge.">Sciencenet' - towards a global search and share engine for all scientific knowledge.</a> Bioinformatics. 27, 1734-1735 (2011).</li><li>Liebel, U., Kindler, B., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harvester':+a+fast+meta+search+engine+of+human+protein+resources.">Harvester': a fast meta search engine of human protein resources.</a> Bioinformatics. 20, 1962-1963 (2004).</li></ol>

الإعلان عن أي تضارب في المصالح.

<p class="jove_content"&gt; نحن هنا تقديم مؤتمتة بالكامل استراتيجية الفرز الفائق الإنتاجية تمكن من تحديد هوية المركبات المناعية تغييري في سياق الحيوانية كلها. مما يسمح لرصد استجابة الالتهابية الحادة<em&gt; في الجسم الحي</em&gt; و في الوقت الحقيقي.</p&gt;<p class="jove_content"&gt; ونحن نقدم لتطبيق بروتوكول الزرد مثل كائن نموذجا قيما لاكتشاف العقاقير أو المخدرات جهود تطويعها لأغراض أخرى، حيث يمكن دراسة آثار المخدرات على تجنيد الكريات البيض على الجروح مع السهولة النسبية في سياق نظام المناعة الفطرية وظيفية بالكامل. ميزة فريدة من نوعها لنهجنا هو حقيقة أن اصابة الكيميائية يجعل دليل تلاعب اليرقات عفا عليها الزمن، مما سمح الأتمتة والفرز في شكل عالية الإنتاجية.</p&gt;<p class="jove_content"&gt; وبالإضافة إلى ذلك، يمكن أن الآثار السلبية للمخدرات أو السامة، فضلا عن آثار قوية من خارج الهدف تقييمها بسهولة مع المقترح انشاء. مكتبة ادارة الاغذية والعقاقير المعتمدة ولكن لا تحتوي على أي مركبات سامة في تركيزات فحص معين. ومع ذلك، فإننا نقترح على مركبات سامة واضح للمقارنة بين مجموعة البيانات المتاحة (مثل النمط الظاهري تغييري المناعي، وتركيز الدواء) بين أفراد من نفس المادة الدوائية الحالي فئة في المكتبة التي تم اختبارها. ويمكن اختبارها أعضاء السامة من عائلة الجوهر، الذي يمارس على خلاف ذلك واعدة النمط الظاهري تغييري المناعي، وبتركيزات أقل، إذا لزم الأمر.</p&gt;<p class="jove_content"&gt; لقد برهنا على امكانية تصنيف المخدرات تلقائيا وفقا لتأثيرها المناعية تغييري المبذولة. ويمكن التحقق من صحة الأدوية التي تنتمي إلى فئة من المركبات المضادة للالتهابات في تجارب مستقلة، فإن معظم المركبات تمارس تأثيرها بطريقة تعتمد على الجرعة. يمكن أن أربع مركبات على النحو المحدد المخدرات المؤيدة للقرار محتمل حتى الآن لم يتم تأكيد هذا النحو. هذا يشير ايضا الى ان هذه الفئة خاصة تغييري المناعي من الصعب وصفها، منذ تم حتى الآن أي أدوية تؤثر فقط قرار وصفها في المؤلفات. ومع ذلك، أظهرت 3 من المخدرات إعادة اختباره 4 اتجاها المضادة للالتهابات في تركيزات أعلى الرحى. وينبغي تحقيق الاستفادة المثلى من معايير عتبة محددة تجريبيا يساعد على الحد من عدد من ايجابيات كاذبة.</p&gt;<p class="jove_content"&gt; علاوة على ذلك، قدمت هذه التقنية توفر إمكانية استخدامها لتحديد الطفرات في الجينات التي تؤثر على سلوك المهاجرة الكريات البيض<sup&gt; 8</sup&gt;.</p&gt;<p class="jove_content"ويمكن&gt; ختامية، الطريقة المقترحة تسهيل وتسريع اكتشاف المخدرات أو المخدرات تطويعها لأغراض أخرى الجهود التي تدر المحتملة هياكل القيادة الجديدة مع المعلومات المتاحة بسهولة على التسامح المخدرات، وسمية، جرعة، فضلا عن الآثار المحتملة خارج الهدف. ولذلك، بمثابة استراتيجية، بسيطة من حيث التكلفة وتوفير الوقت في المراحل الأولى من تطوير الأدوية.</p

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> ثنائي ميثيل sulphoxide </td><td> كارل روث GMBH &amp; المشارك </td><td> A994.2 </td><td></td></tr><tr><td> النحاس (II) كبريتات اللامائية </td><td> كارل روث GMBH &amp; المشارك </td><td> PO23.1 </td><td></td></tr><tr><td> وافقت ادارة الاغذية والعقاقير مكتبة المخدرات </td><td> انزو العلوم الحياتية </td><td> BML-2841-0100 </td><td></td></tr><tr><td> 384 لوحة microwell غير ملزم، μClear </td><td> غرينر </td><td> 781906 </td><td> أسود </td></tr><tr><td> أوليمبوس المسح ^ R </td><td> أوليمبوس </td><td></td><td></td></tr><tr><td> زفير التعامل مع السائل الميثاق محطة </td><td> الفرجار </td><td></td><td></td></tr></tbody>
 </table>

facilitating drug discovery: an automated high-content inflammation assay in zebrafish

الزرد اليرقات قابلة خاصة إلى كل الحيوانات الصغيرة 1،2 شاشات جزيء نظرا لصغر حجمها والسهولة النسبية للتلاعب والمراقبة، فضلا عن حقيقة أن مركبات ببساطة أن تضاف إلى مياه الاستحمام ويمتص بسهولة عندما تدار في &lt;حل DMSO 1٪. يرجع ذلك إلى وضوح بصري اليرقات الزرد، ومدى توافر خطوط المعدلة وراثيا التعبير عن البروتينات الفلورية في الكريات البيض، الزرد توفر ميزة فريدة من نوعها لرصد رد فعل حاد للالتهابات في الجسم الحي. وبناء على ذلك، والاستفادة من الزرد لارتفاع محتوى شاشات جزيء صغير يهدف إلى التعرف على المركبات المناعية تغييري مع إنتاجية عالية وقد اقترح 3-6، مما يشير إلى سيناريوهات الحث على سبيل المثال التهاب النكات المترجمة في الأنسجة الزعانف، والضرر ليزر موجهة إلى السطح من صفار البيض الأجنة (7) أو tailfin بتر 3،5،6. العيب الرئيسي من هذه الأساليب ولكن كانشرط تلاعب يرقة دليل للحث على جرح، وبالتالي منع الفرز الفائق الإنتاجية. القضاء مقدمة من الالتهاب الناجم كيميائيا (الذقن) 8 فحص هذه العقبات. منذ يلحق اصابة كيميائيا عدد الأجنة التي يمكن معالجتها في وقت واحد تكاد تكون غير محدودة. علاج مؤقت من يرقات اسماك الزرد مع كبريتات النحاس يستحث موت الخلايا بشكل انتقائي في خلايا الشعر لنظام الخط الجانبي والنتائج في تجنيد المحببة السريع إلى neuromasts الجرحى. يمكن اتباعها لاستجابة التهابية في الوقت الحقيقي باستخدام مركب المعدلة وراثيا cldnB :: GFP / lysC :: DsRED2 يرقات اسماك الزرد 6،9 تعبر عن ببروتين أخضر في الخلايا neuromast، فضلا عن وضع العلامات الحمراء بروتين فلوري المحببة. من أجل وضع استراتيجية الفرز من شأنها أن تسمح على حد سواء تحليل محتوى عالية وعالية الإنتاجية، وعرض علينا التعامل مع السائل الروبوتية وميكر الآلية المشتركةoscopy مع برنامج نصي مخصص البرمجيات المتقدمة. هذا السيناريو يمكن القياس الكمي الآلي للاستجابة التهابية بتسجيله المنطقة التي تحتلها في المئة الكريات البيض فلوري الحمراء ضمن المنطقة المحددة تجريبيا المحيطة المصابين neuromasts الفلورية الخضراء. وعلاوة على ذلك، ونحن الآلي ومعالجة البيانات، والمناولة، والتصور، وتخزين تقوم على العرف وضعت جميع MATLAB والبرامج النصية بيثون. وباختصار، نحن نقدم الآلي HC / HT الشاشة التي تسمح اختبار المركبات الكيميائية من حيث تأثيرها على التقدم، وبدء أو قرار من استجابة التهابية المحببات. هذا البروتوكول يخدم نقطة انطلاق جيدة لمزيد من التحليل المتعمق لآليات المخدرات وطرق المشاركة في تزامن من استجابة المناعة الفطرية. في المستقبل، قد يساعد تحديد الآثار السامة لا يطاق أو بعيدا عن المرمى، في المراحل السابقة من اكتشاف المخدرات وبالتالي الحد من المخاطر والتكاليف الإجرائية لتطوير الأدوية.

Watch this Scientific Journal Video about Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish at JoVE.com

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

نحن هنا وصف رواية عالية المحتوى فحص الالتهاب الناجم كيميائيا يهدف إلى التعرف على المناعة تغييري bioactives. لقد نجحنا في الجمع بين المجهر الآلي مع العرف مخطوطات البرمجيات المتقدمة تمكين الكمي الآلي للاستجابة التهابية، فضلا عن مزيد من معالجة البيانات وتحليلها، والتعدين، والتخزين.

تسهيل اكتشاف المخدرات: مؤتمتة فحص التهاب عالية المحتوى في الزرد

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

facilitating-drug-discovery-an-automated-high-content-inflammation

Research

JoVE Journal

Immunology and Infection

14.0K Views.  Karlsruhe Institute of Technology, Karlsruhe, Germany. نحن هنا وصف رواية عالية المحتوى فحص الالتهاب الناجم كيميائيا يهدف إلى التعرف على المناعة تغييري bioactives. لقد نجحنا في الجمع بين المجهر الآلي مع العرف مخطوطات البرمجيات المتقدمة تمكين الكمي الآلي للاستجابة التهابية، فضلا عن مزيد من معالجة البيانات وتحليلها، والت...

Video: Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

باستخدام بويضات من<em&gt; البلهارسيا المنسونية</em&gt; ك<em&gt; في الجسم الحي</em&gt; نموذج من التهاب الرئة الناجم عن الديدان الطفيلية

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

فحوصات لتحديد رواية الأدوية المضادة للفيروسات ضد فيروس اللسان الأزرق

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

فحص فينوطيبيك مجهري للقياس الكمي للبكتيريا الفطرية داخل الخلايا المكيفة للفحص عالي الإنتاجية/عالي المحتوى

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

عالية الإنتاجية الكمي في الوقت الحقيقي RT-PCR الفحص لتحديد ملامح التعبير من أنواع الأول والثالث الانترفيرون الأنواع الفرعية

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

جيل ومتعدد المظهري فحص عالية المحتوى من<em&gt; الكوكسيلا البورنيتية</em&gt; المسوخ ينقول

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

والطريقة الأمثل لعزل وتوسيع قسما ثابتا الطبيعية الخلايا التائية القاتلة من الماوس الطحال

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

قياس الأنفلونزا النورامينيداز تثبيط الجسم المضاد التتر من قبل انزيم مرتبط كتين الفحص

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

تحريض استجابة التهابية في الثقافات الابتدائية الكبدية من الفئران

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

ل<em&gt; في المختبر</em&gt; كاسيم ملزمة الفحص الذي يتنبأ اختراق المخدرات في الآفات السل

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

الفائق، والمحتوى السامي، المستندة إلى سائل C. ايليجانس باثوسيستيم

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...