Name: a high-throughput, high-content, liquid-based c. elegans pathosystem
Uploaded: 2018-07-01T15:00:00
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وأيد هذه الدراسة بالوقاية من السرطان ومعهد أبحاث من ولاية تكساس (كبريت) جائزة RR150044، ج المنح البحثية مؤسسة ولش-1930، ومن المعاهد الوطنية من AI110552 K22 على الصحة، منح NVK. وكان الممولين أي دور في تصميم الدراسة أو جمع البيانات والتحليل، وقرار نشر أو إعداد المخطوطة.

تنبيه: P. الزّنجاريّة و faecalis هاء- 2 مستوى السلامة البيولوجية المسببة للأمراض، ويجب اتخاذ احتياطات السلامة المناسبة لمنع الإصابة بعرضي والتقليل من تلوث الأسطح. يجب تعقيم جميع وسائل الإعلام والمواد التي تتلامس مع مسببات الأمراض أو التخلص منها. وتتوفر المزيد من المبادئ التوجيهية من...

<ol>
	<li>Falagas, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandrug-resistant+Klebsiella+pneumoniae,+Pseudomonas+aeruginosa+and+Acinetobacter+baumannii+infections:+Characteristics+and+outcome+in+a+series+of+28+patients.">Pandrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii infections: Characteristics and outcome in a series of 28 patients.</a> International Journal of Antimicrobial Agents. 32 (5), 450-454 (2008).</li><li>Hsueh, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandrug-resistant+Acinetobacter+baumannii+causing+nosocomial+infections+in+a+university+hospital,+Taiwan.">Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan.</a> Emerging Infectious Diseases. 8 (8), 827-832 (2002).</li><li>Wang, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandrug-resistant+Pseudomonas+aeruginosa+among+hospitalised+patients:+clinical+features,+risk-factors+and+outcomes.">Pandrug-resistant Pseudomonas aeruginosa among hospitalised patients: clinical features, risk-factors and outcomes.</a> Clinical Microbiology and Infection. 12 (1), 63-68 (2006).</li><li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Draft+genome+sequences+of+pandrug-resistant+Serratia+marcescens+clinical+isolates+harboring+blaNDM-1.">Draft genome sequences of pandrug-resistant Serratia marcescens clinical isolates harboring blaNDM-1.</a> Genome Announcements. 5 (3), (2017).</li><li>Utari, P. D., Quax, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+reveals+novel+Pseudomonas+aeruginosa+virulence+mechanism.">Caenorhabditis elegans reveals novel Pseudomonas aeruginosa virulence mechanism.</a> Trends in Microbiology. 21 (7), 315-316 (2013).</li><li>Conery, A. L., Larkins-Ford, J., Ausubel, F. M., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+novel+anti-infectives+using+a+C.+elegans+pathogenesis+model.">High-throughput screening for novel anti-infectives using a C. elegans pathogenesis model.</a> Current Protocols in Chemical Biology. 6 (1), 25-37 (2014).</li><li>Kirienko, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+disrupts+Caenorhabditis+elegans+iron+homeostasis,+causing+a+hypoxic+response+and+death.">Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death.</a> Cell Host &amp; Microbe. 13 (4), 406-416 (2013).</li><li>Kirienko, D. R., Revtovich, A. V., Kirienko, N. V. <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-content,+phenotypic+screen+identifies+fluorouridine+as+an+inhibitor+of+pyoverdine+biosynthesis+and+pseudomonas+aeruginosa+virulence.">A high-content, phenotypic screen identifies fluorouridine as an inhibitor of pyoverdine biosynthesis and pseudomonas aeruginosa virulence.</a> mSphere. 1 (4), (2016).</li><li>Manning, A. J., et al. <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+content+microscopy+assay+to+determine+drug+activity+against+intracellular+Mycobacterium+tuberculosis.">A high content microscopy assay to determine drug activity against intracellular Mycobacterium tuberculosis.</a> Methods. 127, 3-11 (2017).</li><li>Marwaha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acylated+derivatives+of+sulfamethoxazole+and+sulfafurazole+inhibit+intracellular+growth+of+Chlamydia+trachomatis.">N-acylated derivatives of sulfamethoxazole and sulfafurazole inhibit intracellular growth of Chlamydia trachomatis.</a> Antimicrobial Agents and Chemotherapy. 58 (5), 2968-2971 (2014).</li><li>Kota, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+high-content+imaging+and+chemical+genetics+to+probe+host+cellular+pathways+critical+for+Yersinia+pestis+infection.">Integrating high-content imaging and chemical genetics to probe host cellular pathways critical for Yersinia pestis infection.</a> PLoS One. 8 (1), e55167 (2013).</li><li>Arif, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+cell+infection+caused+by+Listeria+monocytogenes+invasion.">Quantification of cell infection caused by Listeria monocytogenes invasion.</a> Journal of Biotechnology. 154 (1), 76-83 (2011).</li><li>Jayamani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Francisella+tularensis-Caenorhabditis+elegans+pathosystem+for+the+evaluation+of+therapeutic+compounds.">Characterization of a Francisella tularensis-Caenorhabditis elegans pathosystem for the evaluation of therapeutic compounds.</a> Antimicrobial Agents and Chemotherapy. 61 (9), (2017).</li><li>Moy, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screen+for+novel+antimicrobials+using+a+whole+animal+infection+model.">High-throughput screen for novel antimicrobials using a whole animal infection model.</a> ACS Chemical Biology. 4 (7), 527-533 (2009).</li><li>Rajamuthiah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+automated+platform+for+drug+discovery+against+multi-drug+resistant+Staphylococcus+aureus.">Whole animal automated platform for drug discovery against multi-drug resistant Staphylococcus aureus.</a> PLoS One. 9 (2), e89189 (2014).</li><li>Breger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+chemical+compounds+identified+using+a+C.+elegans+pathogenicity+assay.">Antifungal chemical compounds identified using a C. elegans pathogenicity assay.</a> PLoS Pathogens. 3 (2), e18 (2007).</li><li>Garvis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+semi-automated+liquid+screen+reveals+a+specialized+role+for+the+chemotaxis+gene+cheB2+in+Pseudomonas+aeruginosa+virulence.">Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in Pseudomonas aeruginosa virulence.</a> PLoS Pathogens. 5 (8), e1000540 (2009).</li><li>Zhou, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+novel+screening+model+for+assessing+the+bioactivity+of+extracts+against+multidrug-resistant+Pseudomonas+aeruginosa+using+Caenorhabditis+elegans.">An efficient and novel screening model for assessing the bioactivity of extracts against multidrug-resistant Pseudomonas aeruginosa using Caenorhabditis elegans.</a> Bioscience, Biotechnology, and Biochemistry. 75 (9), 1746-1751 (2011).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+biosensing+with+fluorescent+C.+elegans+strains.">High-throughput screening and biosensing with fluorescent C. elegans strains.</a> Journal of Visual Experiments. (51), (2011).</li><li>Kirienko, N. V., Ausubel, F. M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+confers+resistance+to+siderophore-mediated+killing+by+Pseudomonas+aeruginosa.">Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (6), 1821-1826 (2015).</li><li>Kirienko, N. V., Cezairliyan, B. O., Ausubel, F. M., Powell, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+PA14+pathogenesis+in+Caenorhabditis+elegans.">Pseudomonas aeruginosa PA14 pathogenesis in Caenorhabditis elegans.</a> Methods in Molecular Biology. 1149, 653-669 (2014).</li><li>Kang, D., Kirienko, D. R., Webster, P., Fisher, A. L., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyoverdine,+a+siderophore+from+Pseudomonas+aeruginosa,+translocates+into+C.+elegans,+removes+iron,+and+activates+a+distinct+host+response.">Pyoverdine, a siderophore from Pseudomonas aeruginosa, translocates into C. elegans, removes iron, and activates a distinct host response.</a> Virulence. , 1-41 (2018).</li><li>Garsin, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+C.+elegans+daf-2+mutants+are+resistant+to+bacterial+pathogens.">Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens.</a> Science. 300 (5627), 1921 (2003).</li><li>Estes, K. A., Dunbar, T. L., Powell, J. R., Ausubel, F. M., Troemel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bZIP+transcription+factor+zip-2+mediates+an+early+response+to+Pseudomonas+aeruginosa+infection+in+Caenorhabditis+elegans.">bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (5), 2153-2158 (2010).</li><li>Tjahjono, E., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+mitochondrial+surveillance+pathway+is+required+for+defense+against+Pseudomonas+aeruginosa.">A conserved mitochondrial surveillance pathway is required for defense against Pseudomonas aeruginosa.</a> PLoS Genetics. 13 (6), e1006876 (2017).</li><li>Moy, T. I., et al. <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+novel+antimicrobials+using+a+live-animal+infection+model.">Identification of novel antimicrobials using a live-animal infection model.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (27), 10414-10419 (2006).</li><li>Henderson, S. T., Bonafe, M., Johnson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-16+protects+the+nematode+Caenorhabditis+elegans+during+food+deprivation.">daf-16 protects the nematode Caenorhabditis elegans during food deprivation.</a> The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 61 (5), 444-460 (2006).</li><li>Beanan, M. J., Strome, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+germ-line+proliferation+mutation+in+C.+elegans.">Characterization of a germ-line proliferation mutation in C. elegans.</a> Development. 116 (3), 755-766 (1992).</li></ol>

هذا الإنزيم (أو فحوصات مماثلة حيث يتم استبدال مسببات الأمراض الأخرى عن P. الزّنجاريّة أو هاء-faecalis) مفيدة لمجموعة متنوعة من الأغراض، بما في ذلك اكتشاف المخدرات. كما أنها مفيدة لمعالجة المسائل البيولوجية الأساسية، مثل تحديد عوامل الفوعة، توضيح سبل الدفاع المضيف، وتحديد الآلية الت?...

تحديد وتطوير المضادات الحيوية واسعة الطيف، وفعالة، الآن تقريبا قبل قرن من الزمان، أدت إلى لحظة فاصلة في مجال الصحة العامة حيث كان هناك اعتقاد على نطاق واسع أن المعدية المرض سيكون آفة من الماضي. في بضعة عقود قصيرة، بدأ هذا التفاؤل إلى الزوال، الممرض بعد الممرض وضعت آليات المقاومة التي تحد من هذه العلاجات المعجزة مرة واحدة. لبعض الوقت، ويبدو أن سباق التسلح بين الجهود الرامية إلى اكتشاف المخدرات ومسببات الأمراض متوازنا. ومع ذلك، قد توجت إساءة استخدام مضادات الميكروبات مؤخرا في ظهور سلالات مقاومة للعقاقير عموم الكلبسيله الرئويةو راكدة بوماني، السراتية marcescensو, P. الزّنجاريّة1 2،،من34.
من P. الزّنجاريّة انتهازية، غرام السلبية، متعدد المضيف ممرض أن تهديدا شديدا للمرضى الذين يعانون من حروق شديدة، أولئك الذين هم المناعة أو التليف الكيسي. فإنه يتم تعريف أيضا بصورة متزايدة كعامل المسبّب للمرض في العداوي المستشفوية شديدة، لا سيما بسبب حيازته الجارية لمقاومة مضادات الميكروبات. للبدء في التصدي لهذا التهديد، وقد استخدمنا موثقة توثيقاً جيدا C. ايليجانس-P. الزّنجاريّة عدوى النظام5. وقد الاستدانة لدينا مختبر هذا النظام لتطوير منصة فرز المستندة إلى السائل، والفائق، وعالية المحتوى لتحديد المركبات الرواية التي تحد من قدرة مسببات المرض قتل المضيف6. الفضول، تبدو هذه المركبات تنتمي إلى مالا يقل عن ثلاث فئات عامة، بما في ذلك مضادات الميكروبات7 وضراوة مثبطات8. وردت أخرى فحوصات اكتشاف المخدرات عالية المحتوى في C. ايليجانس لبكتريا توبيركولوسوم، والمتدثرة الحثريه، واليرسينيا بيستيس، الليستريه المستوحدة، فرانسيسيلا تولارنسيس، المكوّرات العنقودية الذهبية، المبيضات البيض، و فايكاليس البرازية، بين أمور أخرى9،10،11،،من1213،14،،من1516. هذه الأنواع من فحوصات لها العديد من المزايا المعترف بها جيدا، مثل الحد من يضرب الإيجابية الكاذبة التي قد تكون سامة للبلد المضيف والممرض، احتمال زيادة التوافر الحيوي بالمقارنة مع شاشة كيميائية، والقدرة على تحديد عدد الزيارات خارج مجرد الحد من نمو الميكروبات، مثل مكافحة فيرولينتس أو جزيئات محفزة محصنة ضد المركبات إلا إمالة توازن التفاعل المضيف الممرض صالح السابقة. بالإضافة إلى ذلك، أن المركبات التي اكتشفت في هذه الشاشات غالباً ما تكون فعالة في الثدييات المضيفين.
تجدر الإشارة إلى أن مالا يقل عن اثنين آخرين فحوصات17،18 متاحة للاضطلاع بشاشات الفائق في C. ايليجانس في السائل. ومع ذلك، كل من هذه الاختبارات هو تعديل يسمح الإنزيم المعوية الاستعمار تنميط، المعروف بالقتل البطيء، يمكن أداؤها في السائل، زيادة الإنتاجية، والسماح لمركبات فحص أكثر سهولة. وقد أظهرت توصيف دقيق قاطع أن آليات الفوعة الجرثومية المختلفة بين هذه الاختبارات ولدينا الشاشة المستندة إلى السائل7. إذ لوحظت كلا النوعين من ضراوة في نظم الثدييات، من المهم النظر المحدد ضراوة الذي هو الأكثر ملاءمة لمصالح المجرب قبل اختيار المقايسة.
هنا نظهر إصدار محسن من السائل على أساس مقايسة C. ايليجانس P. الزّنجاريّة . ونحن أيضا تقرير تكييف أسلوب التحليل القائم على سائل لدينا لاستيعاب الممرضات البكتيرية إيجابية البرازية faecalis. مثل P. الزّنجاريّة، يتم تعريف faecalis هاء- يتزايد كتهديد خطير المستشفيات مع تسلح متزايد لمقاومة مضادات الميكروبات مسارات1. على الرغم من وجود أسلوب سابقة للفرز الفائق من faecalis هاء- 14، فإنه يتطلب بريينفيكشن مع مسببات المرض، مما يزيد من تعقيد الإجراءات ويزيد من احتمال تلويث المعدات مثل فلووسورت COPAS. لدينا بروتوكول يلغي الحاجة لقبل الإصابة، تحسين السلامة الشخصية. أخيرا، نحن التقرير وسيلة بها أي من هذه الاختبارات يمكن دمجها مع تغذية [رني]، مما يتيح للمستخدم البحث عن المضيف من العوامل التي تلعب دوراً في إنشاء، أو مقاومة للعدوى.

<table>
	<tbody>
		<tr>
			<td>COPAS FP BioSorter</td>
			<td>Union Biometrica</td>
			<td></td>
			<td>Large object flow cytometer/worm sorter</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EL406 Washer Dispenser</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron Pro</td>
			<td>Infors HT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 Deep-Well RB Block</td>
			<td>Thermo Fisher Scientific</td>
			<td>CS15124</td>
			<td></td>
		</tr>
		<tr>
			<td>384-Well plate</td>
			<td>Greiner Bio-One</td>
			<td>MPG-781091</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode Growth Media (NGM)</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 2.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) plates</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 18 grams agar, 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phospate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Slow Killing (SK) media</td>
			<td></td>
			<td></td>
			<td>Amount per liter:&#160; 3 grams NaCl, 3.5 grams Peptone, 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL Phosphate buffer, and 973 mL of milli-Q water</td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>USBiological Life Sciences</td>
			<td>L1520</td>
			<td></td>
		</tr>
		<tr>
			<td>Brian Heart Infusion broth (BHI)</td>
			<td>Research Products International Corp</td>
			<td>50-488-526</td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Bleach Solution</td>
			<td></td>
			<td></td>
			<td>Amount per 100 mL: 10 mL of 5 M NaOH solution, 20 mL of 5% Sodium Hypochlorite Solution, and 70 mL of sterile water</td>
		</tr>
		<tr>
			<td>S Basal</td>
			<td></td>
			<td></td>
			<td>Amount per liter: 5.85 grams NaCl, 6 grams KH2PO4, 1 gram K2HPO4, and 1 Liter of milli-Q water</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>USBiological Life Sciences</td>
			<td>A0930</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>USBiological Life Sciences</td>
			<td>S5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>USBiological Life Sciences</td>
			<td>P3300</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>USBiological Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffer</td>
			<td></td>
			<td></td>
			<td>amount per liter: 132 mL of K2HPO4 (1M) and 868 mL of KH2PO4 (1M)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Acros Organics</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>USBiological Life Sciences</td>
			<td>P5100</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Sodium Hypochlorite Solution</td>
			<td>BICCA</td>
			<td>7495.5-32</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH solution</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-easy</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX Orange Nucleic Acid Stain</td>
			<td>Fisher Scientific</td>
			<td>S11368</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P. aeruginosa (PA14)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. faecalis(OG1RF)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli superfood (OP50)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAi expressing bacteria (HT115)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm Strains </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glp-4(bn2) (Beanan and Strome, 1992, PMID: 1289064)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PINK-1::GFP reporter (Kang et al., 2018, PMID: 29532717)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput, high-content, liquid-based c. elegans pathosystem

عدد الأدوية الجديدة التي حددت بالتقليدية، في المختبر شاشات تضاءل، الحد من نجاح هذا النهج في البحث عن أسلحة جديدة لمكافحة مقاومة الأدوية المتعددة. وادي هذا إلى الاستنتاج بأن الباحثين لا تحتاج فقط إلى العثور على أدوية جديدة، ولكن أيضا بحاجة إلى تطوير طرق جديدة للعثور عليهم. بين المرشح الأكثر تبشيرا بالنجاح أساليب مسببة للجامعة، في فيفو فحوصات هذا الاستخدام الفائق، والمظهرية قراءات ويستضيف أن تتراوح بين ايليجانس كاينورهابديتيس دانيو rerio. هذه الأجهزة المضيفة لها العديد من المزايا القوية، بما في ذلك تخفيضات هائلة في عدد الزيارات إيجابية كاذبة، كما يتم عادة إسقاط مركبات سامة للمضيف و/أو بيونافيلابل في الشاشة الأولى، قبل متابعة مكلفة حتى.
هنا نعرض كيف استخدمت لدينا المقايسة لاستجواب تباين المضيف في الموثقة توثيقاً جيدا C. ايليجانس–الزائفة الزّنجاريّة قتل السائل باثوسيستيم. كما نبدي ملحقات عدة لهذا الأسلوب جيدا عملت بها. على سبيل المثال، نحن قادرون على الاضطلاع بشاشات الجينية الفائق باستخدام [رني] في بئر 24 أو 96 لوحة الأشكال لعوامل المضيف الاستعلام في هذا التفاعل المضيف الممرض. باستخدام هذا التحليل، يمكن أن تكتمل شاشات الجينوم كله في بضعة أشهر فقط، التي يمكن تبسيط مهمة تحديد أهداف المخدرات، يحتمل أن تكون دون الحاجة إلى اتباع نهج التنقية البيوكيميائية شاقة بشكل كبير.
نحن أيضا تقرير هنا تلاف أسلوب لدينا بدائل بكتيريا إيجابية faecalis البرازية الممرض الغرام P. الزّنجاريّة. كثير كما الحال بالنسبة P. الزّنجاريّة، قتل faecalis هاء- تعتمد على الوقت. على عكس السابق C. ايليجانس– فحوصاتفايكاليس هاء ، ولدينا المقايسة faecalis هاء- لا يتطلب برينفيكتيون، تحسين صورتها السلامة وتقليل فرص تلوث معدات مناولة السائل. المقايسة قوية جداً، عرض ~ 95% معدلات الوفاة 96 ساعة بعد الإصابة.

المعالم الهامة لتحليل الأداء
فهم سليم للبيولوجيا الكامنة وراء هذا التحليل ضروري لاستكشاف الأخطاء وإصلاحها وتحسين المقايسة. تحقيقا لهذه الغاية، ونشير أولاً إلى عدة ورقات رئيسية توضيح آليات إمراضية P. الزّنجاريّة-بوساطة قتل في ?...

Watch this Scientific Journal Video about A High-throughput, High-content, Liquid-based C. elegans Pathosystem at JoVE.com

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

هنا يمكننا وصف بروتوكول هو المضيف قابلة للتكيف، وكله، أداة فحص عالية المحتوى التي يمكن استخدامها لدراسة التفاعلات بين المضيف الممرض ويمكن استخدامها لاكتشاف المخدرات.

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 ...

This method can answer questions in host pathogen interaction and drug discovery, such as the the importance of various virulence factors and the ability of small molecules to ameliorate with the genesis. The main advantage of this technique is that it takes place in a liquid format with small volumes. To begin, while working inside a BSL 2 biosafety cabinet, streak P.aeruginosa from a frozen stock onto an LB Agar plate.Incubate the plate at 37 degrees C for 16 to 24 hours, then store the plate at four degrees Celsius for up to one week. Two days prior to setting up assay plates, use a single colony from the plate to inoculate three to five milliliters of sterile LB broth. Incubate the culture at 37 degrees Celsius for 12 to 16 hours.After preparing the slow kill or SK media according to the text protocol, with 350 milliliters of p. Aeruginosa from the fresh overnight LB culture, seed each 10 centimeter SK plate. Use a sterile bacterial spreader to evenly spread the bacteria, and allow the plates to dry in the biosafety cabinet.Then, incubate the plates at 37 degrees Celsius for 24 hours. To test multiple RNAi bacterial strains in parallel in a single well of a 24 well deep well plate, inoculate a single colony from each clone of previously prepared RNAi containing bacteria into four milliliters of carbenicillin supplemented LB.Place the plates into a shaking incubator optimized for multi-well plates at 37 degrees Celsius and 950 rpm for 16 hours, then collect the bacteria by centrifugation at 2, 000 g for 5 minutes. Decant the supernatant by inverting the plate and vigorously shaking it.Using 100 microliters of S Basal resuspend the RNAi bacteria. Pipette the resuspended bacteria into an appropriate number of wells of a multi-well NGM plate supplemented with IPTG and carbenicillin and allow the plate to dry. After preparing L1 worms according to the text protocol, prepare plates for basic experiments by pipetting approximately 5, 000 worms per 10 centimeter plate, seeded with RNAi or OP50 superfood.To set up for an RNAi screen, pipette approximately 300 worms per well in a 24 well plate seeded with RNAi. If the strain being used is temperature-sterile, incubate the worms at 15 degrees Celsius for approximately 16 hours and then transfer them to 25 degrees Celsius for 44 hours to complete development and prevent embryogenesis. To carry out a liquid killing assay use a cell scraper to remove the P.aeruginosa from an SK plateand resuspend the bacteria in approximately 5 milliliters of S.Basal.Using a spectrophotometer meaure the OD 600 of the bacterial suspension. Prepare 24 milliliters of diluted stock of P.Aeruginosa in S.Basal at an OD 600 equal to 09, then add 21 milliliters of liquid SK media, and, using a multichannel pipette, transfer 45 microliters of bacteria and media to each well of a 384 well plate. Wash the worms from their source into a 50 milliliter conical tube, and allow them to settle under gravitational force.Aspirate the supernatant to 5 milliliters, then use a total of 50 milliliters of S.Basal to resuspend the worms, and repeat the wash twice more. Using a worm sorter, sort approximately 22 worms into each well of a 384 well plate, according to the text protocol, then use a gas-permeable film to seal the plate and incubate it at 25 degrees Celsius for 24 to 48 hours. At the desired time, use a microplate washer and S.Basal to wash the 384 well plate a total of 5 times.One of the easiest mistakes to make is to aspirate the 384 well plate before worms have completely settled. It takes practice to accurately see whether the worms have settled completely. After the final wash, aspirate the supernatant down to 20 microliters.Then, add 50 microliters of 98 micromolar nucleic acid stain per well, for a final concentration of 0.7 micromolar. Incubate the plate at room temperature for 12 to 16 hours. After the desired incubation period, use the microplate washer to wash the plates a minimum of three times.For a data acquisition use a spectrophotometer or an automated microscope to image both transmitted light and fluorescence. Add one milliliter of S Basal per well of a 24 well plate containing 300 worms per well. Gently agitate the worms by shaking the plate, then transfer worms to an empty, sterile 24 deep well plate.Allow the worms to settle gravitationally, about five minutes. Aspirate the supernatant, leaving approximately one milliliter per well, Then add 7 milliliters of S Basal to each well. Repeat the wash two more times, and then after the final wash aspirate all but approximately 400 microliters of supernatant.After pipetting the bacteria into 384 well plates to avoid starvation, using the resampler function of the worm sorter sort 22 worms into each well of a 384 well plate. Finally, following sorting, add small molecules or other experiment specific materials. As illustrated in this graph, when the steps outlined in this video are followed, a time dependent killing of C.elegans will be observed only in the presence of P.aeruginosa.In contrast, in the absence of key bacterial nutritional supplements, little to no killing will be observed. As shown here, the two step incubation of P.aeruginosa at 37 degrees Celsius and then 25 degrees Celsius, which is critical for conventional slow killing assays, and was originally implemented in liquid killing, is dispensable in this assay. The protocol tolerates a wide range of initial bacterial concentrations, from as low as an OD 600 equal to 0025, and still exhibits time and concentration dependent killing, although the timing does shift.In addition, as few as four wells is frequently sufficient to obtain statistically significant data. An example of the utility of processing is shown here when the signal-to-noise ratio is high, as in this example, analysis is simple and discriminating between positive and negative conditions is trivial. In these cases, even weak hits can be readily identified.Once mastered, this technique can be performed in about 60 to 75 minutes of hands on time per 384 well plate, not including sorting. While attempting this procedure, it's critical to remember to add all the components to your media or virulence will be compromised. This assay simplifies the application of whole organism phenotype based screening to drug discovery in host-pathogen research.After watching this video, you should have a good understanding of how to carry out liquid-based C elegans pathogenesis assays. This procedure can be modified by substituting with other pathogens, such as E.Faecalis for P.Aeruginosa, facilitating the search for treatments for other bacterial infections. Don't forget that working with infectious bacteria like P.Aeruginosa or E.Faecalis can be dangerous.Appropriate precautions, such as proper training, good personal protective equipment, and proper technique, should always be taken while performing this procedure.

a-high-throughput-high-content-liquid-based-c-elegans-pathosystem

Research

JoVE Journal

Immunology and Infection

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

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

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

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 ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

الفصل الآلي لل<em&gt; C. ايليجانس</em&gt; المستعمرة بنسب مختلفة من قبل الممرض الجرثومي

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

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 Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

عالية الإنتاجية الفحص إلى النمط الظاهري<em&gt; السالمونيلا الملهبة لل</em&gt; جمعية التيفية الفأرية، والغزو، والنسخ المتماثل في الضامة

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

إنشاء إعداد إنتاجية عالية لفحص الجزيئات الصغيرة التي تعدل ج-دي-GMP في اشارة<em&gt; الزائفة الزنجارية</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

ومضان القائم على الخلايا الليمفاوية الفحص مناسبة لفحص إنتاجية عالية من الجزيئات الصغيرة

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

الخلية الحرة البيوكيميائية المقايسة الانزيمية فلوروميتريك لقياس الفائق وبيروكسيد في البروتين الدهني العالي الكثافة

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

الآلي التحليل السلوكي لكبير C. ايليجانس السكان استخدام واسعة مجال-من-عرض "تتبع النظام الأساسي"

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

الفائق دوسنتاريا-مقايسة جراثيم معينة

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...