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.

<ol>
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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. 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الكتاب يعلن أن لديهم لا تضارب في المصالح بالكشف عن.

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 ...

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JoVE Journal

Immunology and Infection

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

14.4K Views.  Rice University. 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.

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

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. ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

الإنتاجية العالية جين توصيف في<em&gt; المثقبية البروسية</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

كشف الإنتاجية العالية للجهاز التنفسي مسببات الأمراض في العينات الحيوانية التي كتبها النانو PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

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 ...

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

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الفصل الآلي لل

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

عالية الإنتاجية الفحص إلى النمط الظاهري

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

إنشاء إعداد إنتاجية عالية لفحص الجزيئات الصغيرة التي تعدل ج-دي-GMP في اشارة

الإنتاجية العالية جين توصيف في

كشف الإنتاجية العالية للجهاز التنفسي مسببات الأمراض في العينات الحيوانية التي كتبها النانو PCR

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

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

الفائق دوسنتاريا-مقايسة جراثيم معينة