وأيد هذا العمل من قبل studentship من جامعة كولومبيا البريطانية (لBV) والمنح المقدمة من التليف الكيسي كندا والكندية معاهد أبحاث الصحة (لDPS). نشكر جاكلين تشونغ لإعداد الأولي من البروتوكولات.

<ol>
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L., 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=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol. Cell Proteomics. 3, 1154-1169 (2004).</li><li>Velapati&#241;o, B., Limmathurotsakul, D., Peacock, S. J., Speert, D. P. <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+differentially+expressed+proteins+from+Burkholderia+pseudomallei+isolated+during+primary+and+relapsing+melioidosis.">Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis.</a> Microbes. Infect. 14, 335-340 (2012).</li><li>Chung, J. W., Speert, D. 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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

وقد وصفت طريقة لتحضير البروتينات التي يمكن استخراج معظم البروتينات بيركولديريا مع استنساخ جيدة. ويتجلى ذلك من خلال الحصول على نفس التشكيل الجانبي البروتين من اثنين الاستعدادات مستقلة أجريت في أيام مختلفة باستخدام نفس الثقافة البكتيرية التي تزرع في مرق LB أو YEM ?...

تضم بيركولديريا جنس أكثر من 62 نوعا، والكائنات السلبية الغرام معزولة عن مجموعة واسعة من المنافذ، ويتم تقسيمها في مجموعتين رئيسيتين 1،2. وتشمل المجموعة الأولى phytotrophic الكائنات البشرية والحيوانية و، وركزت معظم الدراسات على الأنواع المسببة للأمراض من هذه المجموعة نظرا لأهميتها السريرية الخاصة بهم. الأعضاء الأكثر الممرضة هي B. الراعومية وباء. المطرقة (الذي يسبب راعوم والرعام على التوالي) 3،4 ومسببات الأمراض الانتهازية (17 أنواع محددة من مجمع الشرهة بيركولديريا، BCC) 5، والتي تسبب المرض في التليف الكيسي (CF) والداء الحبيبي المزمن (CGD) 1. المجموعة الثانية، مع أكثر من 30 نوعا غير إمراضية، وتشمل البكتيريا المرتبطة النباتات أو مع البيئة، وتعتبر قد تكون مفيدة إلى المضيف 2. مضاعفات عديدة تظهر الابعدوى بكتيرية ام مع أعضاء المسببة للأمراض من جنس بيركولديريا، مثل نقل مسببات المرض بين المرضى، وانتشار المرض وفشل العلاج بسبب المقاومة الذاتية أو المكتسبة للمضادات الحيوية مما يجعل من الصعب للقضاء في معظم الحالات 6-9. لذا، والحصول على فهم أوضح لأساس لإنشاء عدوى بكتيرية أمر حيوي لعلاج الأمراض التي تسببها هذه الكائنات. من أجل الحصول على نظرة ثاقبة إنشاء العدوى، وهناك حاجة إلى إجراء تحقيقات واسعة النطاق على المكونات البكتيرية المرتبطة المرضية. وصف الدراسات التي تركز على تحليل البروتين من الكائنات الحية بيركولديريا باستخدام نهج البروتين البروتينات التي قد تورطت في المرضية البكتيرية وكذلك التغيرات في بروتيوم بهم لمحات 10-16. طرق استخراج البروتين باستخدام صوتنة ودورات إذابة تجميد في تحلل العازلة التي تحتوي اضرب عاليةentration من اليوريا، وقد تم تطبيق ثيوريا في تركيبة مع المنظفات وampholytes في بيركولديريا الدراسات البروتين 10-13. على الرغم من اليوريا هي فعالة جدا للبروتين تمسخ، فإنه يمكن إقامة التوازن في محلول مائي مع الإيزوسيانات الأمونيوم، والتي يمكن أن تتفاعل مع مجموعات من الأحماض الأمينية، وبالتالي تشكيل التحف (carbamylation رد فعل) 17. وبالتالي، فمن المستحسن أن تشمل ampholytes الناقل، والتي تكون بمثابة الزبالين سيانات وتجنب درجات الحرارة فوق 37 درجة مئوية 17. علاوة على ذلك، لمنع أي تدخل الكيميائي للتحلل العازلة مع البروتين الكمي، وهو نفس العازلة تحلل يمكن استخدامها لتوليد منحنى القياسية بحيث العينات والمعايير لديهم نفس الخلفية 10. تنطوي منهجيات أخرى استخدام مخازن القلوية والمنظفات مع فترات الحضانة حرارة 17،18، ولكن هذه الظروف قد تحدث تغييرات في بروتيوم وبعض المنظفات غير متوافقة مع العلاقات العامةتطبيق oteomics ما لم يتم تضمين الخطوات اللاحقة إزالة المنظفات 17،18. بعد استخراج والكمي كافية، والتعبير البروتين العالمي لكل فرد البروتين يمكن دراستها باستخدام نهج البروتين مثل ثنائي الأبعاد (2-D) هلام الكهربائي. وقد وصفت هذه التقنية أول مرة من قبل O&#39;Farell 19 وتتكون في فصل البروتينات وفقا لجهة نظرهم كهرساوي بواسطة كهرساوي التركيز في البعد الأول، ومن ثم وفقا لوزنهم الجزيئي بواسطة مادة الأكريلاميد هلام الكهربائي في البعد الثاني. بسبب قرارها والحساسية، وهذا الأسلوب هو أداة قوية لتحليل والكشف عن البروتينات من مصادر بيولوجية معقدة 19،20. هذه التقنية الانفصال هو متاح حاليا في النهج التي تركز البروتين مع ميزة كبيرة في حل الأشكال الإسوية البروتين الناجمة عن التعديلات بعد النسخي أو تجهيز بروتين. التغيرات الكميةويمكن الكشف عن طريق مقارنة شدة البقعة المقابلة بعد تلطيخ من هلام 20. ومع ذلك، لا تناسب هذه التقنية لتحديد البروتينات كبيرة جدا، والبروتينات الغشاء، والبروتينات الأساسية للغاية والحمضية أو مسعور، وتقنية شاقة وتستغرق وقتا طويلا إلى حد ما 20. النهج الببتيد مركزية جديدة (بناء هلام غير) التي هي أكثر قوة وموضوعية تصبح متاحة ويمكن استخدامها للمقارنة الكمي بالطرق وضع العلامات النظائر مستقرة التفاضلية مثل وضع العلامات السيستين من قبل تلوينها نظير تقارب الجغرافية (ICAT) 21، والمجموعة الأمينية وضع العلامات النظائر الجغرافية لتحديد الكميات النسبية والمطلقة (iTRAQ) 22. استخدام تقنية البروتين واحد قد يعطي معلومات كافية، وبالتالي استخدام نهجين البروتين التكميلية ضروري لمعظم التغييرات تقييم كامل في بروتيوم. ومع ذلك، يتم استخدام على نطاق واسع 2-D الكهربائي للهلام ويمكن تطبيقها بشكل روتيني لتشيوانالكمي المنجز التعبير عن العديد من البروتينات في الكائنات الحية المختلفة. نحن هنا وصف استخراج البروتين خلية كاملة و 2-D إجراءات هلام الكهربائي عن الأنواع التي تم تكييفها بيركولديريا والأمثل من الرعاية الصحية GE 2-D المبادئ الكهربائي وطرق كتيب 80-6429-60AC عام 2004 ( <a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a> ). تم تنفيذ استخراج البروتين من استخدام الخافق حبة مع الخرز الزجاجي في وجود برنامج تلفزيوني يحتوي على 5 ملي EDTA (الإيثيلين حمض tetraacetic) و 1 ملي PMSF (phenylmethylsulfonyl الفلورايد). يسمح هذا الإجراء الكمي للبروتينات مع الحد الأدنى من التدهور وغير قابلة للتعديل لنهج البروتين كما ذكرت سابقا 15،23،24. تم تنفيذ 2-D الكهربائي للهلام خارج باستخدام 24 سم يجمد طويلة درجة الحموضة 4-7 التدرج وتم فصل البروتينات وفقا لجهة نظرهم كهرساوي. ثم تم فصل البروتينات وفقا لmolec بهمالوزن جزيئية بواسطة هلام SDS-بولكرلميد. بالإضافة إلى ذلك، وصفنا طريقة الفضة تلطيخ لرؤية البروتينات بقعة، وطريقة وصمة عار الفضة والتي تتوافق لتحليل الطيف الكتلي. معا هذه الإجراءات يمكن أن تسمح بتحديد البروتينات الهامة من الأنواع بيركولديريا التي يمكن أن تشارك في التسبب.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Phenylmethanesulfonyl fluoride (PMSF)</td>
 <td>Sigma</td>
 <td>P7626</td>
 <td>Toxic, corrosive</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher</td>
 <td>A18-1</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>PBS Buffer</td>
 <td>Bioscience</td>
 <td>R028</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher</td>
 <td>BP118-500 </td>
 <td>toxic</td>
 </tr>
 <tr>
 <td>Glass beads (0.1 mm)</td>
 <td>BioSpec Products</td>
 <td>11079101</td>
 <td></td>
 </tr>
 <tr>
 <td>Nalgene Oak Ridge Centrifuge tubes, polycarbonate (50 ml)</td>
 <td>VWR</td>
 <td>21009-342</td>
 <td></td>
 </tr>
 <tr>
 <td>MicroBCA protein extraction kit</td>
 <td>Pierce</td>
 <td>23235</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge Tubes 2.0 ml conical Screw cap Tubes with Cap and O-Ring</td>
 <td>Fisher</td>
 <td>02-681-375</td>
 <td></td>
 </tr>
 <tr>
 <td>2-D Clean-Up kit</td>
 <td>GE Healthcare</td>
 <td>80-6484-51</td>
 <td></td>
 </tr>
 <tr>
 <td>Urea</td>
 <td>Invitrogen</td>
 <td>15505-050</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>Amersham Biosciences</td>
 <td>17-1314-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>MPBiomedical, LCC</td>
 <td>856126</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Immobiline DryStrips pH 4-7 24 cm</td>
 <td>Amersham Biosciences</td>
 <td>176002-46</td>
 <td></td>
 </tr>
 <tr>
 <td>Duracryl</td>
 <td>Proteomic Solutions</td>
 <td>80-0148</td>
 <td>Very toxic, carcinogen</td>
 </tr>
 <tr>
 <td>Ammonium persulfate</td>
 <td>Fisher</td>
 <td>BP179-100</td>
 <td>Flammable, toxic, corrosive</td>
 </tr>
 <tr>
 <td>N,N,N',N'-tetramethylethylenediamine (TEMED)</td>
 <td>Invitrogen</td>
 <td>15524-010</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate (SDS)</td>
 <td>Fisher</td>
 <td>BP166-500</td>
 <td>Acute toxicity, flammable</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Fisher</td>
 <td>HC1100-1GL</td>
 <td>Flammable, toxic</td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Fisher</td>
 <td>A491-212</td>
 <td>Flammable, corrosive</td>
 </tr>
 <tr>
 <td>Glutaraldehyde</td>
 <td>Fisher</td>
 <td>G151-1</td>
 <td>Very toxic, corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Potassium tetrathionate</td>
 <td>Sigma</td>
 <td>P2926</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Sodium acetate</td>
 <td>EM Science</td>
 <td>7510</td>
 <td></td>
 </tr>
 <tr>
 <td>Silver nitrate</td>
 <td>Sigma</td>
 <td>209139</td>
 <td>Corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>252549</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Sodium thiosulfate</td>
 <td>Sigma</td>
 <td>S7026</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>EMD</td>
 <td>9230</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>MPBiomedical, LCC</td>
 <td>808831</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>MPBiomedical, LCC</td>
 <td>800688</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher</td>
 <td>A412-4</td>
 <td>Flammable, toxic, health hazard</td>
 </tr>
 <tr>
 <td>Sodium carbonate anhydrous</td>
 <td>EMD</td>
 <td>SX0400-3</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>ACROS</td>
 <td>415080010</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>JA-20 ultracentrifuge rotor</td>
 <td>Beckman Coulter</td>
 <td>334831</td>
 <td></td>
 </tr>
 <tr>
 <td>Mini Beadbeater</td>
 <td>Biospec Products</td>
 <td>3110BX</td>
 <td></td>
 </tr>
 <tr>
 <td>Ettan IPGphor II Isoelectric Focusing System and accessories</td>
 <td>GE Healthcare</td>
 <td>80-6505-03</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 <tr>
 <td>Ettan DALT Large Vertical electrophoresis system</td>
 <td>GE Healthcare</td>
 <td>80-6485-27</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 </tbody>
</table>

total protein extraction and 2-d gel electrophoresis methods for burkholderia species

التحقيق في مستويات البروتين داخل الخلايا من الأنواع البكتيرية من الأهمية لفهم الآليات المسببة للأمراض من الأمراض التي تسببها هذه الكائنات. نحن هنا وصف الإجراء لاستخراج البروتين من الأنواع بيركولديريا على أساس تحلل الميكانيكية باستخدام الخرز الزجاجي في وجود حمض tetraacetic الإيثيلين وphenylmethylsulfonyl الفلوريد في الفوسفات مخزنة المالحة. هذه الطريقة يمكن استخدامها لأنواع مختلفة بيركولديريا، لظروف النمو المختلفة، وانها مناسبة المحتمل للاستخدام في الدراسات البروتين من البكتيريا الأخرى. بعد استخراج البروتين، ويوصف (2-D) هلام الكهربائي تقنية البروتين ثنائي الأبعاد لدراسة التغيرات العالمية في proteomes من هذه الكائنات. يتكون هذا الأسلوب من فصل البروتينات وفقا لجهة نظرهم كهرساوي بواسطة كهرساوي التركيز في البعد الأول، تليها الفصل على أساس الوزن الجزيئيبواسطة الأكريلاميد هلام الكهربائي في البعد الثاني. ويتم فصل التصور من البروتينات بها الفضة تلطيخ.

وأظهر تحليل مقارن لمحات البروتين المستخرج من نفس الثقافة البكتيرية في مناسبتين مختلفة أنماط مماثلة النطاقات تشير الاستخراج البروتين ناجحة. تراوحت البروتينات الوزن الجزيئي المستخرجة 10-150 كيلو دالتون. الشكل 1 يبين ممثل Coomassie هلام تلطيخ الزرقاء في الاستخراج ا...

Watch this Scientific Journal Video about Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species at JoVE.com

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

أعضاء من جنس بيركولديريا هي مسببات الأمراض ذات الأهمية السريرية. نحن تصف طريقة لاستخراج البروتين الكلي البكتيري، وذلك باستخدام اضطراب الميكانيكية، و 2-D الكهربائي للهلام لتحليل البروتين اللاحقة.

مجموع استخراج بروتين و 2-D طرق جل الكهربائي ل<em&gt; بيركولديريا</em&gt; الأنواع

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

total-protein-extraction-2-d-gel-electrophoresis-methods-for

Research

JoVE Journal

Immunology and Infection

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

28.9K Views.  University of British Columbia.  أعضاء من جنس بيركولديريا هي مسببات الأمراض ذات الأهمية السريرية. نحن تصف طريقة لاستخراج البروتين الكلي البكتيري، وذلك باستخدام اضطراب الميكانيكية، و 2-D الكهربائي للهلام لتحليل البروتين اللاحقة. 

Video: Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

إجراء 1.5 ساعة لتحديد معوي أنواع الثقافات مباشرة من الدم

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

الطيور ترصد الأنفلونزا اتفاقية التجارة الحرة مع بطاقات : أساليب الميدانية ، الأحيائية ، وقضايا النقل محلولة

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

A Chelex بروتوكول بسيط لاستخراج الحمض النووي من<em&gt; الأنوفيلة</em&gt; النيابة.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

إجراءات لتحديد المعدية البريونات بعد المرور عبر الجهاز الهضمي لأنواع الطيور

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

إنشاء والأمثل لإعداد إنتاجية عالية لدراسة<em&gt; المكورات العنقودية البشروية</em&gt; و<em&gt; المتفطرة البحرية</em&gt; عدوى نموذجا لاكتشاف المخدرات

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

البروتين من التنميط الضامة التي كتبها 2D الكهربائي

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

متساوي التضخيم (LAMP) فحوصات بوساطة حلقة للكشف عن أنواع محددة من<em&gt; الأيمرية</em&gt; التي تصيب الدجاج

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

أساليب بديلة في المختبر لتحديد السلامة الهيكلية قفيصه الفيروسية

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

القياس الآلي كبسولة السكاريد الأنواع الفطري وجسم الخلية

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

تشريح مجمعات البروتين متعدد الإشارات بالتكامل Bimolecular انجذاب تطهير (بيكاب)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...