This work was supported by BC Lung Association and Mitacs.

1. سلالة بكتيرية والنمو المتوسطة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جعل سكر العنب الزلال وحل الأسهم الملح (ADS) من خلال الانحلال 25 غرام من الزلال المصل البقري، 10.0 غرام من سكر العنب، و4.05 غرام من كلوريد الصوديوم في 460 مل من الماء منزوع الأيونات. تصفية تعقيم ADS وتخزينها في 4 ° C. </li><li style=";text-align:right;direction:rtl"> جعل 7H9 مرق من خلال إضافة 4.7 غرام من مسحوق 7H9 و 2 مل من الجلسرين 900 مل من الماء النقي. الأوتوكلاف مرق 7H9 على 121 درجة مئوية لمدة 10 دقيقة والسماح لتبرد إلى درجة حرارة الغرفة قبل المتابعة. جعل 7H9ADST بإضافة 100 مل من ADS و 0.5 مل من Tween80 إلى 900 مل من مرق 7H9. تخزينها في 4 ° C. </li><li style=";text-align:right;direction:rtl"> تزن 50 ملغ من كبريتات كانامايسين وتذوب في 1 مل من الماء منزوع الأيونات. التركيز النهائي هو 50 ملغ / مل. تصفية تعقيم وتخزين في -20 ° C. إضافة 0.5 مل من الكانامايسين حل سهم لكل 1 لتر من 7H9ADST. ملاحظة: يجب أن يتم هذا متوسطة جديدة، لذلك حجم وحدات التخزين بشكل مناسب وفقا لحجم الثقافة. </li><li style=";text-align:right;direction:rtl"> تنمو السل M. في 7Hتستكمل 9ADST مع كانامايسين في الثقافة واقفا. يهز الثقافة يوميا وتمييع قبل أن يصل OD600 1.0 لتجنب تراكمها. ملاحظة: سلالة السل M. المستخدمة في تطوير هذه الطريقة كان H37Rv تحولت مع pJAK2.A البلازميد 12. pJAK2.A هو البلازميد التكاملي على أساس ناقلات pMV361، والذي يسمح التعبير رفيع المستوى من الجين يراعة luciferase من المروج hsp60 ويمكن اختيار باستخدام كانامايسين. </li></ol> 2. THP-1 المتوسطة والصيانة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 50 مل من الحرارة المعطل مصل بقري جنيني (FBS) و 5 مل من 200 ملي L-الجلوتامين إلى 500 مل من RPMI 1640 لجعل RPMI المتوسطة ناقصة (حوالي 10٪ FBS و 2 الجلوتامين ملم). </li><li style=";text-align:right;direction:rtl"> الحفاظ على ثقافة الخلية THP-1 وفقا لبروتوكول قياسي 13. لفترة وجيزة، وتنمو THP-1 الخلايا في RPMI المتوسطة ناقصة مع الحفاظ على كثافة خلية من ،2-1٬000٬000 في مل من المتوسط ​​بين نظام تقييم الأداءحكماء. </li></ol> 3. فحص عالية الإنتاجية داخل الخلايا عن طريق معربا عن luciferase المراسل M. السل H37Rv <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قياس الكثافة البصرية لتعليق البكتيرية بنشاط متزايد في معمل عند طول موجي 600 نانومتر. حساب كثافة البكتيرية باستخدام عامل التحويل من 0.1 OD 600 = 3 × 10 7 بكتيريا لكل مليلتر. </li><li style=";text-align:right;direction:rtl"> ماصة البكتيريا كافية لعدد وافر من العدوى (وزارة الداخلية) من 10: 1 في أنبوب الطرد المركزي الجديدة. بيليه في 3000 x ج لمدة 10 دقيقة، ونضح السائل. إضافة 50 ميكرولتر من المصل البشري إلى 450 ميكرولتر من RPMI1640. مقياس حجم تخصيص قيم لهذه التجربة. </li><li style=";text-align:right;direction:rtl"> ليستطهي البكتيريا، إعادة تعليق بيليه في مناطق ذات كثافة من 1 × 10 8 بكتيريا في كل 500 ميليلتر من RPMI1640 المحتوية على مصل الإنسان 10٪. السماح للخليط لاحتضان عند 37 درجة مئوية لمدة 30 دقيقة. تحديد THP-1 خلية كثافة الثقافة عن طريق العد مع عدادة الكريات وmicrosco مقلوببي. </li><li style=";text-align:right;direction:rtl"> بيليه الخلايا في أنابيب الطرد المركزي معقمة في 100 x ج و 37 درجة مئوية لمدة 10 دقيقة. نضح في وطاف resuspend الخلايا في RPMI غير مكتملة في مناطق ذات كثافة من 1 مليون خلية لكل مليلتر. إضافة phorbol 12 ميريستيت-13-خلات (PMA) إلى 40 نانوغرام / مل تركيز النهائي. ملاحظة: سيتم يشار إلى ذلك المزيج التمايز. </li><li style=";text-align:right;direction:rtl"> الجمع بين opsonized السل M. مع مزيج التمايز THP-1 في وزارة الداخلية من 10: 1 وقسامة المزيج النهائي في 100 ميكرولتر لكل بئر في لوحة بيضاء 96-جيدا مسطحة القاع. يحرك الخليط بانتظام لضمان الاتساق. السماح للتمايز وعدوى والمضي قدما ليلا 37 درجة مئوية في حاضنة مرطب يحتوي على 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> يغسل الآبار مرتين مع 100 ميكرولتر من RPMI لكل منهما. إضافة مركبات مخففة للتركيز المطلوب في RPMI غير مكتملة واحتضان لمدة 3 أيام. </li><li style=";text-align:right;direction:rtl"> نضح في المتوسط ​​من الآبار. إضافة 50 ميكرولتر من كاشف فحص وسيفيراز إلى كل بئر. ختم لوحات مع تيransparent السدادات لوحة لاصقة. سماح 5 دقائق من الحضانة عند 22 درجة مئوية ومن ثم الحصول على قراءات في luminometer في 1 ثانية لكل بئر. </li></ol> 4. تحليل السمسة باستخدام 3- (4،5-Dimethylthiazol-2-YL) -2،5-diphenyltetrazolium بروميد (MTT) فحص 14 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> التفريق THP-1 الخلايا في RPMI تستكمل كاملة مع 40 نانوغرام / مل من سلطة النقد الفلسطينية في واضحة لوحات 96-جيدا. الحفاظ على كثافة الخلية 1 مليون لكل مليلتر وقسامة 100 ميكرولتر لكل بئر. السماح التمايز والمضي قدما ليلا 37 درجة مئوية في حاضنة مرطب يحتوي على 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> نضح المتوسط ​​من الآبار وغسلها مرتين مع RPMI 1640. اضافة مركبات مخففة في RPMI غير مكتملة إلى الآبار. احتضان لمدة 3 أيام. </li><li style=";text-align:right;direction:rtl"> حل 0.5 غرام من MTT في 100 مل من الفوسفات مخزنة المالحة (PBS) لجعل حل الأسهم من 5 ملغ / مل. تصفية العقيمة وتخزينها في -20 ° C، بعيدا عن الضوء. فمن الأفضل لجعل هذا الحل الطازجة. </li><li style=";text-align:right;direction:rtl"> 2.5 ساعة قبل احتسابه نهاية فترة الحضانة لمدة 3 أيام، إضافة 25 ميكرولتر من محلول MTT إلى كل بئر واستكمال فترة الحضانة. </li><li style=";text-align:right;direction:rtl"> تجهيز 50٪ N، N -dimethyl الفورماميد (DMF) عن طريق خلط 250 مل من DMF مع 250 مل من الماء منزوع الأيونات. </li><li style=";text-align:right;direction:rtl"> إعداد استخراج العازلة MTT على النحو التالي: وزن 100 غرام من SDS في زجاجة 500 مل وإضافة 300 مل من 50٪ DMF. تطبيق الحرارة المنخفضة للسماح للSDS إلى حل. إضافة 10 مل من حمض الخليك النقي و 12.5 مل من 1 M حمض الهيدروكلوريك. ملء ما يصل الى علامة 500 مل مع 50٪ DMF. التشكيل النهائي لاستخراج العازلة هو 50٪ DMF، و 20٪ SDS، 2.5٪ حامض الخليك، و 2.5٪ 1 M حمض الهيدروكلوريك. </li><li style=";text-align:right;direction:rtl"> في نهاية فترة العلاج، إضافة 100 ميكرولتر من استخراج العازلة (درجة حرارة 45 ° C حل أي بلورات) إلى كل بئر. السماح للخليط لاحتضان ليلا 37 ° C في حاضنة مرطب يحتوي على 5٪ CO 2. قراءة الامتصاصية في 570 نانومتر. ملاحظة: من الأفضل أداء في مقايسة السمسة بالتوازي مع intracellشاشة ular باستخدام نفس الدفعي وعمر THP-1 الخلايا. </li></ol> 5. في ومرق تحليل آخر باستخدام ريسازورين الفحص 3 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنمو السل M. في 7H9ADST إلى مرحلة منتصف السجل (~ 0،5-0،8 OD 600). تمييع الثقافة مع 7H9ADST إلى 0.01 OD 600. تمييع المركبات في 7H9ADST إلى 2x تركيزات الاختبار وقسامة 100 ميكرولتر من كل مجمع المخفف في كل بئر. </li><li style=";text-align:right;direction:rtl"> نقل 100 ميكرولتر من تعليق البكتيرية مخففة في كل بئر. السماح لوحات لاحتضان عند 37 درجة مئوية في حاضنة مرطب لمدة 5 أيام. حل 10 ملغ من ريسازورين في 100 مل من الماء منزوع الأيونات وتصفية العقيمة. </li><li style=";text-align:right;direction:rtl"> إضافة 30 ميكرولتر من محلول ريسازورين ورصد تغير اللون بعد 48 ساعة. يشار نمو البكتيريا عن طريق تحويل اللون من الأزرق إلى اللون الوردي. ملاحظة: يمكن أيضا إجراء تحليل الكمي عن طريق قياس إما مضان في 590 نانومتر مع الإثارة في 530-560نانومتر أو الامتصاصية في 570 نانومتر و 600 نانومتر (15). </li></ol>

<ol>
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The authors declare no competing financial interests for this work.

وكان الهدف من هذه الدراسة هو إيجاد طريقة HTS بسيطة وفعالة من حيث التكلفة باستخدام نموذج العدوى داخل الخلايا البشرية لM. السل. والسل هو مرض يصيب الإنسان تتميز إصابة الضامة السنخية التي كتبها M. السل. بسبب قضايا السلامة الأحيائية، وقد استخدم البحوث التي تنطوي عل...

المتفطرة السلية، العامل المسبب لمرض السل (TB)، هو أحد الأسباب الرئيسية للمراضة والوفيات في جميع أنحاء العالم. TB الحساس للأدوية هو مرض قابل للعلاج يتطلب مضادات حيوية متعددة لمدة 6 أشهر. على الرغم من كونه مرض قابل للعلاج، وقدرت نسبة الوفيات بمرض السل أن يكون 1.5 مليون في عام 2015 1. في السنوات ال 10 الماضية، كانت هناك زيادة المخاوف من انتشار M. السل المقاوم للأدوية. يتم تعريف للأدوية المتعددة مقاومة السل (MDR-TB) كما TB التي هي مقاومة على الأقل أيزونيازيد (INH) وريفامبيسين (RMP)، ومعظم سلالات السل المقاوم للأدوية المتعددة هي أيضا مقاومة لتحديد الأدوية TB الخط الثاني، مما يحد من خيارات العلاج . آثار مقاومة المخدرات تخلق تحديا أكبر لعلاج المرضى الذين يعانون شارك في المصابين بفيروس نقص المناعة البشرية (HIV). 400000 المرضى في جميع أنحاء العالم توفي من مرض السل المرتبطة بفيروس نقص المناعة البشرية في عام 2015 1. مخيبة للآمال، والولايات المتحدة الأمريكية للغذاء والدواء Administوقد وافقت التموينية واحد فقط المخدرات TB جديدة ضد MDR-TB، bedaquiline، في السنوات ال 40 الماضية 2. هناك حاجة ماسة إلى التقدم في إيجاد علاجات أفضل وأقصر TB من أجل البقاء قدما في مكافحة السل وMDR-TB. تقليديا، يتم تنفيذ الشاشات المخدرات TB تحت في المختبر ظروف النمو في متوسط النمو، حيث تتم إضافة مركبات للثقافة متنامية ويتم تحديد فعاليتها في القضاء على الكائنات الحية الدقيقة عن طريق عد مستعمرة (كفو) في وسط صلب. كما تعول CFU هي كثيفة العمالة، تستغرق وقتا طويلا، ومكلفة، وقد وضعت وسائل غير مباشرة مختلفة للتخفيف من حدة هذه المشكلة. وتشمل هذه الأساليب في الامار الأزرق جدوى فحص 3، وتحديد مضان 4 من البروتين الأخضر الفلورسنت (GFP) أو التلألؤ 5 من البكتيريا، معربا عن وسيفيراز، وتقدير مجموع أدينوسين ثلاثي الفوسفات (ATP) 6 و 7. يتميز TB نموذجي من عدوى السل M. في الرئة، حيث تتواجد البكتيريا وتتكاثر داخل جسم بلعمي الضامة السنخية 8. الشاشة المظهرية بسيطة في مرق قد تتناسب مع مسببات الأمراض خارج الخلية. ومع ذلك، في المنظور التاريخي، وأصيب المركبات ضد M. السل التي تم تحديدها باستخدام هذه الطريقة غالبا ما تفشل في الارتقاء إلى مستوى التوقعات خلال خطوات التحقق من صحة المصب في نماذج العدوى. نقترح أن المخدرات TB يتم تنفيذ الأفضل في نموذج عدوى الخلية المضيفة داخل الخلايا. ومع ذلك، ونماذج الخلايا تمتلك العديد من الحواجز التكنولوجية والبيولوجية لفحص (HTS) تطوير الإنتاجية العالية. وهناك عقبة كبيرة هي تعقيد عملية العدوى، والتي تجسدت خطوات عديدة وإزالة متطورة من البكتيريا خارج الخلية التي كتبها في الفترات الفاصلة بين الغسيل. وهناك عقبة رئيسية الثانية هي المرة إعادة طويلةيتطلبه، كما كشف عن النمو، ويتم عادة CFU تعول على لوحات ثقافة، هي العملية التي تستغرق أكثر من 3 أسابيع لإكمال. تم توفير حل واحد ليحل محل التهم CFU بواسطة المجهر الفلورسنت الآلي في تركيبة مع البكتيريا الفلورسنت. ومع ذلك، هذا الحل يتطلب الاستثمار في المعدات الأولي الذي هو خارج عن متناول العديد من مختبرات البحوث. ومن شأن ومنخفضة التكلفة بسيطة، والأمراض ذات الصلة طريقة HTS يعزز إلى حد كبير عملية اكتشاف المخدرات. في هذه الدراسة، ونحن التقرير نظام HTS جديد، وحدات التي تهدف إلى توفير السريع، وتدرجية عالية، بعد اقتصادي، فحص مناسبة لتحديد النشاط من المركبات ضد الخلايا السل M.. ويتكون هذا النظام من ثلاث وحدات: (ط) داخل الخلايا، (ب) السمية الخلوية، و (iii) فحوصات في المرق. توفر النتيجة النهائية مجتمعة وصفا شاملا للخصائص مركب، مع معلومات إضافية عن الوضع المحتمل للعمل. هذا الشورينظام reening استخدمت في العديد من المشاريع مع المكتبات مجمع المختلفة التي تستهدف طريقة العمل، بما في ذلك تحليل التآزر المخدرات 9، وتحفيز الالتهام الذاتي 10، وتثبيط M. السل -secreted عامل الضراوة (غير منشورة). كما تم دراسة المركبات من وضع غير معروف من عمل 11. اعتمد نسخة معدلة من هذا الأسلوب أيضا شريك صناعي لدينا كأسلوب غربلة أولية لتحديد مركبات جديدة ضد الخلايا السل M. 11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R5886</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>Becton, Dickinson and Company</td>
			<td>271210</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween80</td>
			<td>Fisher Scientific</td>
			<td>T164</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine pH7</td>
			<td>Affymetrix</td>
			<td>10857</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>BP350</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide (DMF)</td>
			<td>Fisher Scientific</td>
			<td>D131</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Hydrocholoric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>351279212</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>351269</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin</td>
			<td>Alfa Aesar</td>
			<td>B21187</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1</td>
			<td>American Type Culture Collection</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>M. tuberculosis H37Rv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well flat bottom white plate</td>
			<td>Corning</td>
			<td>3917</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well flat bottom clear plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent plate sealer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-0580</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Biomate 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>Biotek</td>
			<td>Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>luminometer</td>
			<td>Applied Biosystems</td>
			<td>Tropix TR717</td>
			<td></td>
		</tr>
	</tbody>
</table>

system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

الإنتاجية العالية فحص الخلايا باستخدام M. السل التعبير عن الجينات وسيفيراز الشكل 2A والجدول 1 يحتوي على البيانات الخام التي تم جمعها من قبل luminometer، أعرب في وحدات الانارة ال?...

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

لقد قمنا بتطوير نظام الفحص الإنتاجية العالية وحدات لاكتشاف مركبات جديدة ضد السل المتفطرة، تستهدف داخل وفي مرق تزايد البكتيريا وكذلك السمية الخلوية ضد الخلية المضيفة الثدييات.

نظام لفعالية والسمسة فحص مثبطات استهداف بين الخلايا<em&gt; المتفطرة السلية</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

system-for-efficacy-cytotoxicity-screening-inhibitors-targeting

Research

JoVE Journal

Immunology and Infection

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

8.5K Views.  University of British Columbia.  لقد قمنا بتطوير نظام الفحص الإنتاجية العالية وحدات لاكتشاف مركبات جديدة ضد السل المتفطرة، تستهدف داخل وفي مرق تزايد البكتيريا وكذلك السمية الخلوية ضد الخلية المضيفة الثدييات. 

Video: System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

تحضير العينة من<em&gt; المتفطرة السلية</em&gt; خلاصات لدراسات النووية المغناطيسي الرنين Metabolomic

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

نمو المتفطرة السلية الأغشية الحيوية

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

نموذج تجريبي لدراسة الملاريا والسل عدوى مرافقة على انتقال الطبيعية السل المتفطرة و المتصورة berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

الإنتاجية العالية للفحص واسع الطيف الكيميائية مثبطات الحمض النووي الريبي الفيروسات

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

مما يدل على متعدد المخدرات مقاومة<em&gt; السل المتفطرة</em&gt; التضخيم ميكروأري

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

الفحص السريع للفيروس نقص المناعة البشرية والناسخ العكسي مثبطات Integrase

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

نموذج الأنسجة 3D الرئة الإنسان للدراسات وظيفية على<em&gt; المتفطرة السلية</em&gt; العدوى

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

وإنتاجية عالية الفحص المدعم لتقييم فعالية الدواء ضد بلعم Passaged<em&gt; المتفطرة السلية</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

تصوير بكتريا السل في الفئران مع "مراسل إنزيم الأسفار"

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

تحديد "علامات الفوعة" بكتريا أبسسيسوس "النسخ المتماثل داخل الخلايا" في البالعات

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...