ونحن نعترف الدعم المالي من CPRIT (RP100119) ومؤسسة وولش (I-1665).

بروتوكول كامل يستغرق حوالي 4 أيام. 1. إعداد الخلايا لسيرنا ومراسل ترنسفكأيشن وسوف أعرض هنا بروتوكولا لاستجواب المكتبات سيرنا باستخدام مجموعة صغيرة من الرناوات siRNAs (384 مختلفة سيرنا برك مجموعة في 96 شكل جيد مع كل تجمع يتألف من الرناوات siRNAs 4X استهداف جين واحد) من مكتبة سيرنا على نطاق الجينوم لأغراض التوضيح. لهذه الدراسة خاصة، ونحن سوف يستغل HCT116 الخلايا التي تظهر إشارات WNT وKRAS المنحرف، والنشاط P53. فإن خط الخلية المناسبة في الدراسات التي تهدف إلى استجواب العمليات الخلوية الأخرى ذات الاهتمام يتعين تحديدها وتقييمها بطريقة مماثلة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يغسل 5 × 10 سم 2 لوحات من 80-90٪ متموجة HCT116 الخلايا مع برنامج تلفزيوني ثم حصاد الخلايا بواسطة trypsinization باستخدام 1 مل من محلول trypsinization / لوحة تليها تحييد مع 10 مل من Dulbecco لتعديل النسر المتوسط ​​(DMEM) التي تحتوي على 2٪ FEتل المصل البقري (FBS) و 1٪ البنسلين / الستربتوميسين / لوحة. </li><li style=";text-align:right;direction:rtl"> نقل الخلايا علقت في أنبوب مخروطي 50 مل ونباذة في ~ 130 x ج لمدة 5 دقائق، وإزالة طاف، وإعادة تعليق بيليه خلية في 10 مل من DMEM / FBS 2٪ / 1٪ البنسلين / الستربتوميسين. </li><li style=";text-align:right;direction:rtl"> حساب عدد الخلايا مع عداد الخلية، ثم جعل الحل الخلية مع 10 5 خلية / مل، والحفاظ على 150 خلية مل تعليق للخطوة التالية. لوحة 10 4 خلايا (100 ميكرولتر) في كل بئر من 96 لوحة جيدا ثقافة الخلية باستخدام Multidrop موزع السائل الآلي (Multidrop من الآن فصاعدا). يجب أن يكون التعليق الخلية كافية في هذه الحالة لتصفيح 12 × 96 لوحات جيدة. </li><li style=";text-align:right;direction:rtl"> احتضان لوحات مع الخلايا عند 37 درجة مئوية في حاضنة مع 5٪ CO 2 أثناء إعداد خليط ترنسفكأيشن. </li></ol> 2. إعداد مراسل بناء الحل الأسهم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عزل مراسل عالية الجودة بناء الحمض النووي باستخدام معيار midiprep مجموعات (ينتج NucleoBond إكسترا ميدي بواسطةClontech) ويخفف من الحمض النووي إلى تركيز النهائي من 1 ملغ / مل لكل مراسل. </li><li style=";text-align:right;direction:rtl"> إعداد 100 ميكرولتر من محلول المخزون مراسل بناء عن طريق الجمع بين 30 ميكرولتر من P53-FL، 30 ميكرولتر من 8X TCF-RL، 30 ميكرولتر من Elk1-GAL4، 6 ميكرولتر UAS-CL و 4 ميكرولتر من H 2 O لتحقيق مزيج الحمض النووي مع نسبة 1:1:1:0.2 النهائي للصحفيين. تركيز الحمض النووي DNA النهائية لهذا المزيج محلول المخزون هو 0.3 ملغ / مل. </li></ol> 3. إعداد سيرنا / DNA ترنسفكأيشن ميكس في هذا الجزء من البروتوكول، ونحن نستعد سيرنا / DNA يمزج ترنسفكأيشن من شأنها أن تكون كافية لtransfecting 12 × 96 لوحات جيدة. لهذه المكتبة الاختبار من 4 × 96 لوحة جيدا من الرناوات siRNAs، وسوف نقوم بالنقل كل تجمع سيرنا في ثلاث نسخ. كاشف ترنسفكأيشن سوف نستخدم ما يسمى Effectene (QIAGEN). في أيدينا وهذا هو كاشف الوحيدة التي تعمل من أجل تسليم في وقت واحد من سيرنا والحمض النووي في الخلايا المستزرعة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تمييع 80 ميكرولتر من مراسل بناء المخزون سولution في 8 مل من EC العازلة (Effectene كيت) للتوصل إلى حل الحمض النووي مع تركيز النهائي من 3 ميكروغرام / مل. بشكل عام، وإعداد ما يكفي من الحمض النووي حل مزيج للاستخدام الفوري. </li><li style=";text-align:right;direction:rtl"> لوحة 20 ميكرولتر من هذا الحل DNA إلى كل بئر من 96 جيدا / لوحة. فإن عدد 96 لوحات جيدة تعتمد على عدد وسيتم اختبار برك سيرنا. على سبيل المثال، في هذه الحالة سوف نحتاج إلى 4 × 96 لوحات جيدة لتقييم 384 برك سيرنا مختلفة. </li><li style=";text-align:right;direction:rtl"> ينبغي أن الرناوات siRNAs لفحصها يكون في تركيز الأسهم من 5 ميكرومتر. إذا لم يكن كذلك، فإنها يمكن أن تكون متماثلة المطلية وتخفيفه إلى هذا التركيز باستخدام العازلة تخفيف الموصى بها المرتبطة بكل مكتبة. </li><li style=";text-align:right;direction:rtl"> نقل 2 ميكرولتر من كل سيرنا الأسهم 5 ميكرومتر إلى المقابلة بئر من لوحة PCR التي تحتوي على الحمض النووي الأسهم المخفف مع Biomek الآلي معالج السائل. اعتمادا على مقياس الدراسة، وهو pipettor متعدد القنوات قد يكون كافيا. </li><li style=";text-align:right;direction:rtl"> الآن، سوف نقوم بإضافة 1 ميكرولتر من الحل محسن (Effectene عدة) إلى كل بئر من الحمض النووي / سيرنا جيش التحرير الشعبى الصينىالشركة المصرية للاتصالات. هذا مرة أخرى يمكن أن يتحقق إما مع معالج السائل الآلي أو pipettor الأقنية. </li><li style=";text-align:right;direction:rtl"> السماح للتفاعل محسن أن تحدث في RT لمدة 5 دقائق ثم تضاف 3 ميكرولتر من Effectene كاشف ترنسفكأيشن إلى كل بئر واحتضان في RT لمدة 10 دقيقة إضافية. </li><li style=";text-align:right;direction:rtl"> لوقف ترنسفكأيشن تشكيل معقدة، إضافة 10 ميكرولتر من DMEM / FBS 2٪ / 1٪ البنسلين / الستربتوميسين إلى كل بئر من لوحة PCR. </li><li style=";text-align:right;direction:rtl"> نقل 10 ميكرولتر من مزيج ترنسفكأيشن إلى كل بئر من لوحة جيدا 96 التي تحتوي على الخلايا (التي تم استردادها من الخلية الحاضنة). كما سيتم تنفيذ كل ترنسفكأيشن في ثلاث نسخ، وسيتم تطبيق نفس المزيج لمدة 96 لوحات جيدة إضافية تحتوي على خلايا. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا مع مخاليط ترنسفكأيشن أضاف عند 37 درجة مئوية في بيئة ترطيب مع 5٪ CO 2 لمدة 36 ساعة. </li></ol> 4. تحديد الأنشطة luciferase المراسل من عينات الخلايا بعد 36 ساعة، والأنشطة وسيفيراز مستعدون لقياسفي الخلايا transfected. وينبغي تحديد نقطة النهاية على أساس التجربة في. في دراستنا، كان له فترة حضانة 36 ساعة أسفرت سابقا إشارة قوية بما فيه الكفاية لمعنى تقرير النتيجة. كما تتضمن هذه الدراسة للصحفيين متعددة (واحد يفرز في مستنبت، واثنين آخرين أعرب في السيتوبلازم الخلية)، وسوف نقوم حصول على قياسات من كل من مستنبت فضلا عن المحللة الخلوية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الكشف عن النشاط CL في مستنبت: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 20 ميكرولتر من مستنبت هي طبق الاصل مطلية مبهمة في 96 لوحة جيدا أبيض (إما باستخدام معالج السائل Biomek أو pipettor الأقنية). </li><li style=";text-align:right;direction:rtl"> إضافة 20 ميكرولتر من العازلة مقايسة CL (استهداف الأنظمة) إلى كل بئر. </li><li style=";text-align:right;direction:rtl"> إضافة 10 ميكرولتر من Cypridina الركيزة luciferin (استهداف الأنظمة) إلى كل بئر وكشف النشاط CL باستخدام luminometer (القارئ لوحة Pherastar التي تنتجها بي إم جي على سبيل المثال). </li></ol></li><li style=";text-align:right;direction:rtl"> كشف FL والنشاط RL: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ليز جells عن طريق إزالة أول ثقافة المتوسط. ويمكن تحقيق ذلك بسرعة من خلال تحويل لوحة رأسا على عقب والقذف المتوسطة في بالوعة. المتبقية المتوسطة يمكن إزالتها عن طريق التنصت بلطف لوحة رأسا على عقب على مناشف ورقية. إضافة 30 ميكرولتر من 1X سلبية الاحتياطي تحلل (PROMEGA) إلى كل بئر من الخلايا باستخدام Multidrop ومكان على الكرسي الهزاز منصة (Bellco هي شركة واحدة أن يجعل هذه) تعيين سرعة متوسطة لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> إضافة 20 ميكرولتر من كاشف الفحص luciferase المراسل الثاني (جزء من PROMEGA كيت luciferase المراسل المزدوج) باستخدام النشاط FL Multidrop وقياس فورا باستخدام luminometer. المقبل، إضافة ستوب اند جلو الكاشف (PROMEGA) التي من شأنها إخماد النشاط FL ويسمح بقياس النشاط RL. ملاحظة: إلا إذا كنت تستخدم خلطات الركيزة التي تسمح المدة إشارة الموسعة (مثل أطقم luciferase المراسل ثنائي جلو من PROMEGA)، واستخدام فلاش مجموعات يتطلب قياس السريع على إضافة الركيزة (في غضون 5 دقائق). </li></ol></li></ol>

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موافق وLL هي الكتاب على براءات الاختراع في انتظار المتعلقة المضاعفة التكنولوجيا وسيفيراز.

وهناك العديد من مروجي أنظمة المستندة إلى مراسل luciferase المراسل (مثل PROMEGA، نظم الاستهداف، نيو انغلاند Biolabs، والحرارية فيشر) التي توفر الموارد على الخط مفيدة لأولئك مسلية شاشة عالية الإنتاجية للمرة الأولى. وبالإضافة إلى ذلك، يجب أن عددا من الاستعراضات ممتازة بشأن تحقيق ?...

مجموعة متنوعة من الصحفيين على وسيفيراز لرصد مجموعة متنوعة من العمليات البيولوجية خلية متاحة تجاريا. غالبية هذه البروتينات الحمض النووي يبني ترميز وسيفيراز التي يسببها وصريح من قبل المحفزات خلية معينة أو اضطرابات. الأكثر صحفيين قوية مقرها transcriptionally وضع التعبير عن انزيم luciferase المراسل تحت سيطرة عناصر محسن الاصطناعية أو مناطق المروج الجين راسخة لموثوقيتها في الإبلاغ عن ذات الصلة من الناحية الفسيولوجية النسخي 1،2 حدث. وهناك أيضا نظم عبر مراسل luciferase التي تستخدم GAL4-UAS لدفع وسيفيراز تعبير البروتين. GAL4 هي الخميرة النسخي المنشط وUAS هو محسن الذي يربط GAL4 خصيصا لتفعيل النسخ من تسلسل الجينات وضعت أسفل تيار من ذلك 3. وعادة ما يتم اعتماد هذه الأنواع من أنظمة وسيفيراز من قبل اثنين من البلازميدات الحمض النووي - واحد بترميز البروتين GAL4 تنصهر إلى regulatoراي جزء من البروتين من الفائدة، والثاني يشفر بروتين وسيفيراز وضعت تحت سيطرة واحدة أو أكثر UAS متواليات. وهكذا، فإن إشارة وسيفيراز الخلوية يعكس مستوى النشاط من البروتين من الفائدة. آخر الاستخدام المشترك للصحفيين المستندة إلى وسيفيراز هو لرصد استقرار البروتين حيث يتم تنصهر بروتين من الفائدة إلى إنزيم وسيفيراز 4. بغض النظر عن نوع من المراسل، ويلاحظ على مسؤولية المشتري هنا كما لا يستطيع المرء تحمل خصوصية أي مراسل لتم استجوابه بشكل جيد. وبالتالي، مطلوب العناية الواجبة في إدماج بنيات مراسل جديدة في أي استراتيجية للبحوث. اختيار الانزيم وسيفيراز قراءة خارج يمكن أن تكون مهمة مثل الاعتماد على عناصر DNA التي تتحكم في التعبير عنها. في حين luciferase يراعة (FL) هو انزيم الأكثر استخداما في التركيبات المتاحة تجاريا، وظهور أنظمة مراسل luciferase الجديدة التي لا تتطلب ATP (كما في حالةمن ردود الفعل على أساس FL) أو التي تتضمن الإنزيمات أكثر استقرارا التي تنبعث منها إشارات أقوى نعد لتحسين كفاءة وموثوقية منصة الأبحاث هذه. ملاحظة هامة فيما يتعلق باختيار صحفيين وسيفيراز في الدراسات الكيميائية هو قابلية FL على تثبيط المواد الكيميائية التي يمكن أن تؤدي إلى تقارير كاذبة. في تجربتنا، والإنزيمات الأخرى مثل Renilla أو Gaussia وسيفيراز (RL وGL، على التوالي) التي تستخدم coelenterazine باعتبارها الركيزة هي تثبيط أقل بسهولة بواسطة الجزيئات الصغيرة 5. الشكل الأكثر شيوعا لتعدد وسيفيراز قراءة عموميات تنطوي على استخدام FL وRL نظرا إلى حد كبير بتوافر سهلة الاستخدام لمجموعات مع القدرة على قياس بالتسلسل الأنشطة الأنزيمية من عينة واحدة. في معظم مجموعات، ويتعرض العينات الأولى للوسيفيرين للكشف عن مستويات النشاط FL تليها كاشف التبريد التي يتم إيداعها في وقت واحد مع coelenterazine أن تسفر عن الشركة السعوديةدارى RL إشارة. مع إضافة luciferases الأخرى التي يمكن أن يفرز مثل GL أو التي تستخدم حتى الآن الركيزة أخرى مثل Cypridina وسيفيراز (CL)، وهو عدد أكبر من إمكانيات لتوليد ظهرت محتوى البيانات عالية باستخدام luciferases. مثال على شاشة رني الجينوم على نطاق باستخدام هذه التقنية يمكن العثور عليها هنا 6. هذه التقدم التكنولوجي قد بدوره حفز تطوير آليات محددة لإرواء كل نوع من انزيم luciferase المراسل من أجل الحد عبر الحديث 7. في أيدينا، ونحن نلاحظ تردد أكبر من &quot;آثار الحافة&quot; (الاتجاهات لا يمكن تفسيره في النشاط الخلوي المرتبطة حافة عالية لوحات ثقافة عيار)، مع luciferases يفرز مثل GL أو CL. من ناحية أخرى، مثل luciferases يفرز هي مفيدة لفحوصات الحركية كما أنها تمكن أخذ العينات إشارة دون غش بقاء الخلية. لدراستنا المعروضة هنا، وسوف نقوم في نفس الوقت مراقبة النشاط من ثلاثة السرطان ذات الصلةالعمليات الخلوية: وP53، KRAS، وWNT مسارات نقل الإشارة. للصحفيين أدرجت في دراستنا هي FL مراسل pp53-TA-لوك من Clontech (المشار إليه فيما مراسل P53-FL) 8، نظام مراسل Elk1-GAL4/UAS-CL (من الآن فصاعدا إلك-1 المراسل؛ اجيلنت)، وTCF مراسل RL 8X الذي يشتمل على عدة عناصر محسن الاصطناعية معترف بها من قبل WNT مسار المنظمين النسخي المعروف باسم العوامل تي خلية (أو TCFs) 9-11.

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>PathDetect Elk1 Trans-Reporting System</td>
			<td>Agilent Technologies Inc.</td>
			<td>219005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pathway Profiling Luciferase System 4 pp53-TA-Luc Vector</td>
			<td>Clontech Lab Inc.</td>
			<td>631914</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen Inc.</td>
			<td>301427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega Corp.</td>
			<td>E1960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cypridina Luciferase Assay reagent</td>
			<td>Targeting Systems</td>
			<td>VLAR-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCT116 cells</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CytoTox-Fluo Cytotoxicity Assay</td>
			<td>Promega Corp.</td>
			<td>G9260</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>MultiFlo Microplate Dispenser</td>
			<td>Labsystems Inc.</td>
			<td>Model 832</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biomek FXP Laboratory Automation Workstation</td>
			<td>Beckman Coulter Inc.</td>
			<td>A31842</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PHERAstar FS-multi-mode HTS microplate reader</td>
			<td>BMG Labtech Inc.</td>
			<td>0471-101A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bright-Line Reichert Hemacytometers</td>
			<td>Hausser Scientific Company</td>
			<td>1490</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a multiplexed luciferase-based screening platform for interrogating cancer-associated signal transduction in cultured cells

الاستجواب على نطاق الجينوم وظيفة الجين باستخدام تدخل الحمض النووي الريبي (رني) يحمل وعدا هائلا لتحديد السريع للين العريكة كيميائيا نقاط الضعف الخلايا السرطانية. مما يحد من إمكانيات هذه التكنولوجيا هو عدم القدرة على ترسيم بسرعة أساس الآلية من نتائج المظهري وبالتالي يسترشد بها في وضع استراتيجيات علاجية تستهدف جزيئيا. نحن هنا الخطوط العريضة الأساليب لتفكيك الظواهر الخلوية الناجمة عن الجين رني بوساطة استهداف باستخدام أنظمة مراسل المضاعفة التي تسمح رصد سرطان مفتاح عمليات الخلية المرتبطة. هذه المنهجية الفرز نسبة عالية هي متعددة ويمكن تكييفه بسهولة لفحص أنواع أخرى من مكتبات الجزيئية كبيرة.

على الرغم من التقدم في رسم المشهد طفرية من السرطانات المختلفة باستخدام ضخمة جهود تسلسل الجينوم 12-14، ونحن ما زلنا لا نملك في مكان مقاربة منهجية لترجمة هذه الملاحظات إلى استراتيجيات التدخل. في سرطان القولون والمستقيم (CRC)، والطفرات التي من المتوقع أن تؤثر على مكو?...

Watch this Scientific Journal Video about A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells at JoVE.com

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

التوصل إلى فهم مستوى النظم من العمليات الخلوية هو هدف بيولوجيا الخلية في العصر الحديث. نحن هنا وصف استراتيجيات للصحفيين وسيفيراز المتنوعة من مختلف وظيفة الخلوية نقطة نهاية لاستجواب وظائف الجينات باستخدام الجينوم على نطاق والمكتبات رني.

A تعدد في منصة الفرز القائم على luciferase المراسل لاستجواب المرتبطة بالسرطان نقل الإشارة في الخلايا المستزرعة

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

a-multiplexed-luciferase-based-screening-platform-for-interrogating

Research

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Medicine

11.0K Views.  UT Southwestern Medical Center. التوصل إلى فهم مستوى النظم من العمليات الخلوية هو هدف بيولوجيا الخلية في العصر الحديث. نحن هنا وصف استراتيجيات للصحفيين وسيفيراز المتنوعة من مختلف وظيفة الخلوية نقطة نهاية لاستجواب وظائف الجينات باستخدام الجينوم على نطاق والمكتبات رني.

Video: A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

كيفية قياس الطي القشرية من صور MR : برنامج تعليمي خطوة بخطوة لحساب الرقم القياسي المحلي Gyrification

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

High Content Screening in Neurodegenerative Diseases

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be addressed as a matter of great urgency1-4. Multiple complementary 'omics' approaches have provided clues as to the genetic risk factors and pathogenic mechanisms underlying numerous neurodegenerative diseases, but most findings still require functional validation5. For example, a recent genome wide association study for Parkinson's Disease (PD), identified many new loci as risk factors for the disease, but the underlying causative variant(s) or pathogenic mechanism is not known6, 7. As each associated region can contain several genes, the functional evaluation of each of the genes on phenotypes associated with the disease, using traditional cell biology techniques would take too long. 
	There is also a need to understand the molecular networks that link genetic mutations to the phenotypes they cause. It is expected that disease phenotypes are the result of multiple interactions that have been disrupted. Reconstruction of these networks using traditional molecular methods would be time consuming. Moreover, network predictions from independent studies of individual components, the reductionism approach, will probably underestimate the network complexity8. This underestimation could, in part, explain the low success rate of drug approval due to undesirable or toxic side effects. Gaining a network perspective of disease related pathways using HT/HC cellular screening approaches, and identifying key nodes within these pathways, could lead to the identification of targets that are more suited for therapeutic intervention.
	High-throughput screening (HTS) is an ideal methodology to address these issues9-12. but traditional methods were one dimensional whole-well cell assays, that used simplistic readouts for complex biological processes. They were unable to simultaneously quantify the many phenotypes observed in neurodegenerative diseases such as axonal transport deficits or alterations in morphology properties13, 14. This approach could not be used to investigate the dynamic nature of cellular processes or pathogenic events that occur in a subset of cells. To quantify such features one has to move to multi-dimensional phenotypes termed high-content screening (HCS)4, 15-17. HCS is the cell-based quantification of several processes simultaneously, which provides a more detailed representation of the cellular response to various perturbations compared to HTS. 
	HCS has many advantages over HTS18, 19, but conducting a high-throughput (HT)-high-content (HC) screen in neuronal models is problematic due to high cost, environmental variation and human error. In order to detect cellular responses on a 'phenomics' scale using HC imaging one has to reduce variation and error, while increasing sensitivity and reproducibility. 
	Herein we describe a method to accurately and reliably conduct shRNA screens using automated cell culturing20 and HC imaging in neuronal cellular models. We describe how we have used this methodology to identify modulators for one particular protein, DJ1, which when mutated causes autosomal recessive parkinsonism21. 
	Combining the versatility of HC imaging with HT methods, it is possible to accurately quantify a plethora of phenotypes. This could subsequently be utilized to advance our understanding of the genome, the pathways involved in disease pathogenesis as well as identify potential therapeutic targets.

We describe a methodology combining automated cell culturing with high-content imaging to visualize and quantify multiple cellular processes and structures, in a high-throughput manner. Such methods can aid in the further functional annotation of genomes as well as identify disease gene networks and potential drug targets.

فحص محتوى عالية في أمراض الاعصاب

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

استعمال قياس المثانة في القوارض الصغيرة: دراسة في Chemosensation المثانة

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

الكشف عن سرطان الجلد باستخدام المعدلات اسماك الزرد الموديل ورم الأصليين

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

دراسة سرطان البنكرياس الجذعية خصائص الخلايا لتطوير استراتيجيات علاجية جديدة

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

والمحتوى العالي<em&gt; في المختبر</em&gt; البنكرياس جزيرة β خلايا النسخ المتماثل منصة ديسكفري

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

فحص الجزيئات النانوية النشطة بيولوجيا في الخلايا المناعية البلعمة لمثبطات مستقبلات تشبه تول

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

روتين فحص الطريقة المجهرية الدقيقة في عمليات نقل الدم الصفيحات

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

هيدرا، منصة حاسوبية لمساعدة الأطباء في تشخيص وتحليل القلب والأوعية الدموية

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. 
This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

في المختبر أسلوب التحكم بتركيزات غازات المهلجنة في استزراع الخلايا الظهارية السنخية

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The ...