نود أن نشكر الدكتور فيليب Erbs لتزويدنا تسلسل FCU1 الجينات. وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح منظمة العفو الدولية 26649.

1. FCU نظام التحديد السلبية في نسج المتحولة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم إنشاء خط السيطرة ناقلات (HM1 / pKT3M) والخط التجريبي (HM1/pKT3M-FCU1) من 1،2 التقنيات الموصوفة سابقا. البذور خطوط قيد الدراسة من أنابيب الأسهم إلى T - 25 قوارير والحفاظ على الاختيار عن طريق إضافة المضادات الحيوية المناسبة (12 ميكروغرام / مل G418) في المتوسط ​​3 TYI - S - 33. والقوارير وينبغي 70-80 ٪ متموجة بعد 16-18 ساعة من النمو بنسبة 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> اعداد جديدة من محلول المخزون 50mM فلوروسيتوزين - 5 (5 - FC ، سيغما) في المتوسط ​​TYI - S - 33 الدافئة. دوامة بصرامة لعدة دقائق لإذابة 5 - FC تماما. فلتر تعقيم محلول المخزون بتمريرها من خلال 0،22 ميكرون التصفية. </li><li style=";text-align:right;direction:rtl"> النواشط الحصاد بوضع القوارير على الجليد لمدة 3-5 دقائق وجمع الخلايا بواسطة الطرد المركزي في 200xg لمدة 5 دقائق في درجة حرارة الغرفة. Resuspend الكرية في المتوسط ​​TYI الطازجة وعدد الخلايا. </li><li style=";text-align:right;direction:rtl"> تصميم تجربة نموذجية يستخدم 0.5 X 10 4 خلايا لكل بئر من 48 لوحة بشكل جيد في الحجم النهائي من 1 مل ، وتكرر ثلاث نسخ لكل حالة اختبار. بالإضافة إلى ذلك ، لتسهيل القياسات الفلورسنت ، وتستخدم لوحة منفصلة لكل سلالة في نقطة زمنية. </li><li style=";text-align:right;direction:rtl"> البذور 0.5 X 10 4 خلايا في كل اختبار جيد ل5 - FC التركيز ، في ثلاث نسخ. الحفاظ على ضغط اختيار المضادات الحيوية في المتوسط ​​TYI. الآن إضافة المبلغ المطلوب من 5 FC حل الأسهم والنواشط كل بئر. </li><li style=";text-align:right;direction:rtl"> احتضان لوحات في حقيبة اللاهوائي عند 37 درجة مئوية. </li></ol> 2. تحديد اختيار الكفاءة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 2 ملغ / مل حل الأسهم الخضراء CMFDA تعقب الخلية (Invitrogen) في DMSO. الحل لجعل العمل تمييع المخزون 1000 - أضعاف في المتوسط ​​TYI المصل الحرة. </li><li style=";text-align:right;direction:rtl"> إزالة المتوسطة تماما من لوحات واستبدال ثقافة أتروفة مع 150 ميكرولتر CMFDA TYI المصل خالية تحتوي. تستمر الحضانة عند 37 درجة مئوية لمدة 1 ساعة. </li><li style=";text-align:right;direction:rtl"> إزالة محلول الصبغة من الآبار وقراءة لوحة في مقياس التألق الطيفي Spectramax microplate M2. يجب تعيين الطول الموجي 492 نانومتر في الإثارة وانبعاث مضان قراءة في 517 نانومتر. </li><li style=";text-align:right;direction:rtl"> مقارنة القيم مضان للخلايا السيطرة مقابل transfectants الاختبار. </li><li style=";text-align:right;direction:rtl"> مواصلة القراءة لوحات في كل فترة زمنية. الاستخدام الإيجابي المناسب (TYI فقط) والسلبية (25 ميكروغرام / مل هيغروميسين) آبار مراقبة في كل لوحة. </li></ol> 3. ممثل النتائج وهاء وأظهرت transfectants نسج التعبير القوي للFCU1 كودون الأمثل المؤتلف (الشكل 1) ، وعندما يتبع هذا البروتوكول بشكل صحيح النتائج في القضاء الانتقائي للE. معربا عن الخلايا نسج FCU1 في وجود 0.5 مم 5 - فلوروسيتوزين. الخلايا السيطرة ، في المقابل تستمر في النمو عادة ما يصل الى 5 من تركيز ملي FC - 5 والوصول التقاء بين 72 و 96 ساعة (الشكل 2 - A). وlysed تماما الخلايا FCU1 تحمل في 48 ساعة عندما يتم النظر إلى الآبار التي تمت معالجتها تحت المجهر. هذه أيضا تظهر عندما انخفض مضان ملطخة CMFDA صبغ الحيوية التي تستخدم في الدراسات الكمية التي تنطوي على عدد كبير من العينات (الشكل 2 ب). نظام FCU1/5-FC هو أداة فعالة وقوية لاختيار سلبية E. نسج النواشط. <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="الشكل 1" /> الشكل 1. جيل من E. نسج HM1 transfectants معربا عن اختيار علامة سلبية (أ) تم الكشف عن بروتين FCU1 المؤتلف وصمة عار على استخدام الألغام المضادة للغرب ج Myc الأضداد (اللوحة العلوية). كما هو متوقع ، يمكن الكشف عن عصابة 42kDa (الذي أبداه سهم) في مجموع lysate من transfectants FCU ولكن ليس في ناقلات وحدها transfectants السيطرة. لوحة أسفل يظهر نفسه لطخة بحث مع الأضداد المضادة أكتين كعنصر تحكم التحميل. (ب) نتائج PCR الوقت الحقيقي يظهر التعبير عن الكمية FCU1 مرنا محددة لتطبيع lgl4 السيطرة. <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="الشكل 2" /> الشكل 2. الحساسية المخدرات في المختبر من E. transfectants نسج معربا عن اختيار علامة سلبية منحنيات بقاء transfectants التحكم (أ) و (ب) transfectants FCU1 معربا عن تربيتها في 48 لوحة جيدا. زرعت الخلايا في وجود تركيزات المتزايد للدواء مساعد - 5 - فلوروسيتوزين ؛ كان كميا بقاء الخلية تلطيخ CMFDA في كل فترة زمنية كما تدل على محور X. ويمثل المحور Y - الوحدات ومضان هو انعكاس مباشر للسكان الخلية على قيد الحياة. يرجى ملاحظة أن التعبير عن transfectants FCU1 أظهرت حساسية كبيرة في تركيز دواء مساعد 0.5mM مقارنة السيطرة. ولوحظ وجود خلايا في نقطة زمنية 120h في هذه الآبار وCOMقلص لنمو متموجة ينظر في آبار المراقبة المقابلة تحت المجهر (لا تظهر البيانات). الرطوبة يدل 25μg / مل هيغروميسين يستخدم كعنصر تحكم سلبية.

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans. R. Soc. Trop. Med. Hyg. 72, 431-432 (1978).</li><li>Clark, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaerobic parasitic protozoa : genomics and molecular biology. , (2010).</li><li>Linford, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-mediated+knockdown+of+protein+expression+in+Entamoeba+histolytica.">Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.</a> BMC Microbiol. 9, 38-38 (2009).</li><li>Solis, C. F., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+genes+by+RNA+interference+in+the+protozoan+parasite+Entamoeba+histolytica.">Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.</a> Methods Mol. Biol. 442, 113-128 (2008).</li><li>Solis, C. F., Santi-Rocca, J., Perdomo, D., Weber, C., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacterially+expressed+dsRNA+to+downregulate+Entamoeba+histolytica+gene+expression.">Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.</a> PLoS ONE. 4, e8424-e8424 (2009).</li><li>MacFarlane, R. C., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dsRNA-based+gene+silencing+in+Entamoeba+histolytica:+implications+for+approaches+to+genetic+analysis.">Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.</a> Exp. Parasitol. 119, 296-300 (2008).</li><li>Dhar, S. K., Vines, R. R., Bhattacharya, S., Petri, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+DNA+fragments+enhance+the+stability+of+transfected+DNA+in+Entamoeba+histolytica.">Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.</a> J. Eukaryot. 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وقد رعت إنتاج هذا الفيديو من قبل الدريتش سيغما ، المؤتمر الوطني العراقي ، والتي تنتج بعض الكواشف المستخدمة في هذه المقالة.

وإن كانت هناك تقدما ملموسا في مجال البيولوجية الجزيئية الأدوات المتحولة لا توجد طرق الحالية المتاحة لحذف أو تعطيل الجينات 4. أحرز ضربة قاضية للتعبير جين معين عن طريق استخدام دبوس الشعر من RNAs 5 و 6 صغيرة بالتدخل RNA ، وأعرب عن البكتريا الرنا المزدوج الجديلة 7. لكن هذه الأساليب في حين فعالة للجينات الهدف منها ، والعديد من القيود بما في ذلك استخدام المواد الكيميائية مكلفة ، واختيار المستمرة ضد المضادات الحيوية وضرورة التحقق من صحة مستمر بسبب ارتفاع معدل حدوث انعكاس على مر الزمن (أليسيا ينفورد ، اتصال شخصي) 8. يمكن لأي البلازميد في تكرار المتحولة كما أن هناك على ما يبدو ليس شرطا التسلسل الدقيق لأصول تكرار الحمض النووي 9 ، ومرة واحدة حتى transfected ، فإنه عادة ما يكون من الصعب علاج البلازميد. وهذا يحد من إمكانية استخدام البلازميدات سليمة في التجارب خروج المغلوب وإعادة التركيب الجيني. علامات التحديد السلبي هي المكونات الرئيسية لاستهداف الجينات والأساليب التي يمكن استخدامها للقضاء على المجموعات السكانية الفرعية المترابطة. أعربنا عنها بنجاح كودون الأمثل FCU1 الجينات المتحولة في نسج واختبار قدرته على اختيار علامة a سلبية. وقد استخدم هذا الانصهار الجين للعلاج بالجينات انتحار الخلايا السرطانية البشرية والتمثيل الغذائي للدواء مساعد 5 - FC في المنتجات السامة الناتجة المصب في تثبيط تخليق الحمض النووي الريبي و10. وقد استخدمت مؤخرا في FCU1 a علامة سلبية في اختيار المتصورة ، وآخر طفيلي ، وتم العثور على الخلايا المنجلية berghei FCU1 معربا عن أن ما يقرب من 1000 مرات أكثر حساسية للدواء مساعد في المختبر والمجراة في المعاملة مع نادي - 5 عن مقتل أكثر من 99،9 ٪ من اصابة الطفيليات المؤتلف في نموذج الفأر الكريات الحمر في المرحلة 11 من الملاريا. E. وأظهرت الخلايا نسج FCU1 فقط التعبير عن حساسية 30 ضعفا متزايدا لدواء مساعد 5 - FC على النقيض من الحساسية التي لوحظت في P. berghei. يمكن أن الأسباب المحتملة لهذا الأمر مجموعة كبيرة من النيوكليوتيدات المتاحة في وسط النمو تتنافس مع الأيضات السامة. في كلا P. وberghei P. وقد استخدمت علامة FCU1 المنجلية في تعطيل الجينات المستهدفة. دراسات 11،12. ونحن نهدف إلى التلاعب في الجينات المتحولة باستخدام إعادة التركيب مثلي. المصادقة على نظام اختيار السلبية FCU1/5-FC يعد خطوة هامة نحو تحقيق هذا الهدف.

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

المتحولة الحالة للنسج هو العامل المسبب لداء الأميبات وتصيب ما يصل الى 10 ٪ من سكان العالم. التقنيات الجزيئية التي مكنت من المتابعة وأسفل تنظيم التعبير الجيني الاعتماد على ترنسفكأيشن من البلازميدات حافظت بثبات. في حين أن هذه قد زادت من فهمنا للعوامل الفوعة المتحولة ، والقدرة على الاندماج في جينوم الحمض النووي الخارجية ، الأمر الذي سيسمح عكس التجارب علم الوراثة ، وسيكون ميزة كبيرة في دراسة هذا الطفيلي. التحديات التي يمثلها هذا الكائن الحي وتشمل عدم القدرة على تحديد للأحداث ، وصعوبة إعادة التركيب مماثل لعلاج DNA البلازميد episomal من النواشط transfected. النتائج في وقت لاحق على خلفية عالية من الحمض النووي الخارجية ، وهي مشكلة رئيسية في تحديد النواشط فيه بحسن نية الحدث التكامل الجينومية حدث. نحن تقرير وضع نظام اختيار سلبية على أساس التعبير المعدلة وراثيا من خميرة نازعة أمين السيتوزين واليوراسيل phosphoribosyl ترانسفيراز خيمر (FCU1) مع دواء مساعد واختيار 5 - فلوروسيتوزين (5 - FC). انزيم تحويل FCU1 غير سامة 5 - FC السامة في فلورويوراسيل - 5 و 5 ، 5&#39; - fluorouridine الأدينوزين هاء. تم العثور على خطوط نسج معربا عن FCU1 ليكون 30 أضعاف أكثر حساسية للدواء مساعد مقارنة سلالة السيطرة.

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

نحن تقرير وضع نظام اختيار سلبية في<em&gt; E. نسج</em&gt; استنادا إلى التعبير المعدلة وراثيا من البروتين خيالية (FCU1) والاختيار مع دواء مساعد 5 - فلوروسيتوزين. البروتين FCU1 هي عبارة عن اندماج نازعة أمين السيتوزين الخميرة واليوراسيل ناقلة الفسفوريبوزيل. نتج تعبير عن زيادة في FCU1<em&gt; E. نسج</em&gt; الحساسية تجاه فلوروسيتوزين - 5.

وضع علامة سلبية لإختيار a المتحولة الحالة للنسج</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

12.3K Views.  University of Virginia Health System. نحن تقرير وضع نظام اختيار سلبية في E. نسج استنادا إلى التعبير المعدلة وراثيا من البروتين خيالية (FCU1) والاختيار مع دواء مساعد 5 - فلوروسيتوزين. البروتين FCU1 هي عبارة عن اندماج نازعة أمين السيتوزين الخميرة واليوراسيل ناقلة الفسفوريبوزيل. نتج تعبير عن زيادة ف?...

Video: Development of a Negative Selectable Marker for Entamoeba histolytica

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

الفحص في المختبر مثلأيشن الحمض الريبي النووي النقال مع الحمض النووي للنسج المتحولة والحمض الريبي النووي النقال Dnmt2 Methyltransferase (Ehmeth) انزيم

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

دراسة التوتة اختيار الإيجابية والسلبية من قياس التدفق الخلوي

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

باستخدام الكيمياء فوق لقياس تأثير عدوى فيروسية في خلية المضيف RNA التجميعي

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

التعرف السريع على البكتيريا سالبة الجرام من ثقافة الدم في مرق عن طريق MALDI-TOF الطيف الكتلي

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

تحليل الأضرار الظهارية التي تنتجها<em&gt; المتحولة الحالة للنسج</em&gt; عدوى

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will die from the disease. The human leukocyte antigen (HLA) class I samples peptides derived from proteasomal degradation of cellular proteins and presents these fragments on the cell surface for interrogation by circulating cytotoxic T lymphocytes (CTL). Generation of T-cell receptor mimic (TCRm) monoclonal antibodies (mAbs) which recognize breast cancer specific peptide/HLA-A*02:01 complexes such as those derived from macrophage migration inhibitory factor (MIF19-27) and NY-ESO-1157-165 enable detection and destruction of breast cancer cells in the absence of an effective anti-tumor CTL response. Intact class I HLA/peptide complexes are shed by breast cancer cells and represent potentially relevant cancer biomarkers. In this work, a breakthrough biomarker screening system for cancer diagnostics incorporating T-cell receptor mimic monoclonal antibodies combined with a novel, label-free biosensor utilizing guided-mode resonance (GMR) sensor technology is presented. Detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated. The impact of this work could revolutionize personalized medicine through development of companion disease diagnostics for targeted immunotherapies.

Intact class I HLA/peptide complexes are shed by cancer cells, representing a potential relevant cancer biomarker. Utilizing label-free sensor technology and T-cell receptor mimicking monoclonal antibodies, detection of shed MIF/HLA-A*02:01 complexes in MDA-MB-231 cell supernatants, spiked human serum, and patient plasma is demonstrated, enabling development of a novel cancer diagnostic platform.

الكشف عن خلايا الدم البيضاء الإنسان مستضد المؤشرات الحيوية في سرطان الثدي الاستفادة تسمية خالية جهاز الاستشعار البيولوجي تقنية

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

تصور آثار البلغم للتنمية بيوفيلم باستخدام نموذج غرف ساترة

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

تطوير نظام الفيروسات مراسل التهاب الكبد B لرصد المراحل المبكرة من دورة النسخ المتماثل

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

استخدام كبسولات لالتلطيخ السلبي من العينات الفيروسية ضمن بيوكنتينمنت

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

تمييز واحد من مونوسبيسيفيك وأنماط DFS70 مختلطة أثناء الفحص أنا مع الركازة رواية خروج المغلوب النخبة/DFS70 أبو عبدالله 2

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...