الكتاب ترغب في الاعتراف الدعم ذات الصلة لهذه المخطوطة من المفوضية الأوروبية البرنامج الإطاري السابع (PIOF-GA-2009-255466) والايرلندي مجلس البحوث الصحية (HRA_POR/2010/138). وكس الموسومة E. كان القولونية النوع هدية من الدكتور جيهان كورماك، كلية جامعة كورك.

1. ورم الحث <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتحريض الورم روتينية، تم حقن الجرعة مكون للأورام من الخلايا علقت الحد الأدنى في 200 ميكرولتر من مستنبت المصل خالية تحت الجلد (SC) في الجناح الإصابة مجانا 6-8 الاسبوع القديمة الإناث BALB / C أو athymic الفئران MF1-nu/nu ن = 6 (هارلان، أكسفورد، المملكة المتحدة) (1 × 10 6 خلايا 4T1) باستخدام إبرة حقنة 21-قياس. وكان جدوى من الخلايا المستخدمة في التلقيح أكبر من 95٪ كما هو محدد من قبل عدد البصرية باستخدام haemocytometer والتريبان الأزرق استبعاد صبغ (Gibco). </li><li style=";text-align:right;direction:rtl"> بعد إنشاء الورم، وسمح أورام لتنمو وتتطور وتم رصدها مرتين أسبوعيا. تم حساب حجم الورم وفقا لصيغة V = (AB 2) Π / 6، حيث هو أطول قطر الورم وب عمودي هو أطول قطر إلى قطر واسع. </li></ol> 2. إعداد البكتيرية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> سلالة بكتيرية المستخدمة في هذا protocoكان ل E. القولونية K-12 MG1655، غير البروتين السم، معربا عن سلالة أو إيواء البلازميد luxABCDE ترميز التي تمكن ليتم الكشف عن البكتيريا عن طريق BLI. E. كانت تزرع القولونية MG1655 التي تحتوي على luxABCDE المتكاملة هوائيا في 37 درجة مئوية في المتوسط ​​LB (سيغما الدريخ، أيرلندا) تستكمل مع 300 ميكروغرام / مل الاريثروميسين (EM). تم إنشاء مشتقة من bioluminescent MG1655 باستخدام بلازميد p16S لوكس الذي يحتوي على HELP P التأسيسي luxABCDE OPERON 1. </li><li style=";text-align:right;direction:rtl"> الاستعداد للإدارة على الفئران، وحضنت الثقافات في المتوسط ​​LB C ° 37 في في في 200 دورة في الدقيقة شاكر إلى مرحلة منتصف سجل (الكثافة البصرية في 600 نانومتر). تم حصادها البكتيريا عن طريق الطرد المركزي (6،000 × ز لمدة 5 دقائق)، وغسلها مع برنامج تلفزيوني (سيغما)، ومخففة في PBS 1 × 7 10 وحدة مستعمرة تشكيل (كفو) / مل لإدارة IV، أو 1 × 10 10 للتسمين. </li></ol> 3. البكتيرية اإلداريةنشوئها <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم تقسيم الفئران عشوائيا إلى مجموعات تجريبية عندما وصلت الأورام حوالي 100 مم 3 في وحدة التخزين. للإعطاء في الوريد، تلقت الفئران ضبط النفس كل 10 6 خلايا في 100 ميكرولتر، حقن في الوريد مباشرة الذيل الأفقي باستخدام إبرة حقنة 28G. تم تحديد عدد قابلة للحياة من كل من اللقاح الطلاء بأثر رجعي. </li><li style=";text-align:right;direction:rtl"> للدراسات الاستعمار GIT، كانت تدار شفويا 10 9 الخلايا البكتيرية في 100 ميكرولتر في الماوس بالتزقيم، على ثلاثة أيام متتالية. وانخفضت مستويات الموجودة من قبل البكتيريا المتعايشة قبل تغذية من إضافة الستربتومايسين ملغ / 5 مل في مياه الشرب الماوس لمدة 7 أيام قبل بدء بالتزقيم عن طريق الفم 1. </li></ol> 4. تلألؤ بيولوجي التصوير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم إجراء 2D التصوير في الجسم الحي باستخدام BLI IVIS100 (الفرجار). في تعريف إدارة الوقت آخر نقطة البكتيرية، كانت الفئران تخدير باستخدام الفرجار في XGI-8 غاز التخديرتم تنفيذ النظام مع Isofluorane 3٪، وكامل الجسم تحليل الصور في نظام 100 لمدة 2-5 دقيقة IVIS في حساسية عالية. </li><li style=";text-align:right;direction:rtl"> للتصوير 3D، وضعت الفئران في تخدير ماوس المكوك التصوير داخل نظام التصوير الضوئي لتصوير الظهرية (IVIS الطيف، الفرجار). للحصول على الصور من إشارة وسيفيراز البكتيرية للإنشاء البصرية 3D، استخدمت موجات الانبعاثات مرشح تتراوح 500 حتي 580 نانومتر مع أوقات بن اقتناء 16 من دقيقة 3-4 في تصفية لتحقيق أقصى قدر من إشارة إلى نسبة الضوضاء. كجزء من هذه الصورة تسلسل الاستحواذ، تم الحصول على صورة ضوء منظم لتحديد خريطة الارتفاع. كان إدخال هذه الخريطة منتشر ضوء التصوير المقطعي (DLIT) اعادة البناء الخوارزميات التي تم استخدامها لتكوين صورة 3D البصرية باستخدام غير سالب الأمثل المربعات الصغرى 2. </li><li style=";text-align:right;direction:rtl"> وجرى تحديد المناطق ذات الأهمية وكميا باستخدام برنامج السكن صورة (الفرجار): تحليل الصور. </li></ol> 5. ممثلالنتائج في هذه الدراسة، والبكتيريا المسببة للأمراض غير المتعايشة القولونية K-12 MG1655 معربا عن OPERON luxABCDE كان IV تدار على فئران تحمل الاورام SC طعم أجنبي 4T1. تم الكشف عن بكتيريا لوكس إشارة تحديدا في أورام الفئران آخر IV-الإدارة (الشكل 2). انتعاش ثقافة البكتيريا من الفئران عينة بالتحقق من صحة وجود علاقة خطية بين الأعداد البكتيرية قابلة للحياة وكمية ضوء الكشف عن (الشكل 3). في الجسم الحي التصوير شفويا يديرها البكتيريا المتعايشة في GIT ويتحقق أيضا باستخدام 3D BLI. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="الشكل 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> الشكل 1. بروتوكول الجدول الزمني. هي التي يسببها الأورام تحت الجلد في الفئران، والبكتيريا تدار على التنمية الورم (100 مم 3). الفئران الحية هي BLI ايمالذين تتراوح أعمارهم بين الإدارة في مختلف آخر مرة نقاط البكتيرية (الأسهم مرات عرض نموذجي). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="الشكل 2" /> الشكل 2. إدارة E. وقد حثت القولونية MG1655 luxABCDE على الفئران الحاملة للورم. تحت الجلد الأورام 4T1 في MF1 نو / نو الفئران وE. القولونية MG1655 luxABCDE تدار على التنمية الورم. تلقى كل حيوان 10 6 خلايا حقن في الوريد مباشرة الذيل الأفقي. تم تصوير الفئران في غضون أربع نقاط خلال الدراسة (نقط سوداء Z-المحور والصور) مع الانتعاش اللاحقة من البكتيريا قادرة على البقاء (كفو) من أورام الفئران عينة ضحى (الرسم البياني بار). زيادة في أعداد البكتيريا والبلازميد التعبير الجيني في الأورام على وجه التحديد لوحظ على مر الزمن (الماوس ممثل يتضح في نقطة زمنية). <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">اضغط هنا لمشاهدتها بشكل اكبر شخصية</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="الشكل 3" /> الشكل 3. والعلاقة بين تعداد أرقام الجرثومي Intratumoral وضوء بارد. البكتيريا قادرة على البقاء في الجسم الحي من قبل الثقافة الأورام البكتيرية السابقين من الأورام بعد BLI في مختلف نقاط الساعة الرابعة بعد الإدارة. تسجيل قيم أعداد البكتيريا (كفو) نسبة إلى وحدات في الجسم الحي ورسوم بيانية bioluminesce. ويلاحظ وجود علاقة قوية بين البكتريا وإشارات تلألؤ بيولوجي البكتيرية R 2 = 0.9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="الشكل 4" /> الشكل 4. المستعمر 3D IVIS صورة الجهاز الهضمي المسالك الفئران بواسطة E. MG1655 القولونية. واستعمر GIT من الفئران عن طريق الفم من كفو 9 10 من E. كولاي لمدة ثلاثة أيام متتالية. يظهر صورة العينة المعزولة من التصوير المقطعي 3D من الفأرة المستعمر.الصور تظهر 3D أطلس الماوس الرقمية من الهيكل العظمي لتوفير تسجيل التشريحية. E. القولونية MG1655 تلألؤ بيولوجي واضح باللون الأخضر في انخفاض، والأرجواني على مستويات أعلى.

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الإعلان عن أي تضارب في المصالح.

في سياق العلاج الجيني، أظهرت استخدام العوامل البيولوجية لتسليم الجينات العلاجية للمرضى وعدا كبيرا 3-5. مثل الفيروسات، والخصائص البيولوجية الفطرية من البكتيريا تسمح الكفاءة في تنفيذ DNA إلى الخلايا أو الأنسجة، وخاصة في سياق السرطان. وقد تبين أن البكتيريا قادرة ط...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> 4T1 خلية خط </td><td> ATCC </td><td> CRL-2539 </td><td> مسانج نموذج سرطان الثدي المستمدة من ورم BALB / ج الناشئة بصورة عفوية الثديية </td></tr><tr><td> DMEM </td><td> سيغما الدريخ </td><td> D6429 </td><td> Dulbecco في معدلة النسر متوسطة </td></tr><tr><td> PBS </td><td> سيغما الدريخ </td><td> D8537 </td><td> التخزين المؤقت المالحة الفوسفات </td></tr><tr><td> Xenogen IVIS </td><td> الفرجار العلوم الحياتية </td><td></td><td> IVIS 100 ل2D التصوير؛ IVIS الطيف ل3D. </td></tr><tr><td> لوريا مرق ميلر (LB) </td><td> سيغما الدريخ </td><td> L2542 </td><td> متوسطة النمو لE. القولونية </td></TR&gt; <tr><td> الاريثروميسين </td><td> سيغما الدريخ </td><td> E5389 </td><td> مضاد حيوي </td></tr><tr><td> الستربتومايسين عقار طبي </td><td> سيغما الدريخ </td><td> S9137 </td><td> مضاد حيوي </td></tr><tr><td> الفئران MF1nu/nu </td><td> هارلان (UK) </td><td> 069 (نو) / 070 (نو / +) </td><td> HSD: Athymic عاري-Foxn1nu </td></tr><tr><td> BALB / ج الفئران </td><td> هارلان (UK) </td><td> 066 </td><td> النمط الفرداني: H-2 د </td></tr><tr><td> بالتزقيم إبرة </td><td> التعليم والتدريب المهني الحلول التقنية (UK) </td><td> DE009 </td><td> 22G X 38mm إبرة بالتزقيم على التوالي </td></tr><tr><td> حقنة للحقن IV </td><td> BD العلوم البيولوجية </td><td> 309309 حتي 1 مل </td><td> حقنة الأنسولين مع 28 بوصة ½ G X الإبرة الدقيقة الرابعة بشكل جيد. </td></tr><tr><td> حقنة التطعيم للورم </td><td> براون </td><td> 9161376V </td><td> Omnifix 26 G &amp; Xfrac12؛ إبرة بوصة </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

هذا الفيديو يصف استخدام كامل الجسم bioluminesce التصوير (BLI) لدراسة الاتجار البكتيرية في الفئران الحية، مع التركيز على استخدام البكتيريا في الجينات والعلاج بالخلايا لمرض السرطان. البكتيريا تقديم فئة جذابة من ناقلات لعلاج السرطان، وحيازة القدرة الطبيعية على النمو داخل الأورام بعد تفضيلي الإدارة النظامية. بكتيريا مهندسة للتعبير عن الجينات لوكس راديو كاسيت تصريح BLI الكشف عن البكتيريا ومواقع الورم في نفس الوقت. ويمكن تصور موقع ومستويات البكتيريا داخل الأورام مع مرور الوقت درست بسهولة، في اثنين أو ثلاثة أبعاد. هذه الطريقة تنطبق على مجموعة واسعة من الأنواع البكتيرية وأنواع طعم أجنبي الورم. توضح هذه المقالة بروتوكول لتحليل البكتيريا داخل الفئران bioluminescent تحت الجلد تحمل الورم. يوصف أيضا من البكتيريا المتعايشة التصور في القناة الهضمية (GIT) من BLI. هذه قوية، ورخيصة، واستراتيجية في الوقت الحقيقي التصوير تمثيلاTS وسيلة مثالية لدراسة البكتيريا في الجسم الحي في سياق أبحاث السرطان، العلاج الجيني في وجه الخصوص، والأمراض المعدية. هذا الفيديو يوضح الإجراء لدراسة لوكس الموسومة E. القولونية في الفئران الحية، مما يدل على قراءات المكاني والزماني للتحقيق BLI استخدام مع نظام IVIS.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

توضح هذه المقالة إدارة<em&gt; لوكس الموسومة</em&gt; البكتيريا على الفئران واللاحقة<em&gt; في الجسم الحي</em&gt; التحليل باستخدام التصوير تلألؤ بيولوجي IVIS.

البكتيرية التصوير Bioluminescent<em&gt; في الجسم الحي</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K Views.  University College Cork. توضح هذه المقالة إدارة لوكس الموسومة البكتيريا على الفئران واللاحقة في الجسم الحي التحليل باستخدام التصوير تلألؤ بيولوجي IVIS.

Video: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

في الجسم الحي التصوير الطفيليات الليشمانية المعدلة وراثيا في مضيف لايف

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

غير الغازية تصوير الزاجل كريات بيضاء والهجرة في الجسم الحي</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

تقييم الاستجابة للعيش فوهي الخلايا البكتيرية باستخدام التصوير الجامعة ليف

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

باستخدام التصوير Bioluminescent بالتحقيق في التآزر بين العقدية الرئوية والأنفلونزا من الفيروسات في الفئران الرضع

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&#39;Bioluminescent&#39; بالعاثية مراسل للكشف عن مسببات الأمراض البكتيرية الفئة أ

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

في الجسم الحي التصوير كشفها (IVIS) نظم لفيروس العصبية الغازية ملتهب الدماغ

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

في الجسم الحي أسلوب التصوير لتميز الالتهاب الحاد والمزمن

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

جيل ومتعدد المظهري فحص عالية المحتوى من<em&gt; الكوكسيلا البورنيتية</em&gt; المسوخ ينقول

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

في المختبر والمجراة نموذج لدراسة التصاق البكتيريا إلى سفينة ستريت تحت ظروف التدفق

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.