We thank Benjamin Knowles, Yan Wei Lim, Andreas Haas, and members of the Viral Dark Matter consortium for their help and constructive input on this manuscript. This research is funded by the National Science Foundation (DEB-1046413) and is part of the Dimensions: Shedding Light on Viral Dark Matter project.

<ol>
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The authors have nothing to disclose.

وتقدر الفيروسات التي تصيب البكتيريا (ويعرف أيضا باسم الجراثيم أو فج) في الوجود في أكثر من 10 31 فيروس مثل الجسيمات (VLPs) عالميا ويفوق عدد جميع الكائنات الحية الأخرى في بيئة 1،2. وركزت الدراسة الأولى metagenomic التحقيق في المجتمعات الفيروسية المرتبطة البيئات البحرية على قياس التنوع ينظر في جزء الفيروسي 3. بالإضافة إلى ذلك، وجد Breitbart وزملاؤه أن أكثر من 65٪ من تسلسل المجتمع الفيروسية المشتركة لا تناظر إلى أي تسلسل المتاحة في قواعد البيانات العامة. وجدت الدراسات metagenomic اللاحقة أدلة مماثلة: metagenomes من الرواسب البحرية في سان دييغو، كاليفورنيا تحتوي على 75٪ تسلسل الفيروسية مجهولة 4؛ metagenomes من البحيرات ذات الملوحة العالية للبحر سالتون تحتوي على 98٪ تسلسل الفيروسية المجهولة 5؛ وmetagenomes المرتبطة المرجانية تحتوي على 95-98٪ تسلسل الفيروسية مجهولة 6. وقد أدى هذا التراكم من المعلومات غير المشروحة فيفج المادة الوراثية بأنها &quot;المادة المظلمة في الكون البيولوجي&quot; (7). توصيف الجيني من فج يعتمد على تحديد التشابه تسلسل من خلال المقارنة مع الأحماض والبروتينات قواعد بيانات النووية القائمة. لأن المعلومات الوراثية المشفرة فج هي في الغالب غير معروفة، وطرق القائم على التماثل غير فعالة. داخل الجينوم، وفاجات عادة ترميز ثلاثة أنواع رئيسية الجينات: النسخ والتكرار الجينات، الجينات التمثيل الغذائي، والجينات الهيكلية. وتشمل الجينات النسخ والتكرار (فئة I / II الجينات 8) بلمرة، primases، إندو / إكسو-nucleases، وتحركات. يتم حفظها هذه الجينات للغاية نظرا لأهميتها في الإصابة فج، الكتابة وتكرار فج المادة الوراثية. يتم تحديد بلمرة فج بسهولة باستخدام أساليب تناظر تسلسل التقليدية بسبب الحفظ العالمي من 9 ولقد ثبت لخدمة علامات النشوء والتطور فعالة إلى 10.في المقابل، فج التمثيل الغذائي والجينات الهيكلية (الطبقة II / III الجينات 8) بشكل متزايد متباينة، وغالبا ما المشروح في الجينات افتراضية. فج جينات الأيض تؤثر على قدرة التمثيل الغذائي للمضيف وليس مطلوبا بالضرورة لتكاثر الفيروس. ويبدو أن هذه الجينات، وغالبا ما يشار إلى جينات الأيض المساعدة الى 11 (AMGs)، لتعديل التمثيل الغذائي المضيف والسماح التقدم الأمثل للعدوى ونجاح الفيريون النضج. وقد ارتبطت AMGs مع استخدام وامتصاص المواد الغذائية التي تحد أو في مسارات إنتاج الطاقة. وتشمل بعض الأمثلة الجينات الضوئي الموجودة في الجينوم من مختلف cyanophage 12-16، الجينات المتصلة والتي تنظمها استقلاب الفوسفات 17،18، والاستفادة من مسار الفوسفات البنتوز لفج dNTP الحيوي 18،19. في المقابل، الجينات الهيكلية من بين منتصف إلى أواخر الجينات التي تنتج أثناء العدوى وتختلف عبر مختلف فج حونظم الحادي والعشرين. إنتاج البروتينات الهيكلية تعتمد على توافر dNTP الفيروسي، وبرك الطاقة الخاصة بهم النسخ والترجمة، والتجمع 8. تعتبر البروتينات قفيصة والألياف ذيل هيكلية إذ أن معظم متباينة من كل جينات ترميز البروتينات الفيروسية وهناك حاجة لإنتاج الفيريون ناجحة. وعادة ما يعزى الاختلاف على الدور الفعال الذي تلعبه في تشكيل coevolution الفيروس في استضافة 20. البروتينات المختلفة، بغض النظر عن الطبقة الجينات، ويتم التغاضي بسهولة عند استخدام تقنيات التماثل والتوافق تسلسل التقليدية. وقد أدت محاولة لتصحيح القيود رأينا مع مقارنات تسلسل صارمة في أدوات المعلوماتية الحيوية قادرة على استخدام الخصائص تسلسل لتحديد تكوين الجمعيات، مثل الشبكات العصبية الاصطناعية 21. الشبكات العصبية الاصطناعية (الشبكات العصبية الصناعية) تسمح للتنبؤ الجينات الهيكلية والتمثيل الغذائي، ومع ذلك، تتطلب التحقق التجريبي المصب لتوصيف مباشرةوظيفة الجين. والهدف من هذا المخطوط هو توفير بروتوكولات مظهري قادرة على رصد كل عملية الأيض تقويضي والمنشطة للبكتيريا المضيف خلال التعبير عن الجينات فج الرواية، وتوقع وظيفيا من خلال الشبكات العصبية الصناعية. مجال phenomics، البيولوجيا المرتبطة الظواهر الخلوية، راسخة في بيولوجيا النظم للمساعدة في التحقيق في البروتينات مع وظيفة غير معروفة أو عديد المظاهر. وتستخدم أدوات مظهري لربط المعلومات المظهرية على المعلومات الوراثية. نحن نفترض للجينات فج المفترضة أن وظيفة (ق) يمكن تحديدها من خلال مراقبة المضيف الآثار الفسيولوجية خلال التعبير فج الجينات. للتحقيق في هذه الفرضية، وقد تم اختيار اثنين من الأساليب الكمية. واستخدمت متعددة النمط الظاهري الفحص لوحات (خرائط) لمراقبة المضيف استخدام الركيزة وتشكيل الكتلة الحيوية لاحق في حين الايض قياس التنوع الأيض المضيفة والوفرة النسبية خلال النمو في البيئى محددةالظروف النفسية. تم overexpressed البروتينات الهيكلية والتمثيل الغذائي المفترضة في القولونية وتتم مقارنة نتائج ممثلة من كل التجارب. يتم عرض تقنيات بصرية عديدة وخطوط الأنابيب وتجهيز إنتاجية عالية لتسهيل النسخ التجريبية. وأخيرا، وتناقش استنساخ ودقة الأساليب المعروضة في سياق الآثار الفسيولوجية المتوقع للبروتين المشروح قفيصة وفج البروتين الأيض، thioredoxin، بالإضافة إلى اثنين AMGs المفترضة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.22&#181;m Sterivex Filter</td>
			<td>Fisher Scientific</td>
			<td>SVGP01050</td>
			<td>Millipore</td>
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			<td>0.22&#181;m Millex Filters</td>
			<td>Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td>Millipore</td>
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			<td>0.22&#181;m SteriCap Filter</td>
			<td>Fisher Scientific</td>
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			<td>Millipore</td>
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			<td>0.22 &#181;m Omnipore membrane filters</td>
			<td>Millipore</td>
			<td>JHWP02500</td>
			<td>Millipore</td>
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			<td>96 well micro-titer plates&#160;</td>
			<td>VWR</td>
			<td>82050-764</td>
			<td>Standard F-Bottom 96 well Microplates</td>
		</tr>
		<tr>
			<td>2 mL 96 well plate</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Seal Plate Film</td>
			<td>Sigma-Aldrich</td>
			<td>Z369667</td>
			<td></td>
		</tr>
		<tr>
			<td>2 L Nalgene square bottles</td>
			<td>Cole Parmer</td>
			<td>T-06040-70</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Nalgene square bottles</td>
			<td>Cole Parmer</td>
			<td>T-06040-50</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4inch Panel Mount Lock Nut, black nylon&#160;</td>
			<td>Cole Parmer</td>
			<td>EW-45509-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Luer Thread Style Panel Mount to 200 Series Barb&#160; 1/16inch&#160;</td>
			<td>Cole Parmer</td>
			<td>EW-45500-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Luer Thread Style Panel Mount to 200 Series Barb, 1/8inch</td>
			<td>Cole Parmer</td>
			<td>EW-45500-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Luer Integral Lock Ring to 500 Series Barb, 1/16inch ID tubing</td>
			<td>Cole Parmer</td>
			<td>EW-45505-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Luer with Lock Ring x Female Luer Coupler</td>
			<td>Cole Parmer</td>
			<td>T-45508-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Barbed Bulkhead Fittings 1/4inch OD&#160;</td>
			<td>Fisher Scientific</td>
			<td>6149-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sanipure Tubing 1/16inch ID x 1/8inch OD&#160;</td>
			<td>SaniPure</td>
			<td>AR400002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sanipure Tubing 1/4inch OD x 1/8inch ID&#160;</td>
			<td>SaniPure</td>
			<td>AR400007</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable Flow Mini Pump (Peristaltic pump)</td>
			<td>Fisher Scientific</td>
			<td>13-876-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stirrer</td>
			<td>Velp Scientifica</td>
			<td>F203A0160</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>14-512-141</td>
			<td>&#160;Millipore* Filter Forceps&#160;</td>
		</tr>
		<tr>
			<td>Multi-plate spectrophotometer plate reader</td>
			<td>Molecular Devices Analyst GT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter manifold</td>
			<td>Fisher Scientific</td>
			<td>&#160;XX10 025 02</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Python version 2.7.5</td>
			<td></td>
			<td></td>
			<td><a href="http://www.python.org/">http://www.python.org/</a></td>
		</tr>
		<tr>
			<td>PyLab module</td>
			<td></td>
			<td></td>
			<td><a href="http://wiki.scipy.org/PyLab">http://wiki.scipy.org/PyLab</a></td>
		</tr>
		<tr>
			<td>R version 3.0.1&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.r-project.org/">http://www.r-project.org/</a></td>
		</tr>
		<tr>
			<td>reshape2 library</td>
			<td></td>
			<td></td>
			<td><a href="http://had.co.nz/reshape">http://had.co.nz/reshape</a></td>
		</tr>
		<tr>
			<td>ggplot2 library</td>
			<td></td>
			<td></td>
			<td><a href="http://ggplot2.org/">http://ggplot2.org/</a></td>
		</tr>
		<tr>
			<td>Gene Composer</td>
			<td>PSI Tech Portal</td>
			<td></td>
			<td>http://www.genecomposer.net</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Services:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>West Coast Metabolomics Center</td>
			<td>UC Davis</td>
			<td></td>
			<td><a href="http://metabolomics.ucdavis.edu/">http://metabolomics.ucdavis.edu</a></td>
		</tr>
		<tr>
			<td>DNA 2.0</td>
			<td></td>
			<td></td>
			<td>https://www.dna20.com</td>
		</tr>
	</tbody>
</table>

phage phenomics: physiological approaches to characterize novel viral proteins

التحقيقات الجارية في التفاعلات فج في استضافة تعتمد على استقراء المعرفة من (الفوقية) الجينوم. ومن المثير للاهتمام، 60-95٪ من كل فج تسلسل حصة لا تناظر للبروتينات المشروح الحالية. ونتيجة لذلك، يتم شرحها نسبة كبيرة من الجينات فج كما افتراضية. هذا الواقع يؤثر بشكل كبير على الشرح كل من جينات الأيض الهيكلية ومساعدة. نحن هنا نقدم طرق مظهري صمم للقبض على الاستجابة الفسيولوجية (ق) من مجموعة مختارة خلال التعبير عن واحد من هذه الجينات فج غير معروفة. وتستخدم متعددة النمط الظاهري الفحص لوحات (خرائط) لرصد تنوع استخدام الركيزة المضيفة وتشكيل الكتلة الحيوية لاحق، في حين توفر الايض تحليل ثنائي المنتج من خلال مراقبة وفرة الأيض والتنوع. وتستخدم كل من الأدوات في وقت واحد لتقديم لمحة المظهري المرتبطة تعبير عن فج المفترض إطار القراءة المفتوحة واحد (ORF). تتم مقارنة نتائج ممثلة لكلتا الطريقتين، HIGHLighting الاختلافات المظهرية الشخصي من مجموعة تحمل إما المفترضة الجينات فج الهيكلية أو التمثيل الغذائي. بالإضافة إلى ذلك، يتم عرض تقنيات التصور وإنتاجية عالية أنابيب الحسابية التي سهلت التحليل التجريبي.

Watch this Scientific Journal Video about Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins at JoVE.com

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Phenomics فج: مقاربات الفسيولوجية لتوصيف رواية الفيروسية البروتينات

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

phage-phenomics-physiological-approaches-to-characterize-novel-viral

Research

JoVE Journal

Immunology and Infection

12.0K Views.  San Diego State University. Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Video: Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

باستخدام الفحص ميكروأري عموم الفيروسي (Virochip) إلى الشاشة العينات السريرية لمسببات الأمراض الفيروسية

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

استخدام مضاد للفيروسات، γ الإنزيم المرتبط الفحص Immunospot لوصف الرواية تي خلية الحواتم من فيروس الورم الحليمي البشري

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

إرساء ثقافة السائل المغطاة من مجرى الهواء الإنسان المستقطب طلائي Calu-3 خلايا لدراسة استجابة الخلية المضيفة لمسببات الأمراض التنفسية<em&gt; في المختبر</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

النهج الوراثية إلى الأمام في<em&gt; الكلاميديا ​​المتدثرة</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

تحديد تركيز الفيروسي من خلال البلاك فحوصات: نظم تراكب عن طريق التقليدية والرواية

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

المواد الجزيئية الحيوية لوصف التفاعلات البكتيريا في استضافة

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

تطوير بوساطة حلقة-المجففة بالتبريد متساوي التضخيم الكواشف للكشف عن<em&gt; الكوكسيلا البورنيتية</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

قياس الأنفلونزا النورامينيداز تثبيط الجسم المضاد التتر من قبل انزيم مرتبط كتين الفحص

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

فحص فحوصات لوصف رواية غشائي المنظمين المتورطين في الاستجابة الالتهابية

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.