עבודה זו נתמכה על ידי מלגת לימודים מאוניברסיטת בריטיש קולומביה (לBV) ומענקים מסיסטיק פיברוזיס מכוני קנדה וקנדיים לבריאות מחקר (לDPS). אנו מודים לז&#39;קלין צ&#39;ונג להכנה הראשונית של פרוטוקולים.

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

שיטה להכנת חלבונים שתוארה שיכול לחלץ את רוב חלבוני Burkholderia עם שחזור טוב. זו באה לידי הביטוי על ידי קבלת באותו פרופיל חלבון משני תכשירים עצמאיים שבוצעו בימים שונים תוך שימוש באותה התרבות חיידקים גדלה בLB או מרק YEM כפי שמוצג באיור 1. החילוץ היה יעיל לחיידקים ...

Burkholderia הסוג מונה יותר מ 62 מינים, אורגניזמים שליליים גרם מבודדים ממגוון רחב של נישות, והוא מחולק לשני אשכולות עיקריים 1,2. האשכול הראשון כולל בני אדם, בעלי חיים ואורגניזמים phytotrophic, ורוב המחקרים התמקדו במינים פתוגניים של קבוצה זו בשל החשיבות הקלינית שלהם. החברים פתוגניים ביותר הם B. pseudomallei ו-B mallei (מה שגורם melioidosis ונחרת בהתאמה) 3,4 ופתוגנים אופורטוניסטיים (17 מינים המוגדרים של מתחם Burkholderia cepacia, BCC) 5, הגורמים למחלות בסיסטיק פיברוזיס (CF) ומחלת granulomatous כרונית (CGD) 1. האשכול השני, עם יותר מ 30 מינים nonpathogenic, כולל חיידקים הקשורים צמחים או בסביבה, והם נחשבים למועילים פוטנציאליים למארח 2. סיבוכים רבים לצאת frזיהום חיידקי אום עם החברים פתוגניים של סוג Burkholderia, כגון העברת של הפתוגן בין חולים, התפשטות המחלה וכישלון טיפול בשל ההתנגדות הפנימית או נרכשה לאנטיביוטיקה הופכת קשה למגר ברוב המקרים 6-9. לכן, צובר הבנה ברורה יותר של הבסיס להקמתה של זיהום חיידקים הוא חיוני לטיפול במחלות הנגרמות על ידי אורגניזמים אלה. על מנת לקבל תובנות לגבי הקמתה של זיהום, חקירות נרחבות על רכיבי חיידקים הקשורים בפתוגנזה יש צורך. לימודים מתמקדים בניתוח proteomic של אורגניזמים Burkholderia תוך שימוש בגישות proteomic תיארו חלבונים שהיו מעורבים בפתוגנזה של חיידקים, כמו גם שינויים בproteome פרופילי 10-16. שיטות הפקת חלבון באמצעות sonication ומחזורי הקפאה מופשרת במאגר תמוגה המכיל גבוה קונצרט כלשהוentration של אוריאה, thiourea בשילוב עם חומרי ניקוי וampholytes כבר יושם במחקרי proteomic Burkholderia 10-13. למרות האוריאה היא די יעילה לdenaturation חלבון, זה יכול לבסס שיווי משקל בתמיסה מימית עם איזוציאניד אמוניום, אשר יכול להגיב עם קבוצות חומצת אמינו, ובכך יוצר חפצים (תגובת carbamylation) 17. לכן, מומלץ לכלול ampholytes המוביל, אשר פועל כמו אוכלי נבלות cyanate ולהימנע מטמפרטורות מעל 37 מעלות צלזיוס 17. יתר על כן, כדי למנוע כל הפרעה כימית של מאגר תמוגה עם כימות חלבון, אותו המאגר תמוגה יכול לשמש כדי ליצור עקומת הסטנדרט, כדי שהדגימות והסטנדרטים יש לו הרקע 10. מתודולוגיות אחרות כרוכות בשימוש במאגרים וחומרי ניקוי בסיסיים עם תקופות דגירה חום 17,18, אולם תנאים אלה עלולים לגרום לשינויים בproteome ושחומרי ניקוי מסוימים אינם תואמים ליחסי ציבוריישום oteomics אלא אם כן צעדים להסרת חומר ניקוי שלאחר מכן כלולים 17,18. לאחר חילוץ וכימות נאותים, ביטוי חלבון הגלובלי של כל חלבון בודד ניתן ללמוד באמצעות גישות proteomic כגון דו ממדים (2-D) ג&#39;ל אלקטרופורזה. טכניקה זו תוארה לראשונה על ידי O&#39;Farell 19 ומורכב בהפרדת חלבונים לפי הנקודה איזואלקטרית על ידי איזואלקטרית התמקדות בממד הראשון, ולאחר מכן בהתאם למשקל המולקולרי שלהם על ידי ג&#39;ל אלקטרופורזה acrylamide בממד השני. בשל הרזולוציה והרגישות שלה, טכניקה זו היא כלי רב עוצמה לניתוח וזיהוי של חלבונים ממקורות ביולוגיים מורכבים 19,20. טכניקת הפרדה זו זמינה כיום בגישות חלבון ממוקד עם היתרון הגדול של פתרון isoforms החלבון נגרם על ידי שינויים לאחר תעתיק או עיבוד פרוטאוליטי. שינויים הכמותייםיכול להיות מזוהה על ידי השוואת עוצמת הנקודה המקבילה לאחר הצביעה של ג&#39;ל 20. עם זאת, טכניקה זו אינה מתאימה לזיהוי של חלבונים גדולים מאוד, חלבונים בממברנה, חלבונים מאוד בסיסיים וחומצי או הידרופובי, והיא טכניקה קצת מייגע וגוזל זמן 20. גישות פפטיד הממוקד חדשות (המבוסס על ג&#39;ל שאינו), כי הם יותר חזקים והמטרה להיות זמינות וניתן להשתמש בם להשוואת כמותית בשיטות תיוג איזוטופ היציב ההפרש כגון תיוג ציסטאין ידי זיקה מקודדת איזוטופ תיוג (ICAT) 21, וקבוצת אמינו תיוג על ידי תיוג איזוטופ כדי לכמת יחסי ומוחלט (iTRAQ) 22. השימוש בטכניקת proteomic אחת עשויה לתת מידע מספיק, ולכן השימוש בשני גישות proteomic משלימות הוא הכרחי עבור רוב השינויים בהערכה באופן מלא בproteome. עם זאת, ג&#39;ל אלקטרופורזה 2-D נעשתה שימוש נרחב ויכולה להיות מיושמת באופן שיגרתי לקוואןtitative ביטוי של מספר חלבונים באורגניזמים שונים. כאן אנו מתארים מיצוי חלבון כל התא ונהלי ג&#39;ל אלקטרופורזה 2-D למיני Burkholderia שהותאמו ומותאמים מ2-D עקרונות אלקטרופורזה GE Healthcare ושיטות Handbook 80-6429-60AC 2004 <a href="http://www.amershambiosciences.com" target="_blank">(www.amershambiosciences.com).</a> הפקת החלבון בוצעה באמצעות מקצף חרוז בחרוזי זכוכית בנוכחות של PBS המכילה 5 מ&quot;מ EDTA (חומצת tetraacetic ethylenediamine) ו1 מ&quot;מ PMSF (פלואוריד phenylmethylsulfonyl). הליך זה מאפשר כימות של חלבונים עם השפלה מינימלית הוא amendable לגישות proteomic כ15,23,24 שדווחו בעבר. ג&#39;ל אלקטרופורזה 2-D בוצעה באמצעות 24 סנטימטר ארוך משותק pH 4-7 שיפוע וחלבונים הופרדו על פי נקודה איזואלקטרית. אז חלבונים הופרדו על פי Molecמשקל תואר בלבד על ידי ג&#39;ל SDS-polyacrylamide. בנוסף, אנו תיארתי שיטת צביעת כסף להדמיה של חלבוני מקום, ושיטת כתם כסף שהוא תואם לניתוח ספקטרומטריית מסה. יחד נהלים אלה יכולים לאפשר זיהוי של חלבונים חשובים ממינים Burkholderia שיכול להיות מעורב בפתוגנזה.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Phenylmethanesulfonyl fluoride (PMSF)</td>
 <td>Sigma</td>
 <td>P7626</td>
 <td>Toxic, corrosive</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher</td>
 <td>A18-1</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>PBS Buffer</td>
 <td>Bioscience</td>
 <td>R028</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher</td>
 <td>BP118-500 </td>
 <td>toxic</td>
 </tr>
 <tr>
 <td>Glass beads (0.1 mm)</td>
 <td>BioSpec Products</td>
 <td>11079101</td>
 <td></td>
 </tr>
 <tr>
 <td>Nalgene Oak Ridge Centrifuge tubes, polycarbonate (50 ml)</td>
 <td>VWR</td>
 <td>21009-342</td>
 <td></td>
 </tr>
 <tr>
 <td>MicroBCA protein extraction kit</td>
 <td>Pierce</td>
 <td>23235</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcentrifuge Tubes 2.0 ml conical Screw cap Tubes with Cap and O-Ring</td>
 <td>Fisher</td>
 <td>02-681-375</td>
 <td></td>
 </tr>
 <tr>
 <td>2-D Clean-Up kit</td>
 <td>GE Healthcare</td>
 <td>80-6484-51</td>
 <td></td>
 </tr>
 <tr>
 <td>Urea</td>
 <td>Invitrogen</td>
 <td>15505-050</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>Amersham Biosciences</td>
 <td>17-1314-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>MPBiomedical, LCC</td>
 <td>856126</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Immobiline DryStrips pH 4-7 24 cm</td>
 <td>Amersham Biosciences</td>
 <td>176002-46</td>
 <td></td>
 </tr>
 <tr>
 <td>Duracryl</td>
 <td>Proteomic Solutions</td>
 <td>80-0148</td>
 <td>Very toxic, carcinogen</td>
 </tr>
 <tr>
 <td>Ammonium persulfate</td>
 <td>Fisher</td>
 <td>BP179-100</td>
 <td>Flammable, toxic, corrosive</td>
 </tr>
 <tr>
 <td>N,N,N',N'-tetramethylethylenediamine (TEMED)</td>
 <td>Invitrogen</td>
 <td>15524-010</td>
 <td>Flammable</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate (SDS)</td>
 <td>Fisher</td>
 <td>BP166-500</td>
 <td>Acute toxicity, flammable</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Fisher</td>
 <td>HC1100-1GL</td>
 <td>Flammable, toxic</td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Fisher</td>
 <td>A491-212</td>
 <td>Flammable, corrosive</td>
 </tr>
 <tr>
 <td>Glutaraldehyde</td>
 <td>Fisher</td>
 <td>G151-1</td>
 <td>Very toxic, corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Potassium tetrathionate</td>
 <td>Sigma</td>
 <td>P2926</td>
 <td>Irritant</td>
 </tr>
 <tr>
 <td>Sodium acetate</td>
 <td>EM Science</td>
 <td>7510</td>
 <td></td>
 </tr>
 <tr>
 <td>Silver nitrate</td>
 <td>Sigma</td>
 <td>209139</td>
 <td>Corrosive, dangerous for the environment</td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma</td>
 <td>252549</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Sodium thiosulfate</td>
 <td>Sigma</td>
 <td>S7026</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>EMD</td>
 <td>9230</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>MPBiomedical, LCC</td>
 <td>808831</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>MPBiomedical, LCC</td>
 <td>800688</td>
 <td></td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Fisher</td>
 <td>A412-4</td>
 <td>Flammable, toxic, health hazard</td>
 </tr>
 <tr>
 <td>Sodium carbonate anhydrous</td>
 <td>EMD</td>
 <td>SX0400-3</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>Mineral oil</td>
 <td>ACROS</td>
 <td>415080010</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>JA-20 ultracentrifuge rotor</td>
 <td>Beckman Coulter</td>
 <td>334831</td>
 <td></td>
 </tr>
 <tr>
 <td>Mini Beadbeater</td>
 <td>Biospec Products</td>
 <td>3110BX</td>
 <td></td>
 </tr>
 <tr>
 <td>Ettan IPGphor II Isoelectric Focusing System and accessories</td>
 <td>GE Healthcare</td>
 <td>80-6505-03</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 <tr>
 <td>Ettan DALT Large Vertical electrophoresis system</td>
 <td>GE Healthcare</td>
 <td>80-6485-27</td>
 <td><a href="http://www.amershambiosciences.com" target="_blank">www.amershambiosciences.com</a></td>
 </tr>
 </tbody>
</table>

total protein extraction and 2-d gel electrophoresis methods for burkholderia species

החקירה של רמות החלבון תאית של מיני חיידקים היא בעלת חשיבות להבנת המנגנונים פתוגניים במחלות הנגרמים על ידי אורגניזמים אלה. כאן אנו מתארים הליך להפקת חלבון ממינים Burkholderia מבוססת על תמוגה מכאנית באמצעות חרוזי זכוכית בנוכחות חומצת ethylenediamine tetraacetic ופלואוריד phenylmethylsulfonyl בופר פוספט. שיטה זו יכולה לשמש למיני Burkholderia שונים, לתנאי גידול שונים, וסביר להניח מתאים לשימוש במחקרי proteomic של חיידקים אחרים. לאחר מיצוי חלבון, טכניקת proteomic דו ממדים (2-D) ג&#39;ל אלקטרופורזה מתוארת ללמוד שינויים הגלובליים בproteomes של אורגניזמים אלה. שיטה זו מורכבת של הפרדת חלבונים לפי הנקודה איזואלקטרית על ידי איזואלקטרית התמקדות בממד הראשון, ואחריו הפרדה על בסיס משקל מולקולריעל ידי ג&#39;ל אלקטרופורזה acrylamide בממד השני. הדמיה של חלבונים מופרדים מתבצעת על ידי צביעת כסף.

ניתוח השוואתי של פרופילי החלבון המופקים מאותה התרבות חיידקים בשתי הזדמנויות שונות הראה פסי דפוסים דומים המצביע על עקירות חלבון מוצלחות. חלבונים במשקל מולקולריים שחולצו נעו 10-150 kDa. איור 1 מציג נציג ג&#39;ל Coomassie כתמי כחולים של עקירות חלבון כל התא מmultivorans Burkholde...

Watch this Scientific Journal Video about Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species at JoVE.com

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

חברים של סוג Burkholderia הם פתוגנים חשיבות קלינית. אנו מתארים שיטה לסך מיצוי חלבון חיידקים, תוך שימוש בהפרעה מכאנית, וג&#39;ל אלקטרופורזה 2-D לניתוח proteomic שלאחר מכן.

הפקת חלבון סה&quot;כ ו2-D שיטות ג&#39;ל אלקטרופורזה עבור<em&gt; Burkholderia</em&gt; מינים

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

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Research

JoVE Journal

Immunology and Infection

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

28.9K Views.  University of British Columbia.  חברים של סוג Burkholderia הם פתוגנים חשיבות קלינית. אנו מתארים שיטה לסך מיצוי חלבון חיידקים, תוך שימוש בהפרעה מכאנית, וג'ל אלקטרופורזה 2-D לניתוח proteomic שלאחר מכן. 

Video: Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

פשוט פרוטוקול Chelex למיצוי DNA מ<em&gt; אנופלס</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

שתי שיטות של גיבוש Heterokaryon לגלות גורמים הגבלת HCV

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

נהלים לזיהוי פריוני זיהומיות לאחר המעבר דרך מערכת העיכול של מינים עופות

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

הקמה ואופטימיזציה של הגדרת תפוקה גבוהה ללמוד<em&gt; סטפילוקוקוס epidermidis</em&gt; ו<em&gt; Marinum Mycobacterium</em&gt; זיהום כמודל לגילוי תרופות

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteomic פרופיל של המקרופאגים על ידי 2D אלקטרופורזה

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isothermal הגברה מבחני בתיווך לולאה (LAMP) לאיתור המינים ספציפי של<em&gt; Eimeria</em&gt; כי להדביק תרנגולות

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

שיטות אלטרנטיביות במבחנה מצפני Capsid ויראלי המבנית

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

מדידה אוטומטיות של Cryptococcal מינים רב-סוכר כמוסה וגוף התא

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

ויבתר חלבון רב מתחמי איתות על ידי טיהור זיקה קומפלמנטציה ריאקציה דו-מולקולרית (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

מקורי פוליאקרילאמיד ג'ל לטיפול חיסוני כתמי באנליזה של אנדודוגני IRF5 דימרזציה

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...