המחברים מודים לכל חברי במעבדות Ule, לאסקומב וזופאן לדיון סיוע ניסיוני. אנו מודים ג&#39;יימס Hadfield ואת ניק מתיוס עבור סידור תפוקה גבוהה. ברצוננו לציין כי השיטה המתוארת כאן iCLIP מניות בכמה צעדים עם פרוטוקול CLIP המקורית, שפותחה על ידי ג&#39;נסן קירק ו JU במעבדה של רוברט דרנל. עבודה זו נתמכה על ידי המועצה האירופית למחקר מענק 206,726-קליפ JU וגם לטווח ארוך גבולות תוכנית אנוש המדע מלגה כדי JK

<ol>
	<li>Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+regulons:+coordination+of+post-transcriptional+events.">RNA regulons: coordination of post-transcriptional events.</a> Nat Rev Genet. 8, 533-543 (2007).</li><li>Wang, Z., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+regulation:+from+a+parts+list+of+regulatory+elements+to+an+integrated+splicing+code.">Splicing regulation: from a parts list of regulatory elements to an integrated splicing code.</a> RNA. 14, 802-813 (2008).</li><li>Trifillis, P., Day, N., Kiledjian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+right+RNA:+identification+of+cellular+mRNA+substrates+for+RNA-binding+proteins.">Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.</a> RNA. 5, 1071-1082 (1999).</li><li>Brooks, S. A., Rigby, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+mRNA+ligands+bound+by+the+RNA+binding+protein+hnRNP+A2+utilizing+a+novel+in+vivo+technique.">Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique.</a> Nucleic Acids Res. 28, E49-E49 (2000).</li><li>Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc Natl Acad Sci. 97, 14085-14090 (2000).</li><li>Mili, S., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reassociation+of+RNA-binding+proteins+after+cell+lysis:+implications+for+the+interpretation+of+immunoprecipitation+analyses.">Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses.</a> RNA. 10, 1692-1694 (2004).</li><li>Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP+identifies+Nova-regulated+RNA+networks+in+the+brain.">CLIP identifies Nova-regulated RNA networks in the brain.</a> Science. 302, 1212-1215 (2003).</li><li>Ule, J., Jensen, K., Mele, A., Darnell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP:+A+method+for+identifying+protein-RNA+interaction+sites+in+living+cells.">CLIP: A method for identifying protein-RNA interaction sites in living cells.</a> Methods. 37, 376-386 (2005).</li><li>Licatalosi, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing.">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456, 464-469 (2008).</li><li>Yeo, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+code+for+the+FOX2+splicing+regulator+revealed+by+mapping+RNA-protein+interactions+in+stem+cells.">An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells.</a> Nat Struct Mol Biol. 16, 130-137 (2009).</li><li>Urlaub, H., Hartmuth, K., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-tracked+approach+to+analyze+RNA-protein+crosslinking+sites+in+native,+nonlabeled+small+nuclear+ribonucleoprotein+particles.">A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles.</a> Methods. 26, 170-181 (2002).</li><li>K&#246;nig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iCLIP+reveals+the+function+of+hnRNP+particles+in+splicing+at+individual+nucleotide+resolution.">iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.</a> Nat Struct Mol Biol. 17, 909-915 (2010).</li></ol>

אין ניגודי אינטרסים הכריז.

מאז פרוטוקול iCLIP מכיל מגוון רחב של תגובות אנזימטיות צעדים טיהור, זה לא תמיד קל לזהות בעיה כאשר ניסוי נכשל. כדי לשלוט על סגוליות של זיהו RNA צולבות קישור לאתרים, אחד או יותר שולטת שלילי יש לשמור לאורך כל הניסוי להשלים ומנתח חישובית הבאים. בקרות אלה יכולים להיות המדגם ללא נוגדנים, הלא צולבים תאים או immunoprecipitation מתאי בנוקאאוט או רקמה. באופן אידיאלי, ניסויים אלו השליטה לא צריך לטהר את כל מתחמי חלבון-RNA, ולכן צריך לתת שום אות על ג&#39;ל SDS-PAGE, ואין מוצרים לזיהוי לאחר הגברה PCR. תפוקה גבוהה רצף של ספריות אלה לשלוט צריך לחזור רצפים ייחודיים מעט מאוד. תאים מציאה אינם מומלצים כביקורת ריצוף, שכן רצפים וכתוצאה מכך עדיין מתאימות צולבות קישור לאתרים של אותו חלבון, אשר מטוהרים מתאי מציאה בכמויות קטנות יותר. אמצעי זהירות צריך גם לנקוט כדי למנוע זיהום עם מוצרי ה-PCR מניסויים קודמים. הדרך הטובה ביותר כדי לצמצם בעיה זו היא מרחבית נפרד צעדים לפני ואחרי-PCR. באופן אידיאלי, הניתוח של מוצרי ה-PCR וכל השלבים הבאים יש לבצע בחדר נפרד. יתר על כן, כל חבר צריך להשתמש במעבדה קובע את buffers ו ריאגנטים אחרים. בדרך זו, מקורות זיהום ניתן לזהות בקלות.

A correction was made to <a href="http://www.jove.com/details.php?id=2638">iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution</a>. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to: "...as well as 2 &#x03BC;l Turbo DNase..." instead of: "...as well as 2 ml Turbo DNase..."

A correction to the article iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution was made.

Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

עבור ג&#39;ל אלקטרופורזה והעברת הממברנה אנו ממליצים לא הוא השימוש XCell SureLock ® Mini-Cell ו XCell II ™ כתם מודול ערכת CE Mark (Invitrogen, EI0002), אשר תואם את השימוש minigels precast שונים שצוינו בכל פרוטוקול . מספר המותג והסדר של כל החומרים המשמשים מוזכר במהלך הפרוטוקול. רשימה של אנזימים שימוש בפרוטוקול מוצג בטבלה שלהלן. <table border="1" style=";text-align:right;direction:rtl"><tr><td> שם מגיב </td><td> חברה </td><td> קטלוג # </td><td> תגובות </td></tr><tr><td> חלבון Dynabeads </td><td> Invitrogen </td><td> 10001D </td><td> להשתמש חלבון G עבור נוגדן עכבר או עז </td></tr><tr><td> RNase אני </td><td> Ambion </td><td> AM2295 </td><td> פעילות יכול לשנות מן אצווה אצווה </td></tr><tr><td> T4-RNA האנזים אני </td><td> חוד </td><td> M0204S </td><td> </td></tr><tr><td> PNK </td><td> חוד </td><td> M0201S </td><td> </td></tr><tr><td> proteinase K </td><td> רוש </td><td> 03115828001 </td><td> </td></tr><tr><td> כתב עילי III הפוך transcriptase </td><td> Invitrogen </td><td> 18080044 </td><td> </td></tr><tr><td> Circligase II </td><td> Epicentre </td><td> CL9021K </td><td> </td></tr><tr><td> FastDigest ® BamHI </td><td> Fermentas </td><td> FD0054 </td><td> </td></tr><tr><td> AccuPrime ™ SuperMix אני </td><td> Invitrogen </td><td> 12342010 </td><td> זו תערובת PCR נותן את התוצאות הטובות ביותר בידיים שלנו </td></tr></table>

iclip - transcriptome-wide mapping of protein-rna interactions with individual nucleotide resolution

הרכב ייחודי הסדר המרחבי של RNA מחייב חלבונים (RBPs) על תעתיק המדריך היבטים מגוונים של תקנה שלאחר תעתיק 1. לכן, צעד חיוני לקראת הבנה תקנה תמליל ברמה המולקולרית היא להשיג מידע מיקומית על אתרי הקישור של RBPs 2. חלבון-RNA אינטראקציות ניתן ללמוד בשיטות ביוכימיות, אך גישות אלה אינן כתובת RNA מחייב בהקשר הסלולר המקורית שלו. ניסיונות ראשוניים ללמוד חלבון-RNA מתחמי בסביבה הסלולרי שלהם מועסקים טיהור זיקה או immunoprecipitation בשילוב עם תצוגת ההפרש או ניתוח microarray (RIP-Chip), 3-5. גישות אלה היו מועדים בזיהוי אינטראקציות עקיפה או שאינם פיזיולוגיים 6. על מנת להגביר את הספציפיות ופתרון מיקומית, אסטרטגיה המכונה CLIP (UV cross-linking ו immunoprecipitation) הוצג 7,8. CLIP משלב cross-linking UV של חלבונים ומולקולות RNA עם ערכות טיהור קפדני כולל ג&#39;ל אלקטרופורזה denaturing polyacrylamide. בשילוב עם תפוקה גבוהה טכנולוגיות רצף, CLIP הוכח ככלי רב עוצמה כדי לחקור אינטראקציות חלבון-RNA בקנה מידה הגנום כולו (המכונה CLIP-היטים או CLIP-seq) 9,10. לאחרונה, PAR-CLIP הוצג המשתמשת אנלוגים ribonucleoside photoreactive עבור cross-linking 11,12. למרות הספציפיות הגבוהה של נתונים המתקבלים, ניסויים CLIP לעתים קרובות ליצור ספריות cDNA של מורכבות רצף מוגבל. זה נובע בחלקו לכמות מוגבלת של שיתוף מטוהרים RNA ושני התגובות יעיל RNA קשירת הדרוש לשם הכנת הספרייה. בנוסף, תחל הרחבת מבחני ציינו כי cDNAs רבים לחתוך בטרם עת על 13 נוקליאוטידים crosslinked. CDNAs מקוצץ כאלה הם איבדו במהלך פרוטוקול סטנדרטי CLIP ספריה הכנה. פיתחנו לאחרונה iCLIP (נוקלאוטיד יחיד-CLIP רזולוציה), אשר לוכדת את cDNAs מקוצץ ידי החלפת אחד מולקולאריים הצעדים יעיל RNA קשירת עם circularization יעיל יותר cDNA intramolecular (איור 1) 14. חשוב לציין, רצף cDNAs מקוצץ מספק תובנות לגבי המיקום של אתר cross-הקישור ברזולוציה של נוקלאוטיד. אנו ליישם בהצלחה iCLIP ללמוד hnRNP ארגון C החלקיקים בקנה מידה הגנום כולו ולהעריך את תפקידה בתקנה שחבור 14.

Watch this Scientific Journal Video about iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution at JoVE.com

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

הסידור המרחבי של חלבונים RNA מחייב על תעתיק הוא הקובע המפתח של תקנה שלאחר תעתיק. לכן, פיתחנו הפרט נוקלאוטיד UV ברזולוציה crosslinking ו immunoprecipitation (iCLIP) המאפשר מיפוי מדויק גנום רחב של אתרי הקישור של חלבון-RNA מחייב.

iCLIP - Transcriptome רחב מיפוי של חלבון-RNA אינטראקציות עם רזולוציה נוקלאוטיד יחיד

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

iclip-transcriptome-wide-mapping-protein-rna-interactions-with

Research

JoVE Journal

Biology

58.1K Views.  Medical Research Council - MRC. הסידור המרחבי של חלבונים RNA מחייב על תעתיק הוא הקובע המפתח של תקנה שלאחר תעתיק. לכן, פיתחנו הפרט נוקלאוטיד UV ברזולוציה crosslinking ו immunoprecipitation (iCLIP) המאפשר מיפוי מדויק גנום רחב של אתרי הקישור של חלבון-RNA מחייב.

Video: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

לא פולשני-3D ויזואליזציה עם תת מיקרון החלטה שימוש Synchrotron-X-ray-טומוגרפיה

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

שחרור הדופמין ב מסופי Presynaptic הפרט דמיינו עם FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - שיטה לזהות Transcriptome רחב אתרי קישור של חלבונים RNA הכבילה

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

הכרומטין ניתוח אינטראקציה עם מנבא סוף תג הרצף (Chia-PET) עבור מיפוי אינטראקציות הכרומטין הבנת תקנה תמלול

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

ניטור בזמן אמת של אינטראקציות יגנד רצפטור עם פלואורסצנטי תהודת העברת אנרגיה

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

מיפוי לאחר הלוואי של תטא Burst גירוי בקורטקס השמיעתי האנושי בהדמיה תפקודית

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

מהירה ויעילה דג הזברה Genotyping באמצעות PCR עם רזולוציה גבוהה ממיסים ניתוח

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

מיפוי הגנום של תרופות-DNA בתאי אינטראקציות עם COSMIC (Crosslinking של מולקולות קטנות לבודד הכרומטין)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-seq: מיפוי הגנום של אינטראקציות החלבון-DNA על ידי תפוקה גבוהה רצף של קטעי DNA-מפוגל אדנין

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.