עבודה זו נתמכה על ידי הקרן הלאומית למדע של ארה&quot;ב מענקים # MCB-0741914 ו MCB-0843627, תמיכה תשתית נוספת נמסר במכללת ניו יורק על ידי המכונים הלאומיים לבריאות 2 RR03060 G12-26 של המרכז הלאומי למשאבי מחקר. אנו בתודה להכיר JKC רוז קבוצת במחלקה לביולוגיה באוניברסיטת קורנל הצמח למתן M82 (סוג בר) ו CM15 (מוטציה) cuticles עגבניות. אנו מודים לד&quot;ר Spyros Monastiriotis מקבוצת הנדסה כימית CCNY של פרופ &#39;אלכסנדר Couzis לעזרה נדיבה עם הניסויים AFM. אנו מודים לגב &#39;לורן Gohara לתמיכה עיצוב גרפי.

<ol>
	<li>Dom#237;nguez, E., Heredia-Guerrero, J. A., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysical+design+of+plant+cuticles:+an+overview.">The biophysical design of plant cuticles: an overview.</a> New Phytologist. 189, 938-949 (2011).</li><li>Bargel, H., Koch, K., Cerman, Z., Neinhuis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+the+plant+cuticle+and+cuticular+waxes+-+a+smart+material?.">Structure-function relationships of the plant cuticle and cuticular waxes - a smart material?.</a> Functional Plant Biol. 33, 893-910 (2006).</li><li>Jetter, R., Sch&#228;ffer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Composition+of+the+Prunus+laurocerasus+Leaf+Surface.+Dynamic+Changes+of+the+Epicuticular+Wax+Film+during+Leaf+Development.">Chemical Composition of the Prunus laurocerasus Leaf Surface. Dynamic Changes of the Epicuticular Wax Film during Leaf Development.</a> Plant Physiol. 126, 1725-1737 (2001).</li><li>Vogg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+fruit+cuticular+waxes+and+their+effects+on+transpiration+barrier+properties:+functional+characterization+of+a+mutant+deficient+in+a+very-long-chain+fatty+acid+Î²-ketoacyl-CoA+synthase.">Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid Î²-ketoacyl-CoA synthase.</a> J. Exper. Botany. 55, 1401-1410 (2004).</li><li>Isaacson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutin+deficiency+in+the+tomato+fruit+cuticle+consistently+affects+resistance+to+microbial+infection+and+biomechanical+properties,+but+not+transpirational+water+loss.">Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss.</a> Plant J. 60, 363-377 (2009).</li><li>Round, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Water+on+the+Nanomechanical+Behavior+of+the+Plant+Biopolyester+Cutin+as+Studied+by+AFM+and+Solid-State+NMR.">The Influence of Water on the Nanomechanical Behavior of the Plant Biopolyester Cutin as Studied by AFM and Solid-State NMR.</a> Biophysical J. 79, 2761-2767 (2000).</li><li>Fung, B. M., Khitrin, A. K., Ermolaev, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Broadband+Decoupling+Sequence+for+Liquid+Crystals+and+Solids.">An Improved Broadband Decoupling Sequence for Liquid Crystals and Solids.</a> J. Magn. Reson. 142, 97-101 (2000).</li><li>Morcombe, C. R., Zilm, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+shift+referencing+in+MAS+solid+state+NMR.">Chemical shift referencing in MAS solid state NMR.</a> J. Magn. Reson. 162, 479-486 (2003).</li><li>Fang, S. J., Haplepete, S., Chen, W., Helms, C. R., Edwards, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+atomic+force+microscopy+images+using+spectral+methods.">Analyzing atomic force microscopy images using spectral methods.</a> J. App. Phys. 82, 5891-5898 (1997).</li><li>Pollard, M., Beisson, F., Li, Y., Ohlrogge, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+lipid+barriers:+biosynthesis+of+cutin+and+suberin.">Building lipid barriers: biosynthesis of cutin and suberin.</a> Trends. Plant Sci. 13, 236-246 (2008).</li><li>Stark, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+studies+of+structure+and+dynamics+in+fruit+cuticle+polyesters.">NMR studies of structure and dynamics in fruit cuticle polyesters.</a> Solid State Nucl. Mag. 16, 37-45 (2000).</li><li>Schaefer, J., Stejskal, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon-13+nuclear+magnetic+resonance+of+polymers+spinning+at+the+magic+angle.">Carbon-13 nuclear magnetic resonance of polymers spinning at the magic angle.</a> J. Amer. Chem. Soc. 98, 1031-1032 (1976).</li><li>Sachleben, J. R., Chefetz, B., Deshmukh, A., Hatcher, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-State+NMR+Characterization+of+Pyrene-Cuticular+Matter+Interactions.">Solid-State NMR Characterization of Pyrene-Cuticular Matter Interactions.</a> Envir. Sci. &#38; Tech. 38, 4369-4376 (2004).</li><li>Zlotnik-Mazori, T., Stark, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+studies+of+cutin,+an+insoluble+plant+polyester.">Nuclear magnetic resonance studies of cutin, an insoluble plant polyester.</a> Macromolecules. 21, 2412-2417 (1988).</li><li>Stark, R. E., Garbow, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+relaxation+studies+of+plant+polyester+dynamics.+2.+Suberized+potato+cell+wall.">Nuclear magnetic resonance relaxation studies of plant polyester dynamics. 2. Suberized potato cell wall.</a> Macromolecules. 25, 149-154 (1992).</li><li>Bennett, A. E., Rienstra, C. M., Auger, M., Lakshmi, K. V., Griffin, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteronuclear+Decoupling+in+Rotating+Solids.">Heteronuclear Decoupling in Rotating Solids.</a> J. Chem. Phys. 103, 6951-6958 (1995).</li><li>Metz, G., Wu, X. L., Smith, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ramped-Amplitude+Cross-Polarization+in+Magic-Angle-Spinning+NMR.">Ramped-Amplitude Cross-Polarization in Magic-Angle-Spinning NMR.</a> J. Magn. Reson. Ser. A. 110, 219-227 (1994).</li><li>Peersen, O. B., Wu, X. L., Smith, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+CP-MAS+Signals+by+Variable-Amplitude+Cross-Polarization+-+Compensation+for+Inhomogeneous+B-1+Fields.">Enhancement of CP-MAS Signals by Variable-Amplitude Cross-Polarization - Compensation for Inhomogeneous B-1 Fields.</a> J. Magn. Reson. Ser. A. 106, 127-131 (1994).</li><li>lessandrini, A., Facci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+a+versatile+tool+in+biophysics.">AFM: a versatile tool in biophysics.</a> Measurement Sci. &#38; Tech. 16, 10-1088 (2005).</li><li>Ben&#237;tez, J. J., Matas, A. J., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+plant+biopolyester+cutin+by+AFM+and+spectroscopic+techniques.">Molecular characterization of the plant biopolyester cutin by AFM and spectroscopic techniques.</a> J. Struct. Biol. 147, 179-184 (2004).</li><li>Koch, K., Neinhuis, C., Ensikat, H. J., Barthlott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+assembly+of+epicuticular+waxes+on+living+plant+surfaces+imaged+by+atomic+force+microscopy+(AFM).">Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM).</a> J. Exper. Bot. 55, 711-718 (2004).</li><li>Muller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+A+Nanotool+in+Membrane+Biology.">AFM: A Nanotool in Membrane Biology.</a> Biochemistry. 47, 7986-7998 (2008).</li><li>Last, J. A., Russell, P., Nealey, P. F., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Applications+of+Atomic+Force+Microscopy+to+Vision+Science.">The Applications of Atomic Force Microscopy to Vision Science.</a> Investigative Ophthalmology &#38; Visual Science. 51, 6083-6094 (2010).</li></ol>

אין ניגוד עניינים הצהיר.

פרוטוקולים שתוארו לעיל מאפשרים אפיון מולקולרית microscale מפורטת של חומר צמחי מורכבת סורר ללא צורך פירוק כימי הרסני. כדי לחקור את השילוב של biopolyester cutin עם שומנים שונים (שעווה) השולטים הארגון המבני של האסיפה cuticular, 10 קיימנו פיקוח הליכים להסרת סלקטיבית של שעוות epicuticular ו intracuticular מתערובת cuticular הטרוגנית. מצב מוצק 13 C NMR נעשה שימוש כדי לאמוד החילוץ של רכיבים מולקולריים שעווה, וכן במיקרוסקופ כוח אטומי שימש לבחון שינויים מקבילה חספוס פני השטח. 6,11 להשוות את cross-linking היכולות של cutins מן הגן, אחת מעובדות wild-type ו פירות עגבנייה מוטנטים, מצב מוצק 13 C NMR שימש גם כדי להעריך את היחס בין CHO ו-CH 2 O moieties כימיים. מספר תכונת עיצובים של פרוטוקול זה בולטים. כמו חומרי שעווה תקיף מגוון רחב של שומנים, טיפול לציפורן פירות עם סדרה של ממסים שיש קטבים מסתעף הוא חיוני על מנת להשיג dewaxing ממצה. בנוסף, זמן dewaxing יכול לנוע בין 8 שעות ל -24 שעות בהתאם לאופי של דגימות לציפורן. כדי לחלץ שעוות epicuticular בעקביות לציפורן פירות שלמים, חובה ליישם ציפוי דבק באופן אחיד על פני השטח. מצב מוצק CPMAS 13 C NMR 12 היא שיטה מהירה איכותית לזיהוי רכיבים מבניים שונים של צמחים biopolymers הטרוגנית מאוד מסיס תוך שמירה על המאפיינים הפיזיים מוצאם; 13 NMR המסורתית פתרון של מדינה יכול לשמש גם כדי לאפיין את תערובות שעווה שחולצו. אם הערכה כמותית של קבוצות פונקציונליות הרצוי עבור פולימרים צמחיים שלמים, 5 באיכות גבוהה קיטוב ישיר קסם זווית ספינינג (ד &#39;PMAS) 13 C NMR 5,14 צריך לשמש כאמצעי משלים. Quantitation מדויק של קבוצות פונקציונליות דורש אופטימיזציה זהירה של פעמים למחזר, דופק אורכי עירור ואת העוצמה של decoupling heteronuclear. 15 decoupling heteronuclear ניתן להגדיר כוח H 1 בתחום החל kHz 50 עד 185 kHz באמצעות 16 או TPPM עמוד השדרה 7 מתודולוגיות. בנוסף פרמטרים אלה, את הרגישות של המדידות CPMAS תלוי זמן ספין המנעול הרטמן, האן במצב תואם. 15 במקום CPMAS מסורתיים, ramped-משרעת CP (Ramp-CP) הטכניקה ניתן ליישם על מנת למקסם את הצלב הקיטוב, יעילות על ידי שינוי האמפליטודה 1 H באופן ליניארי (~ 20-50%) או בנקודת השקה תוך שמירה על משרעת של כוח C 13 בשדה קבוע בתקופה ספין המנעול (או להיפך). 17,18 בביצוע מדידות CPMAS ב מינימום של RO 2 שונהTor-ספינינג תדרים חובה להבחין sidebands ספינינג מן פסגות רפאים העיקריים. במקביל מדידות שנערכו AFM במצב מגע לאפשר הדמיה ישירה של מצב פני השטח לציפורן עם מהירויות סריקה גבוהות ברזולוציה גבוהה, 19 למשל במהלך הסרת רציפה של המרכיבים דונגי. הפעלה ב AFM הקשה (ללא מגע) במצב ניתן להשתמש כתחליף לאפיון פני השטח של חומרים עדינים &quot;רכים&quot; צמחים, נמנע נזק אפשרי בשל (גזירה) כוחות בצירי גירוד של מדגם השטח. 5,20 בכל מקרה , רכישה רציפה של מספר רב של תמונות של נקודה על פני השטח משמש לזיהוי נזק לפני השטח בשל &quot;החללית על פני קרום אינטראקציות&quot; במדידות AFM. 6,21 עבור שחזור אופטימאלי, בדיקות AFM עם קבועי קפיץ מתאים משטחים רכים cuticular צריך להיות שימוש ולאחר קביעות של טמפרטורה ולחות יש לשמור. 6,15,20 </sאת&gt; בעוד התמ&quot;ג של מצב מוצק מציע הפרופיל המולקולרי של ההרכב הממוצע (בתפזורת) נכסים cuticles פירות עגבניות, הדמיה כוח אטומי מספק בדיקה פולשנית משלימה 22,23 למעקב אחר הטופוגרפיה משטח של מכלולים אלה macromolecular מורכבים להפליא. 1,2

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog no.</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pectinase</td>
			<td>TCI America</td>
			<td>P0026</td>
			<td>EC 3.2.1.15; 10 U ml-1, store in refrigerator</td>
		</tr>
		<tr>
			<td>Cellulase</td>
			<td>Sigma-Aldrich</td>
			<td>C1184-100KU</td>
			<td>EC232.734.4; 1.3 units/mg, store in refrigerator</td>
		</tr>
		<tr>
			<td>Glacial Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-100G</td>
			<td>Extremely hazardous</td>
		</tr>
		<tr>
			<td>Incubator/shaker</td>
			<td>New Brunswick Scientific Co.</td>
			<td>Model No.G24</td>
			<td>MFG No.M1036-000G</td>
		</tr>
		<tr>
			<td>Vacuum Oven</td>
			<td>Precision Scientific</td>
			<td>31566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Variac Controller</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sintered glass thimble (85 mm/25mm)</td>
			<td>VWR</td>
			<td>89056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable extraction thimble ( 80 mm/ 25 mm)</td>
			<td>VWR</td>
			<td>28320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>EMD-MX0485-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>VWR</td>
			<td>RK20789</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>VWR</td>
			<td>82027-452</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>EM-CX1050-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>H302-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>VWR</td>
			<td>89002-984</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kim wipes</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma</td>
			<td>G9752</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.6 mm fastMAS zirconia rotor</td>
			<td>Varian (Agilent)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NMR spectrometer</td>
			<td>Varian 600 NMRS</td>
			<td>standard bore magnet</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>49419</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Adamantane</td>
			<td>Sigma-Aldrich</td>
			<td>100277</td>
			<td>To calibrate 90&deg; pulse in NMR</td>
		</tr>
		<tr>
			<td>Multimode Scanning Probe Microscope (Nanoscope IIIA)</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>Version 5.30r3sr3 (2005)</td>
		</tr>
		<tr>
			<td>AFM probe (Nonconductive silicon nitride tip)</td>
			<td>Veeco (Bruker AXS)</td>
			<td>Model NP-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnetic puck</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>VWR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fruit Peeler</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>VWR</td>
			<td>89038</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and biophysical study of fruit cuticles

לציפורן, שכבת מגן הידרופובי על החלקים אוויר של הצמחים היבשתיים, משמש כמחסום מגן צדדי מדגיש ביוטיים אביוטי שונים וכן מסדיר זרימת מים מן הסביבה החיצונית. 1 biopolyester (cutin) וארוך (שרשרת חומצות שומן שעוות) יוצרים את המסגרת המבנית העיקרית של לציפורן, על שלמות תפקודית של שכבת cuticular תלוי השכבה החיצונית &quot;epicuticular&quot;, כמו גם את תערובת המורכבת biopolymer cutin ו &#39;intracuticular של שעוות 2 במסמך זה, אנו מתארים פרוטוקול מקיף. כדי לחלץ שעוות ממצה של עגבניות מסחרי (Solanum lycopersicum) cuticles פירות או להסיר שעווה epicuticular ו intracuticular ברצף סלקטיבי מורכב מ לציפורן. השיטה של Jetter ו שפר (2001) הותאם להפקת בשלבים של שעוות epicuticular ו intracuticular מ לציפורן פירות. 3,4 כדי לפקח עלתהליך של הסרת שעווה רציף, מצב מוצק בין הקיטוב קסם זווית-ספינינג (CPMAS) 13 C NMR ספקטרוסקופיה שימש במקביל במיקרוסקופ כוח אטומי (AFM), מתן מולקולרית ברמת פרופילים מבניים של חומרים בתפזורת משלימים מידע על הטופוגרפיה microscale וחספוס של פני השטח cuticular. כדי להעריך את היכולות cross-linking של cuticles dewaxed של wild-type ו יחיד הגן פירות מעובדים עגבניות מוטציה, MAS 13 C NMR נעשה שימוש כדי להשוות את הפרופורציות היחסיות של aliphatic מחומצן (CHO ו-CH 2 O) moieties כימיים. Dewaxing ממצה על ידי מיצוי Soxhlet בשלבים עם פאנל של ממיסים של הקוטביות משתנה מספק אמצעי יעיל לבודד moieties שעווה על בסיס המאפיינים הידרופובי של aliphatic שלהם ומרכיבים ארומטיים, תוך שמירה על המבנה הכימי של biopolyester cutin. מיצוי מכני של שעוות epicuticular ו Seleהסרת ctive של שעוות intracuticular, כאשר בפיקוח על ידי שיטות פיזיות משלימים, מספק אמצעים חסרי תקדים כדי לחקור את מכלול לציפורן: גישה זו חושפת את הארגון סופראמולקולרית אינטגרציה מבנית של סוגים שונים של שעווה, הארכיטקטורה של מטריקס cutin שעווה, וכן חומר כימי ההרכב של כל מרכיב. בנוסף, מצב מוצק 13 C NMR מגלה הבדלים במספרים היחסיים של CHO ו-CH 2 moieties הו כימיים עבור פירות אדומים wild-type ו מוטציה עגבניות בשלות. את שיטות תמ&quot;ג מציעים כלים יוצאי דופן כדי טביעות אצבע המבנה המולקולרי של חומרים cuticular כי הם לא מסיסים, אמורפי, ו הטרוגנית מבחינה כימית. כטכניקה קרקע סלקטיבית לא פולשנית הדמיה, AFM מספק אמצעי יעיל וישיר לחקור את הארגון המבני של האסיפה cuticular בהיקף ננומטר-מיקרומטר אורך.

Watch this Scientific Journal Video about Isolation and Biophysical Study of Fruit Cuticles at JoVE.com

Isolation and Biophysical Study of Fruit Cuticles

אברי הצמח אוויר מוגנים על ידי לציפורן, הרכבה סופראמולקולרית biopolyester שעווה. אנו מציגים פרוטוקולים כדי לפקח על הסרת סלקטיבי של EPI-ושעוות intracuticular מ cuticles פירות עגבניות על סולמות מולקולרית מיקרו ידי NMR מצב מוצק וכן במיקרוסקופ כוח אטומי, בהתאמה, על מנת להעריך את יכולת cross-linking של biopolyesters cuticular מהונדסים.

בידוד מחקר Biophysical של cuticles פירות

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

isolation-and-biophysical-study-of-fruit-cuticles

Nanyang Technological University

Research

JoVE Journal

Biology

16.6K Views.  City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies. אברי הצמח אוויר מוגנים על ידי לציפורן, הרכבה סופראמולקולרית biopolyester שעווה. אנו מציגים פרוטוקולים כדי לפקח על הסרת סלקטיבי של EPI-ושעוות intracuticular מ cuticles פירות עגבניות על סולמות מולקולרית מיקרו ידי NMR מצב מוצק וכן במיקרוסקופ כוח אטומי, בהתאמה, על מנת להעריך את יכולת cro...

Video: Isolation and Biophysical Study of Fruit Cuticles

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

בדיקת רגישות חזותית על מהירות ועל כיוון התנועה של לטאות

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

לימוד תוקפנות תסיסנית (זבובי פירות)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

מחשב שנוצר מודל החיה גירויים

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

בידוד טיהור תסיסנית נוירונים היקפיים על ידי מיון ביד מגנטי

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

מדורים פרפין-Embedded קפואים של תסיסנית סקס שרירים

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

פרי ניתוח נדיפים באמצעות האף האלקטרוני

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

לכידה סלקטיבית של 5-hydroxymethylcytosine מהדנ&quot;א הגנומי

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

הדמית סופר רזולוציה של מכונות אגף קטריאלי

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

הפקה וטיהור יעילה של רקומביננטי Murine Kindlin-3 מתאי חרקים לBiophysical לימודים

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.