Biomod/2013/OSU/meetingnotes: Difference between revisions

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<img src="http://openwetware.org/images/1/15/Ohiomod2013.jpg" height="60%" width="60%" style="margin-left:-40px;"> <div id="navbar" style="margin-left:auto; margin-right:auto;text-align:center;width:650px;height:auto;" > <ul id="homebutton" class="navigation-bar"> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU">Home</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/about">About</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/abstract">Abstract</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/goals">Goals</a></li>

<li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/about">Description</a> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/experiments">Project</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/experiments">Life of Project</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/labnotes">Lab Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/meetingnotes">Meeting Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/resources">Resources</a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/results">Results</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/results">Experimental Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/data">Data</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/futurework">Future Work</a></li> </li> </ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/media">Media</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/projectvideo">Project Video</a><li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/animations">Animations</a><li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/videoblogs">Video Blog</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/photos">Photos</a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/team">Team</a></li> </ul>

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<div id="lab-notes" style="width:65%;margin-left:auto;margin-right:auto;margin-top:-110px;"> <h1>Meeting Notes</h1>

Drugs Update Doxorubicin TNF Alpha – using nanoparticles to localize Which type of cancer are we targeting? Already have leukemia cells (different types) available to us Direction for continued research : Pick 3 (maybe 5) top candidates of drugs for testing that have already been tested in leukemia Will test in vitro for now, maybe in vivo down the road Shapes Update Shape involvement seems to be related to loading ability Ideas: sphere, dense vs. non-dense, surface area Use shape for delivery, ligand for targeting Nanoparticle or liposomal delivery Is uptake efficiency affected by shape? Direction for continued research : Limit to 3 designs, tube as starting point, then change aspect ratio or number of layers (density) Already have 6-helix bundle that could be used Maximize intercalation efficiency and cell uptake efficiency Targeting Update Ideas: <ol> <li>folate targeting – overexpressed in tumor site Liposomes, pei, peg (folate receptors). Size is an issue for penetrating membrane.

</li>

<li>magnetic nanoparticles – induced magnetic field at tumor site ferrofluids mixed with drug</li>

<li>peptide ligand targeting – peptides on outside of tumor capillary growth hormones</li> </ol> Direction for continued research: surface bound receptor (as opposed to secreted) Goal may be B-cell targeting, even if we can’t target malignant B-cell > proof of principal CD 20 receptor Design Direction: Focus on leukemia · Easier to test by delivering into blood stream, easily reaches cancer <ul> <li> Leukemia cells available </li> </ul> · Drugs (daunorubicin, idarubicin) already tested in leukemia Plans & Targets <ol> <li>More focused literature search on drugs and targeting</li>

<li>Look for ligands</li> <ol type="a"> <li>What is overexpressed on leukemia cell surface?</li> <li>What is commercially available?</li> </ol> <li>Begin designing and drawing cross-sections for shape ideas</li> <ol type="a"> <li>Look at degradation – If shape lasts longer, does it get closer to nucleus, etc</li> <li>Focus on targeting, effects of size/shape on uptake</li> <li>Potential mechanisms to explain difference in results between tube and triangle</li> <li>How did they measure loading efficiency?</li> <li>What shapes do we already have?</li> <li>Preliminary tests to check hypothesis for loading efficiency if no data is available</li> <li>Does intercalation affect folding?</li> <li>Hypothesis – dense would have lower loading efficiency because layers are inaccessible to solution</li> <li>Incubation period?</li> </ol> </ol> <h2>5/21/2013</h2> Drugs Update Intercalating agents: Daunorubicin (doxorubicin is a derivative of this) Mitoxantrone Used in combination with vincristine, inhibits mytosis Anthrapyrazols Rituximab commonly used for CLL but not an intercalating agent Direction: research drug mechanisms Targeting Update ROR-1 is overexpressed in CLL Monoclonal antibodies used to target How do we attach antibodies to structure? Direction : research how to target ROR-1 Shapes Update Plan: use structures already available in the lab for prelimary tests Tube (6 helix bundle) as control, adjust diameter (6, 12, 18 helix bundles), extended cylinder, density, layers (bennett linkage) From test results, develop 3 structures to test Measure intercalation loading efficiency by intensity Direction: investigate DNA structures in the lab that would be useful, research how loading efficiency was measured in doxorubicin study <h2>5/23/2013</h2> Drugs Update Drug possibilities: Daunorubicin Epirubicin Mitoxantrone All intercalating agents, none approved for treatment of CLL, mostly in combination with other drugs, few studies about efficacies on their own Possibility of using a combination of drugs? Targeting Update New research at OSU regarding dual-ligand immunoliposome using CD20 and CD37 In combination even more selective than ROR-1 Anti CD20 is commonly known as rituximab and is widely used for CLL treatment ROR-1 in 2012 study was found on healthy frozen reactive lymph nodes, but overall effective for targeting Will need biotinylated antibodies if we can get them Candidates: CD37 and ROR-1 Will attempt to contact Robert Lee’s lab for dissertation on dual ligand liposome Shapes Update Selected 5 or 6 structures with varying densities, surface areas Examples: 18 helix bundle of 2 different lengths, Bennett linkage (64 helices thick, can be held in open or closed configuration), soak Bennett linkage in open position, and then close it as a possibility <h2>5/28/2013</h2> Drugs Update Direction: Which of the three drugs is most readily used in CLL? Is it commercially available and what is the cost? Will have selection for next meeting on Thursday May 30 Targeting Update Group has selected ROR-1 for targeting It is commercially available but not biotinylated This is pending information from the OSU dual-ligand liposome study Per Chris, our lab is already working with them and collaboration is possible Direction: Read dissertation on CD20 & CD37 once available Shapes Update Presented important follow-up questions regarding time in blood stream and renal filtering Selected structures per presentation Direction: will wait for drug selection to begin loading efficiency testing Need to verify that measurement process from doxorubicin study will work for selected drug <h2>5/30/2013</h2> Targeting Update ROR1 would require characterizations of the cells o Can do with antibodies o Cannot find biotinylated ROR1 so the other option would consist of attaching streptavidin on the origami if we cannot find biotinylated ROR1. Shapes Update Shapes depend on loading efficiency – keeping in mind the concentration of drug. o The concentration of drug that will be intercalated within the origami. o The goal is the control the concentration of the drug to be less cytotoxic. Drugs Update Daunorubicin has been selected as the drug of choice between Mitoxantrone and Epirubicin. Daunorubisin has the most trials (around 146 in CLL) and epirubisin had around 11. Mioxantrone was ruled out because it attacks T-cells while CLL is in B-cells Other: We will be using MEC1 and OSUCLL cells Questions that we want answered: - Does the shape depend on uptake? - How long does Daunorubicin take to kill cells? Plans for upcoming weeks: - Tuesday: Doug and Emily will be presenting article about Doxorubicin with DNA origami. - Other days: Start thinking about WetLab, Design team, Analysis, and Modeling groups. <h2 id="june">6/4/2013</h2> Drugs Update Daunorubicin has been ordered, should arrive any day Direction: Start experimentation of cell viability with/without drugs Design: Can start viability assays without drugs immediately now as controls Options: <ol> <li>M(13) and cells</li> <li>M(13) and dauno and cells (single stranded and double stranded)</li> <li>Daunorubicin and cells</li> <li>6-helix and cells</li> <li>Bennett linkage and cells</li> <li>6-helix and dauno and cells</li> <li>bennett and dauno and cells</li> </ol> culture cells in presence of these conditions for 24 hours and then take read-outs for every 4 hours (6, 8, etc) What can we expect to see as results for these tests? Cell viability should decrease with increasing drug concentration but should not be affected by dna strand

Resistant cells? Don’t have that cell available to us at this point Intercalation loading efficiency with structures How can we control loading? <h2>6/6/2013</h2> LLS Proposal – Dr Castro is further revising, will request letter of support from Dr. Byrd to send to LLS along with our proposal Presentation on Doxorubicin Study: Doug Powerpoint will be uploaded to google drive Supporting figures: tested many of the same controls we suggested Discussed in depth how concentrations of drug were calculated (i.e. once intercalated into origami structures) Groups Wet Lab - will be soaking and preparing the different combinations we want to test, performing cytotoxicity assays Analysis/Microscope – will be looking at results under microscope, imaging Design – design origami structures, models, experiment protocol Structures are ready for testing Need to determine how to measure final concentration of supernatant Doug will write up simple beginning protocol for control testing 6/18/2013 WetLab Progress: <ul> <li> Made solution out of daunorubicin </li> <li> Purified Bennett linkage structures </li> </ul> Next Steps: · Once structures are purified, they will be soaked and incubated with drug solution for 24+ hours · May look at the effect of daunorubicin on the DNA after extended incubation times · May have difficulties measuring concentration of dense structures, will estimate if necessary · Will discuss amounts and specific protocol for incubation at meeting tonight (June 18) Direction for Microscope Group: <ul> <li> will meet to research imaging done in Bing study </li> </ul> · training on microscope with Molly – available every day at 5pm Direction for Design Group: <ul> <li> create logo for OHIOmod </li> <li> maybe go through Union for t-shirts and designs </li> <li> design protocol for making resistant CLL cells </li> </ul> <h2>6/20/2013</h2> Upcoming BIOmod deadlines <ul> <li> Abstract due in September </li> </ul> WetLab Progress: · So far, closed Bennett linkage is showing most promising results <ul> <li> 24, and 36 hour batches have been completed </li> </ul> Microscope Progress: <ul> <li> Doug and Josh have started training with Molly </li> <li> Will start next week </li> </ul> Direction for Design Group: · Dr Byrd has protocol on resistant cell line, Paul will contact him or Molly · Dr Byrd published paper with good results regarding CLL and ibrutinib – will need to research · The reason DNA origami MDR-1 receptor, transmembrane protein, stops daunorubicin and doxorubicin, DNA may be shielding the drug from this protein – this will be an important mechanism to research, especially for wiki and presentation <h2>6/27/2013</h2> WetLab Update: <ul> <li> Have tested CBL, OBL, hinge, and 18 HB </li> <li> Need to test 12 and 6 HB </li> </ul> · Need a baseline for PCR: absorbance reading after dilution is different than expected · Test absorbance with different buffers: water, FOB, PBS · Sodium levels may be dropping with dilution in water and may be causing different results · Alex already has curve from buffer excited at 488 <ul> <li> Possible reaction from PEG, odor </li> </ul> · Paul got liposomes from Dr Lee’s lab that can be used Microscope Update: · Will meet with Chris today regarding cell lines and imaging ideas <ul> <li> Chris recommends 7-AAD cell viability assays </li> </ul> <h2 id="july">7/2/2013</h2> Design Updates: Wiki is set-up, will continue to add info – Mike will send link Spreadsheet on google drive for member bio information that will go on the website Video – vote on top ideas in meeting Scope Updates : Next week: Start to look at Daunorubicin (free drug) on both cell lines (cell viability) at multiple incubation times (24, 36, 48 or 24, 48, 72) Need baseline daunorubicin concentration for comparison After: Unloaded origami structures and cell lines Loaded origami and cell lines In the meantime, start resistant cell line Will try to schedule in the morning to avoid conflicts WetLab Updates: <ul> <li> Gel baseline results should be in tomorrow </li> <li> TEM – start next week </li> </ul> Video blog – under 60 seconds each, What is OhioMod? (Pat), What is our project? (Vidhi), What is DNA origami? (Tyler) 7/9/2013 Scope Updates : <ul> <li> Started free drug experiments this week </li> </ul> · Incubating with 5 different concentrations of daunorubicin and cells only, two different incubation times (24 and 48) · At 24 hours, most of the cells are dying (MEC1 and OSU CLL cells lines) · Next will be origami controls (plain origami and cells) · After, drug-loaded structures by the end of next week, still need to establish baseline concentrations <ul> <li> Explore multiple ways to test cell viability </li> <li> Need to order more daunorubicin </li> </ul> WetLab Updates: <ul> <li> Met with Dr. Lucas from Dr. Byrd’s lab </li> </ul> · Already have resistant line that should be resistant to daunorubicin, developing drug resistant cell lines can take months · Work extensively with Silvestrol – may try to attach to origami (covalently, or intercalation) <ul> <li> They are on board with our project </li> </ul> · TEM – view loaded structures tomorrow from 12-2pm · Friday – loading efficiency results, Bennett linkages are loading more than helix bundles on average Video Blog: Paul - why cll? Mike (biochem) - how big is nano? <h2>7/11/2013</h2> Chris ordered more daunorubicin Design Updates: · Plain double stranded dna for controls: Need to lay out scaffold, staples that follow path of scaffold, no crossovers, 42 bases long, will have to force the staple crossovers to be at the same place as the scaffold crossovers Scope Updates : <ul> <li> Running free daunorubicin on controls </li> <li> Changes: may add 12 hour incubation time </li> <li> Structure controls planned for next week </li> <li> Need people available later morning </li> </ul> WetLab Updates: · Gel results: 400 micromolar – 900 micromolar (what was absorbed by structure) found using plate reader <ul> <li> TEM – reviewed images of loaded structures </li> </ul> · No sure if shapes are due to crystallization or the actual structure <ul> <li> Flat 18 helix bundles stuck together a lot </li> <li> unexplained blobs </li> <li> images on the google drive </li> </ul> · Need to determine if structures are breaking down after being stored in PBS · Will try with additional salt (Carlos suggested adding 10 millimolar mgcl) <ul> <li> Will try varying concentration of daunorubicin </li> </ul> · Image structures by themselves before for comparison <h2>7/16/2013</h2> Design Updates: <ul> <li> Went over movie ideas </li> <li> Limited to 4 minutes </li> </ul> Scope Updates : <ul> <li> Completed cell viability with structures and CLL </li> <li> 18 helix bundles killed some CLL cells </li> </ul> WetLab Updates: · 3 ready for imaging on Friday, other lab may be able to do additional imaging · making changes – structures seemed to have degreaded in the PBS by hour 40 <ul> <li> TIRF images this week </li> </ul> <h2>7/18/2013</h2> Design Updates: <ul> <li> Will start working on new structure </li> </ul> · Pat’s idea is something more square shaped and densely packed · Square lattice is more densely packed than honeycomb Scope Updates : · Resistant cells now growing – 5 different cell lines (HL60 – doxo resistant and other strains that are silvestrol resistant) · Showed images from cell viability with MEC 1 and daunorubicin at different concentrations at 24 and 48 hours <ul> <li> Now attempting a 12 hour incubation and culture </li> <li> Also ran 2 cell only controls </li> <li> Will complete counts next week </li> </ul> · Will need to consider concentration of drug delivery clinically and how our test concentrations compare WetLab Updates: <ul> <li> have more 6 and 12 helix bundles to retest </li> <li> Chris has updates to make process easier </li> </ul> <h2>7/25/2013</h2> Scope Updates : · When imaging resistant cells, they spotted something unexpected, possibly contamination of the cell line · HL60 cells are supposed to be resistant at 2-2.5 micromolar, going to adjust concentrations around this range WetLab Updates: <ul> <li> Scheduling additional TEM time </li> </ul> <h2 id="august">8/1/2013</h2> Design Updates: <ul> <li> Copyright meeting this week </li> <li> Cando – maya toolkit for animation </li> </ul> Scope Updates : · When imaging resistant cells, they spotted something unexpected, possibly contamination of the cell line · HL60 cells are supposed to be resistant at 2-2.5 micromolar, going to adjust concentrations around this range WetLab Updates: · If people are available and want to work in the lab/learn protocols before school starts, contact Pat <h2>8/15/2013</h2> Design Updates: · Script is mostly complete with movie clip/animations/etc in place · Copyright meeting last week – doesn’t seem to be a problem · Structure is mostly complete (5x6 square lattice cross-section with 4 holes) Scope Updates : · OTT (cell viability) assay yesterday, free daunorubicin at 5 different concentrations with and without structures · Bennett linkage appeared to have an impact on results relative to free daunorubicin at same concentration (10-15% difference) <ul> <li> None of the other cells had that affect </li> </ul> · Removing .1 micromolar and adding 50 micromolar for next trial, Dr Castro suggests factors of 10 (bigger change is less senstie to variability) WetLab Updates: <ul> <li> Currently incubating more structures for imaging </li> <li> TEM tomorrow </li> </ul> <h2>8/20/2013</h2> Design Updates: · Adding staple overhangs to structure and then will be ready to order <ul> <li> Need lab notes, pictures for website </li> </ul> Scope Updates : · MTT – will re run with different concentrations and without 12hb · MTT cell viability results are different than TIRF results with same concentrations WetLab Updates: <ul> <li> New folded structures have polymer </li> <li> Will wait for new structure </li> </ul> <h2>8/27/2013</h2> Design Updates: <ul> <li> Need lab notes, pictures for website </li> </ul> Scope Updates : <ul> <li> Running TIRF on Bennett linkages now </li> </ul> · Want to run MTT in the next week with more Bennett linkages WetLab Updates: <ul> <li> 488 and 480 absorbance and fluorescence </li> <li> 6 helix bundle had the highest concentration </li> <li> inconsistent results for drug loading efficiency </li> </ul> · may continue to use the 6 helix bundle for comparison to original paper <ul> <li> control double stranded scaffold is folded </li> </ul> Carl gave scaffold presentation <h2 id="september">9/5/2013</h2> General: <ul type="disc"> <li> Abstract due Sept 13, first draft is finished </li> </ul> Design Updates: <ul> <li> Website – split lab notes into two sections </li> </ul> TEAM GOALS a. More structures (Bennett linkage, double stranded, checkered structure, filled structure) i. Rachel will design second structure ASAP b. Evidence that shows circumvention of drug resistance i. MTT ii. TIRF c. Comparing shapes i. Plate reader d. Attach antibodies e. Proposed Mechanism (MDR I, or concentration) i. Site literature (Mike Haupt) f. Research DNA structures in vivo - Molly Alex gave folding presentation <h2>9/12/2013</h2> Plan for next week’s meeting: compile all results and decide next steps <h2 id="october">Week of 10/7/2013</h2>

<table border="0" cellspacing="0" cellpadding="0" align="left" width="844"> <tbody> <tr> <td width="844" colspan="7" valign="bottom"> Ohio MOD October 2013 Weeks of 10/7 10/14 </td> </tr> <tr> <td width="121" valign="bottom"> M </td> <td width="126" valign="bottom"> T </td> <td width="138" valign="bottom"> W </td> <td width="126" valign="bottom"> T </td> <td width="126" valign="bottom"> F </td> <td width="66" valign="bottom"> S </td> <td width="141" valign="bottom"> S </td> </tr> <tr> <td width="121" valign="top"> 7 <ul> <li> Gel on BL Structures (setup 11 AM – Needs cut 4PM) </li> </ul> </td> <td width="126" valign="top"> 8 <ul> <li> Seed cell plate for TIRF imaging (10:30 P.M.-12:00 A.M.) </li> <li> Gel </li> <li> Plate Reader Concentrations </li> <li> Structure incubation </li> </ul> </td> <td width="138" valign="top"> 9 <ul> <li> Wash cells with PBS (11:30-1 P.M.) </li> </ul> </td> <td width="126" valign="top"> 10 <ul> <li> Meeting 8:00AM </li> <li> End Incubation </li> <li> New Video Day 4PM-??? </li> </ul> · Seed Cell plate for TIRF on double stranded DNA and daunorubicin(~10:30 P.M. -12) </td> <td width="126" valign="top"> 11 · 11:30-12:45 Wash cells from Thursday night with PBS and put back in cell plate <ul> <li> 12:30-3 PM TIRF image cells from loaded structures </li> </ul> </td> <td width="66" valign="top"> 12 </td> <td width="141" valign="top"> 13 · 11:30 A.M.-2 P.M. TIRF image of double stranded DNA and daunorubicin controls </td> </tr> </tbody> </table> <h2>10/17/2013</h2> Lab Work Day: 10/20/2013 Goals: Last folding batch Running 3 TIRF and analysis Finalizing Presentation outline Editing Website Wet Lab Update: Pat finalized loading control experiment Higher daunorubicin concentration (10 mM) – loaded 3 times standard Thermal ramp from 37 C down – loaded double of standard incubation Room temperature (25 C) Refrigerated (4 C) Scope Update: Because of high loading efficiency, once structures are diluted to match free drug concentrations, there are not enough structures in solution to get consistent results Will test undiluted loaded structures

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+		<td colspan="2" align="left" valign="top">
+			<a href="http://openwetware.org/wiki/Biomod/2013/OSU/meetingnotes#august">
+				<img src="http://openwetware.org/images/9/9f/Calendar_03.gif" width="160" height="159" border="0" alt=""></a></td>
+		<td colspan="3" align="left" valign="top">
+			<a href="http://openwetware.org/wiki/Biomod/2013/OSU/meetingnotes#september">
+				<img src="http://openwetware.org/images/e/e7/Calendar_04.gif" width="149" height="159" border="0" alt=""></a></td>
+		<td colspan="3" align="left" valign="top">
+			<a href="http://openwetware.org/wiki/Biomod/2013/OSU/meetingnotes#october">
+				<img src="http://openwetware.org/images/8/8d/Calendar_05.gif" width="168" height="159" border="0" alt=""></a></td>
+	</tr>
+</table>
+</center>
+<div id="lab-notes" style="width:65%;margin-left:auto;margin-right:auto;margin-top:-110px;">
 <h1>Meeting Notes</h1>
@@ Line 1,097: / Line 1,121: @@
 	Plan for next week’s meeting: compile all results and decide next steps
 </p>
-<h2>9/19/2013</h2>
+<h2 id="october">Week of 10/7/2013</h2>
-<p id="october">
-	Week of 10/7/2013
-</p>
 <table border="0" cellspacing="0" cellpadding="0" align="left" width="844">
 	<tbody>
@@ Line 1,302: / Line 1,324: @@
 {{OSUFooter}}
-[http://openwetware.org/index.php?title=Biomod/2013/OSU/meetingnotes&action=edit Edit]