Friday, August 20, 2010
A visit to Robo Lab was a great way to start the week. As someone who is interested in high-throughput operations, this lab offered a glimpse into the way hundreds of thousands of samples can be tracked and processed every day. Although the system works fairly well, there are still major issues that need to be addressed, and the efficiency should go up significantly with renovations and upgrades scheduled for next year. Some of the problems with the current system were obvious (e.g. physically moving samples across long distances rather than drawing multiple samples from tubes in a single location), while other problems were more difficult to spot (e.g. using mechanical tracks that are prone to malfunctioning or contain parts that readily wear out, and using systems from multiple suppliers/manufacturers so cohesiveness becomes an issue and technical support can be difficult to receive). It was also interesting to see the way the samples are transported through the hospital via a vacuum tube system. It would be nice to see the updated and condensed Robo Lab a few years from now.
Before selecting the genes of interest for my qPCR, I spoke with a researcher in the lab who has done studies on wound healing in mouse lung and liver samples. Based on our conversation, nearly a dozen genes were identified for analysis. The first step in ordering primers is to consult the National Center for Biotechnology Information (NCBI) database in order to identify the loci of genes as well as to find the sequence. The Harvard Primer Bank is then used to identify primers that others have reported as being useful when amplifying the gene of interest. Since others in the lab will likely use these stocks of primers for cell cultures containing both human and mouse cells, it is important to use the Basic Local Alignment Search Tool (BLAST) to verify that both the forward and reverse primer for each gene is a 100% match for the mouse gene and at least one of the two primers is not a match for the human gene. Once this is done, the primer can be ordered. Once the primers arrived, the stocks were diluted to a standard concentration, the concentration of the RNA was analyzed, cDNA was made from the RNA, and the qPCR was run. Eleven genes were analyzed along with one house-keeping gene for normalization of the results.
A preliminary review of the data has revealed which genes were up regulated and which were down regulated as a result of 45 hours in the bioreactor, and a third tissue set, involving a different media, has provided even more interesting results. Unfortunately, the company hold the components of the media as a trade secret, so it will be difficult to form a strong hypotheses as to why the change in media resulted in such a dramatic change in genetic regulation.
Summer Immersion has been a wonderful experience that has helped shape my outlook on biomedical engineering. Thank you to everyone who has helped make this possible. I look forward to hearing about the experiences next year's class will have during immersion.
In addition to lab work Tuesday I was able to attend medicine grand rounds in which Dr. Muthukumar presented a lecture on "Why Organs Fail? Molecular mechanisms of Fibrosis". His discussion of cellular differentiation complemented what I had been learning in the lab, so it was well worth skipping lunch in order to attend the lecture. Much of the rest of the day was spent reading papers.
On Wednesday I was able to make histology slides from the frozen lung tissue, and I learned that it is best to inject the lungs prior to freezing to ensure they do not fall apart or shred during slicing. Unfortunately, everyone I had spoken to had omitted this step when describing the protocol, so I was left with samples that provided a bit of a challenge. Thankfully, although the process became more time consuming, good sections appear to have been produced, and any slides I make in the future will likely seem easy by comparison. This is probably the best way to learn.
Wednesday evening I was able to attend a second MRI class in which we learned how to scan the head and abdomen. It was very interesting to see the limits of the "breath hold" technique, and I now have a greater appreciation for the work done by programmers in order to reduce motion artifacts. It was nice to learn more about the fundamentals of MRI (T1 vs T2, how to get Susceptibility SPGR images and Axial FLAIR scans, in-phase vs. out-of-phase, etc.). Again, the manual for the course was very easy to follow and made the laboratory portion of the course quite enjoyable.
Thursday I was able to treat the slides with hematoxylin and eosin (H&E) stain then leave them overnight to dry, and Friday I was able to look at the slides and plan out the next stages of the project with my research mentor in better detail.
Note: The above image is from http://drsvenkatesan.files.wordpress.com/2009/04/gunther-tulip-ivc-filter.jpg
Saturday, August 14, 2010
Tuesday, August 10, 2010
The experience was not as fun as the rest of summer immersion; apparently watching surgery, imaging, and lab tests are more fun than having them performed on you. Over the course of my treatment I ended up receiving a CT Scan, an MRI, and several x-rays. While all of these imaging procedures were painless and easy the entire process of getting all the appointments did take weeks to complete. I’m not sure where the engineering solution lies, but it seems like this whole process could be streamlined and made more efficient by somehow providing patients quicker access to these tools.
After eventually obtaining all of the imaging needed to decide on a proper course of action two surgeons both suggested the same procedure. They told me my wrist was most likely to heal quickly and correctly if an autograft from my hip was taken and inserted into the space between the fractured bones. This whole mess would then be screwed closed and I’d be left with some cool new hardware and a sweet scar. After going through the information I consented to the procedure and scheduled to have it done.
The process of actually going for the surgery wasn’t too bad. The only difficult part was actually walking into the OR and seeing everybody surrounding me. Having been on the opposite side I never realized how overwhelming the number of people present for a surgery could be. With everyone busy around me doing all sorts of different things; including sticking me with needles and getting scalpels ready it was difficult to remain calm. Eventually they put me under anesthesia though and I’m a little fuzzy on the details from there on.
Recovery has progressed quickly considering all that was done. My wrist is already painless, though the bone will take months in a cast to heal fully. My hip hurt a lot at first, but is also rapidly returning to normal. After surgery I was able to return to work the next day; albeit in a little more pain; and accomplish some paperwork on my research project. While the project is not completely finished I’ve made some considerable progress towards completing it and both advisors in New York and Ithaca seem happy with my progress.
Finally receiving the surgery I needed made an excellent end to summer immersion showing me medicine from the other side. As a patient myself it was immediately much easier to relate. My research project with Dr. Cesarman provided me access to a more clinical side of research then I’ve ever seen and taught me a toolbox of new techniques. Overall my progress has been successful and hopefully with a little more work will lead to publication. Further, we’ve received a small pilot grant through the Weill Cornell Clinical and Translational Pilot center to continue the project.
This program has provided an excellent opportunity and served as a great crash course in medicine. During these seven weeks I was able to learn much more than if I had remained in Ithaca and the experience has helped me develop as a researcher. Thanks to Dr. Wang, Dr. Frayer, Belinda Floyd, and Mitch Cooper for all their time setting this program up; it ran great!
The first task didn’t appear too difficult, but as I began I realized it was going to be hard to detect whether or not my oligonucleotides attached to the gold nanoparticles. I had thought the added mass of the oligonucleotides would cause a shift in the SPR of the particles, but realized because my oligonucleotides were short this wasn’t going to be the case. The aggregation of gold particles would still work; I just needed another method of determining whether the first step of my preparation had completed.
Ultimately I came up with another test to determine whether the oligonucleotides attached to the gold nanoparticles. Specifically, gold nanoparticles react with salt and precipitate out of solution. However, if the surface of the particles is protected by oligonucleotides the particles remain stable in suspension. This stability is also particularly important because DNA hybridization is normally performed in the presence of salt, and will be required for the next step. By running an array of samples changing the concentration of salt and whether or not oligonucleotides had been bound to the gold nanoparticles I was able to confirm their presence. Looking at the figure the gold nanoparticles with no nucleotides rapidly precipitate out of suspension and the solution loses its color. However, once a monolayer of oligonucleotides is attached to the particles they remain in suspension for days.
The next step is to actually test these nanoparticles with DNA from cell lines infected with the virus and see if I can get a color change reaction. So far I’ve been unsuccessful, but I have another week to try. I think the problem might be the proximity of the gold nanoparticles; even though the lengths of DNA I chose closely match the literature I’m worried the particles are still too far. I have some particles with larger radii which I plan on trying which might help alleviate this problem.
This week I also visited the Emergency Room which so far has been one of my favorite places in the hospital. I really enjoyed the fast pace of and was able to see how quick, easy to use technology would really benefit the patients here. I continued in pathology too and observed more patients and cases. I also saw a mortality conference on one case I had followed and was really interested to see all of the information presented together. Another great week in New York!
By week five enough of my materials had arrived to start experiments. I had obtained my gold nanoparticles and my PCR Primers, though not some specific thiol modified oligonucleotides. Just a recap, I’m working on a detection mechanism for Kaposi’s Sarcoma causing herpes virus based on changes in the optical properties of gold nano particles when they aggregate and are packed tightly together. By functionalizing the gold nanoparticles with two different oligonucleotides which are non complimentary and then introducing a third oligonucleotide which is complimentary to both the particles can be caused to aggregate. If this third oligonucleotide is from a virus such a scheme could be used to determine whether or not the virus is present.
Because the thiol modified oligonucleotides hadn’t arrived yet I started with regular oligonucleotides I had ordered with the same sequences which could be used for PCR. Using these PCR Primers I should be able to detect viral DNA using more conventional means and test whether or not my DNA sequences will actually detect KSHV and nothing else. DNA was extracted from two cell lines, one positive for KSHV and one not, and also from a mouse tail clipping. The DNA was prepared for PCR with my primers and the results were run through a gel.
The results came out positive; only the cell line positive for KSHV showed the PCR product while all other cell lines and a water control were negative. The product was the right size given the primers and was seen in both KSHV positive samples.
My clinical experiences continued on as normal this week and I observed more brain dissections and histology. Overall this was another successful week and I was happy to begin making real progress in my research.