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August 27, 2005
Missourified
I've been down in Missouri for the past week. And oh man...the experiences. There is a lot to catch up on... my visit to the Tiburon, my visit to Fuji-Ya, the farewell party...
I'll get to those reviews in time; when I have the time to contemplate my navel. So yeah... Missouri:
Big news! The speed limit on the interstate in Iowa has been raised to 70! Finally I can drive 80 m.p.h. and not have to look over my shoulder constantly.
So I decided to make a visit to Columbia to see Adam at Mizzou. All in all it was a nice trip. I got to see the campus, I ran into my cousin, we played some wiffleball, and I got to hear the word "faggot" used more in one day than I have heard in the entire year. Didn't see too many homos out and about on campus. Well, not like on the campus at Minnesota where I can just sit down in front of Coffman and run into at least one homo I know in less than 5 minutes on a good day.
Despite the lack of a gay presence on campus, I'm making a bet that I was staying with a homosexual (we'll call him T for short). Yes, he has a girlfriend. But here's why he's gay: (1) We had unusual eye contact when we first met (2) His girlfriend is a spaz, they've been dating a year, she's the first girl he's kissed and he's never had sex with her and they don't even sleep in the same bed (3) He's not bad looking, I'd even say he's attractive (4) He's in engineering, and I think I would have been in the same boat as he is if I had gone to Mizzou given the lack of visible homosexuality there and (5) He is from Cape Girardeau and really has never experienced an urban center i.e. Chicago or even Minneapolis.
Does he love his girlfriend? Yes. Is he sexually attracted to her? Hmmm...
Yes all of this is circumstantial so please dont' tell me I'm trying to out this guy, because I'm not. He is what he is, he is what he says he is, and he is what he believes himself to be. But then again...I call 'em like I see 'em.
While Adam went to a meeting for Campus Crusade for Christ I decied to check out the local mall. Yes, it's one story and anchored on one end by a Target. But they have all the stores that have become typical: Express, A&F, AE, Hollister, and so forth. The lack of coffee stores in this town really upset me. I had to go to the weirdiest coffee shop I've ever seen to get a latte because I was getting drowzy. This was a low class coffee shop folks. I guess it's representative of their level of reading capacity in this part of the state.
When I went into Abercrombie & Fitch I probably flirted the most out of the whole time I was there. There was a guy working there and we played the 'looking game' with each other for a while. I wonder what it must be like for him; he has to live out here in the middle of nowhere. Maybe he can get transferred somewhere gayer. I should have given him my number.
After the mall I went to SoCo Club to experience Mid-Missouri's biggest gay club. I went on a Thursday night, all by myself (Yikes!) I'll have to check out 18+ night on Friday some time when I'm back. And be sure to get there early because I had to wait because they reached max capacity.
The drag show itself was okay, worthy of something at the 90s. However...the clientele was just a little lesbionic. Like, it was almost 70% lesbians to gay men. And at that, the gay men were rather odd. Which is to be expected in small towns such as these. Another shocker was the amount of smoke in the building. It's nice to leave these cities to appreciate the clean air of the bars in Minneapolis.
And Happy Birthday Kristina! It's gonna be a rockin' time I know. Twenty-One is always a rockin' year.
Posted by piep0058 at 06:04 PM | Comments (2)
August 16, 2005
Finally A Break
It's been a long time. I've been really busy with my internship and just getting things done and going out and having fun that I haven't had the down time to actually just sit at my computer.
So yeah, what's been up?
I finally made it to A Rebours Bistro. (sorry they don't have a website). It's on St. Peter Street in downtown St. Paul inside the renovated Hamm building. Did I like it? That is quite the understatement. Of course I liked it! There was so much food though! The next time I go back (and I will go back soon) I will have to order less.
The food in general was very well put together. The portions were larger than what you might find at Levain or Cosmos or Red. One entree here will fill you up. I got the salmon, the wiener schnitzel, the foie gras, and the lax. Too much food indeed. And I think I'm going to give up on foie gras, for a while at least. My co-worker at the pasta bar feels it's too inhumane. And so I did some very credible research on Google and many of the websites make it appear to be rather inhumane. (It involves force-feeding ducks and geese to make their liver grotesquely large.) Yes, it tastes good. But I think I need to find out some more facts than just doing a Google search.
Then came my alcoholism. Yes, folks, the past two weeks have seen some of my heaviest drinking. Thirty per week. Going out every night. Having a whole bottle of wine to myself. Forgetting what I did. It needs to stop. And it will. Yeah, I don't have any responsibilities at all really, I just need to cut back on the booze for when I actually do have responsibilities. And for the safety of my liver.
I really just wanted to get rid of my alcohol. And I did. Except for my black cherry smirnoff and my wine. I think I'll pawn off my vodka to my roommates for the party this weekend and just stick to wine/beer when school starts again. So I might pop open a bottle this week before I leave for Missouri. But I'm in no rush to get back to drinking.
I went to Valley Fair yesterday to get the life back into me. Nothing says ENJOY LIFE OR ELSE than feeling like you're falling fast to your death. I went through this whole acceptance that I am going to die at least seven times yesterday. And that whole process of letting go once things are out of your control was quite enjoyable. That might not be the allure for other people of roller coasters and thrill rides, but that's why I like them.
Posted by piep0058 at 12:13 PM | Comments (5)
August 11, 2005
DONE FOREVER!
Okay so, here's my final paper! Is it crap? Yes. Am I too tired to care? Yes. Sorry you might not be able to see the figures...
Increasing Antibody-Antigen Binding Affinity of Humanized Murine Antibody 11E6
Joseph Pieper, Jennifer Maynard
Bioinformatic Summer Institute, University of Minnesota – Twin Cities
Introduction
Despite the availability of vaccines for whooping cough in the past 50 years, it still remains a childhood illness with no available therapy for the established disease. The disease itself is caused by a toxin secreted by the Bordetella pertussis bacterium. The overall goal aims to find appropriately engineered anti-pertussis toxin antibodies that will be effective in ameliorating the disease. The belief that antibodies are capable of neutralizing and reversing the effects of the toxin is supported by three lines of evidence: (i) reduced virulence of bacteria lacking pertussis toxin genes; (ii) demonstrated efficacy of the acellular pertussis vaccines (comprised of pertussis toxin and 0-4 additional virulence factors); and (iii) passive immunotherapy studies which have demonstrated protection and even reversal of disease post-infection. For this project in particular, the nature of antibody-antigen binding will be elucidated in order increase the affinity of the engineered antibodies, specifically humanized antibodies that have shown high affinity to the pertussis toxin.
Currently, two murine antibodies, 1B7 and 11E6 have been found to have high affinity to the pertussis toxin. The second stage of the project, humanization of the murine antibodies has also been completed. However, the humanized versions of both antibodies do not retain the same binding affinity to the toxin that were exhibited in the murine versions. Correcting this loss of affinity in the humanized version will require an in-depth examination of the nature of the sequence of amino acids in the antibody framework (FR) and the amino acids in the complementary determining region (CDR).
Because of the immunogenic result of administering murine antibodies for human use, humanization of these murine antibodies is necessary for human use. Human hosts generate human anti-murine antibodies (HAMA) as the result of the introduction of murine antibodies. This can eventually result in the elimination of these antibodies from the body or dangerous reactions with other cellular functions. Humanized antibodies exhibit less immunogenic effects than their murine counterparts.
The process of humanizing murine monoclonal antibodies begins with identifying murine antibodies that are successful in binding to a specific pathogen. The specific amino acid sequence of the antibody is subsequently analyzed and the CDRs are identified. The portions of the framework in the mouse antibody are then replaced with human framework (see figure 1). Good candidates for human framework are ones that are shown to express and fold well in E. coli and framework sequences in already FDA-approved therapeutics.
Figure 1: Humanization of Murine Antibody (source: Maynard).
Figure 2: ELISA results for m11E6 and hu11E6. ELISA plates were coated with scAb titration series and detection with goat-anti-huCk or goat anti-mouse IgG-HRP. The humanized version has lost the reactivity seen in the murine version. (source: Maynard)
In this specific investigation, the two murine antibodies, 1B7 and 11E6, were shown to have sufficient binding affinity to the pertussis toxin. In addition, the administration of the two antibodies to mice infected with the toxin was shown to have promising results. When 1B7 was administered to 25 infected mice, 25 survived compared to one in the control. Twenty-three of the 25 infected mice survived with 11E6 (Maynard). However, the humanized versions of these antibodies lost much of their affinity to the toxin and therefore lost their effectiveness as potential therapeutics (see figure 2).
Many factors affect antibody-antigen binding starting from the primary amino acid sequence all the way to the tertiary structure of the antibody. The amino acids of interest are those that are exposed and see interaction with solvents and antigens rather than amino acids that are buried in the framework.
The solution to the lost affinity of the humanized versions of 1B7 and 11E6 lies in identifying key amino acids in the framework which indirectly affect antibody-antigen binding in the CDRs. The correct amino acid substitutions at these sites will allow the humanized version of the antibodies to mimic the affinity of the murine version.
Selecting proper amino acid substitutions fell under five criteria: (i) the amino acid is closer to the CDR and therefore more exposed to antigen-interaction (ii) the amino acid conserves the hydrophobicity of the original amino acid; (iii) if it is a charged species, the amino acid conserves the charge of the original amino acid; (iv) the amino acid is comparable in size to the original amino acid; (v) the proposed amino acid substitution returns matching human results after the proposed sequence is entered into BLAST and the Thornton group’s structural alignment server (SAS). When submitting a sequence, only one variable region is used, either light or heavy, not both.
Results and Discussion
This project in particular focused on the correcting the substitutions in 11E6. The following sequences show matches after submitting both 11E6 amino acid sequences into BLAST.
11E6 VL (first two are the mouse and human sequence, respectively)
DIVMTQSTSSLSASLGDRVTISCRASQDIDNYLSWFQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISSLDQEDIATYFCQQGNTFPWTFGGGTKLEIKRT
DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLSWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTFPWTFGQGTKVEIKRT
BLAST matches
DIQLTQSPSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK
DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIK
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEHEDIATYFCQQGSTLPRTFGGGTKLEIKR
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQKKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDIATYFCQQGYTLPYTFGGGTKLEIKR
DIVMTQSTSSLSASLGDRVTISCRASQDIDNYLSWFQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISSLDQEDIATYFCQQGNTFPWTFGGGTKLEIKRT
DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIHYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK
DIQMTQSPSSLSASVGDRVTITCRASQDINNYLNWYQQKPGKAPKLLIYYTSTLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPPTFGAGTKVEIKRT
DIQMTQSPSSLSASVGDRVTITCRASRDIKSYLNWYQQKPGKAPKVLIYYATSLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGQGTKVEIKRT
DIQMTQSPSSVSASVGDRVTITCRASQDISTWLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFSLTINSLQPEDFATYYCQQANSF-FTFGGGTKVEIKRT
DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQHGESPWTFGQGTKVEIKRT
DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSAPRTFGQGTKVEIKRT
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
11E6 VH (first two are mouse and human sequence, respectively)
EVKVVESGGGLVQPGGSLRLSCTTSGFTFTDYYVSWVRQPPGKALEWLGFIRNKVNGYTTEFSSSVKGRFTISRDNSQSILYLQMNTLRVEDSATYYCARVSYYGRGWYFDYWGQGTTLTVSS
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYVSWVRQAPGKGLEWVSFIRNKVNGYTTEFSSSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSYYGRGWYFDYWGQGTLVTVSS
BLAST matches
EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQPPGKALEWLGFIGNKANGYTTEYSASVKGRFTISRDKSQSILYLQMNTLRAEDSATYYCTRDR--GLRFYFDYWGQGTTLTVSS
EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKADGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCTRDPYGPAA----YWGQGTLVTVSS
EVKLVESGGGLGQPGGSLRLSCATSGFTFTDYYFNWARQPPGKALEWLGFIRNKAKGYTTEYSASVKGRFTISRDNSQGILYLQMNTLRAEDSATYYCARWGSYA----MDYWGQGTSVTVSS
EVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKGNKYTTEYSASVKGRFIVSRDTSQSILYLQMNALRAEDTAIYYCAR-NYYGSTWYFDVWGAGTTVTVSS
EVKVVESGGGLVQPGGSLRLSCTTSGFTFTDYYVSWVRQPPGKALEWLGFIRNKVNGYTTEFSSSVKGRFTISRDNSQSILYLQMNTLRVEDSATYYCARVSYYGRGWYFDYWGQGTTLTVSS
QVQLVESGGGLVQPGGSLRLSCATSGFTFTDYYMSWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDGSYA----MDYWGQGTSVTVSS
EVQLLESGGGLVQPGGSLRLSCATSGFTFTDYYMNWVRQAPGKGLEWLGFIGNKANGYTTEYSASVKGRFTISRDKSKSTLYLQMNTLQAEDSAIYYCTRDR--GLRFYFDYWGQGTLVTVSS
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKEREIVSAVSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLKKYA----FDYWGQGTLVTVSS
EVQLVESGGGVVQPGRSLRLSCSTSGFTFSDYYMYWVRQAPGKGLEWVAYMSNVGAITDYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGTRDGS--WFAYWGQGTPVTVSS
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCAR--NLGPSFYFDYWGQGTLVTVSS
QVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVALISYDESNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVKFYDPTAPNDYWGQGTLVTVSS
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
EVHLLESGGNLVQPGGSLRLSCAASGFTFNIFVMSWVRQAPGKGLEWVSGVFGSGGNTDYADAVKGRFTITRDNSKNTLYLQMNSLRAEDTAIYYCAKHRVSYVLTGFDSWGQGTLVTVSS
Color Key:
Black – CDR
Red – E/D (aspartic acid & glutamic acid) (polar/acidic)
Blue – H/K/R (histidine & lysine & arginine) (polar/basic)
Orange – N/Q (asparagine & glutamine)
Turquoise – T/S (threonine & serine)
Gold – M/C (methionine & cystine)
Green – F/Y/W (phenylalanine & tyrosine & tryptophan)
Dark green – P (proline)
Violet – G/A/L/V/I (glycine & alanine & leucine & valine & isoleucine)
Notable Substitutions
For the light variable region, in FR4 a glycine in the murine version changes to a glutamine in the humanized version. In addtion, the heavy chain had the following amendments: (i) in FR1 two threonines in the murine version changed to two alanines; (ii) in FR2 a leucine and a glycine changed to a valine and a serine; (iii) in FR3 a threonine is changed to a valine. While the other substitutions were not ignored, they were deemed less important because they did not meet the pre-stated criteria. Many were ignored because they cause no structural changes to the antibody’s CDRs. Other substitutions were ignored due no clear pattern in the BLAST hits, or the amino acid that was substituted still conserved the chemical properties of the original amino acid.
Antibody Modeling
Antibody modeling was done through the use of the University of Bath’s web antibody modeling (WAM) server: http://antibody.bath.ac.uk/abmod.html. For this project, WAM files were created and displayed in Swiss PDB viewer.
Above: the original humanized 11E6; CDRs are in green. Below: Revised humanized 11E6; CDRs are in blue. Frameworks for both antibodies are in white.
Below: Carbon backbone alignment of both humanized 11E6 antibody variable regions using Swiss PDB viewer. CDRs for original humanized version is in green; revised humanized version CDRs is in green. Notable substitutions have been labeled.
The revised antibody retains the similarities in the framework of the humanized 11E6 while mimicking the murine CDR regions (as seen above). The optimized version should retain the less immunogenic characteristic of the original humanized version, while still being effective at neutralizing the effects of the pertussis toxin.
The next step of this project is the creation of clones of the new humanized 11E6 antibody and the insert the genetic sequence of the plasmid into E. coli. After protein expression, the binding affinity of these new antibodies will then be measured using enzyme-linked immunosorbent assay (ELISA). If the proposed revisions to the humanized version increase the affinity, further clinical trials on its effectiveness in host will then be studied.
Acknowlegements
Jennifer Maynard, University of Minnesota
Susan Crennell, University of Bath
Yiannis Kaznessis, University of Minnesota
Posted by piep0058 at 02:06 PM | Comments (6)
August 01, 2005
Restaurant Levain
Friday night I was allowed the privilege to enjoy a dinner at Restaurant Levain with some great company. We opted for the five-course dinner. It's been a while since I've enjoyed such great food. While everything I tasted was definitely superb in quality, I must say that the duck [with a whole bunch of other stuff going on] was my favorite part of the meal. The only thing preventing me from going back here anytime soon would be the price. This is a once-or-twice-a-year kind of restaurant, at least for me. Well, I guess if I end up having fifty dollars lying around the house every week, it wouldn't be a bad thing to stop in.
The quality is definitely worth the money though. The service was great, the style was great (simple, but definitely refined), the food was great, and the wine was great. We brought our own wine, a cabernet sauvignon from a winery near Walla Walla, WA, but I'm sure the wines that they supply would be excellent as well.
The only critique I would give, and perhaps it's just because I am by no means an expert epicurean, is that there was so much going on with each dish. Sure, it was either duck, bass, tomatoes, whatever the dish was, but it had a minute long trail of extra flavor that was put in, or what exactly was on the side. I stopped listening after I heard "duck" and whatever the sides in the sauce she said I simply ignored, mainly because listening to whatever our server said would mean nothing. Again, I am but a lowly midwestern boy, who knows nothing of such haute cuisine. Bottom line: try to condense what comes after the word "duck" or "bass" or whatever the main part of the dish is [like, cut the time in half].
Will I go back? YES! But only for a special occasion. Like if a family member were to come and visit me. Or I get hired somewhere.
Posted by piep0058 at 11:02 AM | Comments (0)