January 2009


Humor me while I brainstorm about my current project. I know that publishing this places my thoughts in the public domain, but since I haven’t a hope of getting something like this into a lab setting for years, and it’s just an untested intellectual exercise without an actual sequence attached, I don’t mind.

The aim is to design an antisense oligonucleotide (AS-ON) for HIV reverse transcriptase to be used as chemotherapy for one strain of the disease. Reverse transcriptase is the enzyme that transcribes the single-stranded RNA viral genome into double-stranded DNA, like the host’s. Blocking the production of reverse transcriptase, then, would block the reproduction of the virus. The AS-ON binds to the mRNA of reverse transcriptase to block translation in one of two ways: a. it hitches onto the 5′ end to block the attachment of ribozymes, or, b. it binds and activates RNAse H, which degrades the mRNA.

In designing an AS-ON treatment, there’s a lot to take into account. First of all, you need to get it into the cells for it to be effective, but straight up DNA or RNA will be degraded before it gets there. One gets around this by using either a lipid vector or using one of the various generations of modified DNA and RNA types that have been developed to cope with this. Then you have to worry about binding specificity, immune response, toxicity, efficiency, and target availability.

Immune response becomes troubling when the AS-ON contains a high percentage of guanine and cytosine residues. GC pairs are found most commonly in bacteria and viruses, and our immune system knows it. Four or more consecutive guanines can bind back on themselves, too, removing the AS-ON from circulation. 

Targeting is complicated by the sometimes complex secondary structures of mRNA molecules, which blocks much of the molecule’s sequence. One either has to run a series of tests to determine available binding sites or just target the 5′ end and hope for the best. Unfortunately, I have to do the latter. Furthermore, to avoid side effects from misbinding, you have to blast your designed sequence against the patient species’ mRNA to make sure there are no overlaps with native proteins.

What I’ve been looking into involves Locked DNA (LNA), which is basically DNA with a methyl bridge. This confers a number of great properties: increased affinity or the mRNA substrate, high potency, resistance to degradation, and apparent lack of toxicity and enhanced cellular substrate. However, it can not activate RNAse H. We also don’t really know how much increased affinity detracts from target specificity.

To remedy that problem, I’ve decided to use a chimeric strand of LNA-DNA. This will activate RNAse H, and the LNA confers all of its good qualities while the DNA center may increase specificity (again, I can’t run in vivo trials to find out). Now, I could have used phosphorothioate (PS) DNA or 2′-O-methoxy-ethyl (MOE) RNA for the internal stretch. More trials have been run on those sorts of hybrids. PS DNA would increase the rate of cellular uptake to fantastic levels; however, it also gives increased toxicity from (it is thought) binding nuclear proteins. LNA-MOE RNA chimeric AS-ONs have been tested, and also have increased cellular uptake and effectiveness, but it won’t activate RNAse H, either, and that’s a tool I really need to exploit. There are a bunch of other 3rd generation monomers that have been developed for AS-ONs, but none of them quite offers the range of benefits that follows an LNA-DNA chimera strand.

So, LNA-DNA it is.

In other news, Fulham handily won today’s match with Portsmouth. Yeah!

As some of you surely know, this past Sunday was the 250th anniversary of Robert Burns’ birth. This is a pretty special year, actually, what with the bicentennial of Charles Darwin coming up and all. The Royal Mail is putting out stamps for both, and I’ll probably end up giving them as holiday presents, because that’s just the type of friends I have. I’m a geek in good company (what would that be: a gaggle of geese, so a google of geeks?).

All that aside, I had a fantastic time at Burns Night. Westminster Abbey held a special evening service with the Burns Society of London to honor the poet, so I took full advantage of my proximity and got there early. Every evening service is preceded by an organ concert (this one by Philip Berg, Master of the Music at The Queen’s Chapel of the Savoy. I have never heard an organ sound that articulate and melodic), but there was a piper wandering about the abbey before and during the service. As much as I love organs in big stone vaults, I have to say the pipes were the most enchanting.

Much of the service was Burn’s poetry, and all but the opening hymn were his metricizations. We may not have sung them all that well, but the enthusiasm compensated for our lack of technical skill. Fortunately there was a soloist for O My Luve’s Like A Red, Red Rose, because the wee little lad sitting in front of me was supposed to be placing the wreath before Burns’ statue, but no one had told him which wreath or which statue.*  His grandda and the society president were able to guide him along, and it was pretty endearing, but with the prompting and whatnot the singer had to add a few verses.

One of the more interesting readings was of The Cottar’s Saturday Night. It is a very astute poem, and contains the stanza:

Compared with this, how poor Religion’s pride,
in all the pomp of method, and of art;
when men display to congregations wide
devotion’s every grace, except the heart!
The Power, incensed, the pageant will desert,
the pompous strain, the sacerdotal stole;
but haply, in some cottage far apart,
may hear, well-pleased, the language of the soul;
and in his Book of Life the inmates poor enroll.

This didn’t seem to perturb the clergy any – the abbey people seem much more laid back than most – but it was not exactly what I expected to hear in that setting. The fact that the stanza made it into the program was quite refreshing and, along with the pipes playing A Man’s A Man, made me smile. Happy Birthday, Mr. Burns!

*If you’ve never been there, take my word that there are a lot of tombs and statues back in the poets’ corner. Just to get to Burns and out again I had to tread on Dickens, Kipling, Tennyson, Hardy, Browning, Carroll, and more that I didn’t have time to read. Burns’ bust is high up on the East wall, looking over Shakespeare’s left shoulder.

I am inexpressibly sorry for the young woman who was murdered at VT this past week. For her sake, I hope there is an afterlife where she can find peace. For the witnesses, too, I am sorry. I don’t know what to say. Know that the thoughts of many are with you.

Guess who got full marks on today’s geohelminth MCQ? That’s right, I just know my parasitic nematodes that well. Honestly, Nematoda (the roundworms) isn’t nearly the most disturbing phylum of parasitic worms. A few of its members are global health problems, such as Ascaris (an intestinal worm which has over a billion infections worldwide) and Wuchereria or Brugia, which both cause lymphatic filariasis (the most globally damaging infection by the W.H.O.’s Disability Adjusted Life Years measure for impact – you may be familiar with pictures of elephantiasis). However, as glad as I was that I skipped breakfast today, the phylum Platyhelminthes is more frightening. That’s the one that has digeneans (flukes) and cestodes (tapeworms), which are responsible for schistosomiasis, cysticercosis, and hydatid disease. Trust me, if you like your supper on the inside, you won’t wiki those.

After being a good student and doing my reading, I spent most of last night dreaming that I’d gotten ascariasis and hydatid disease. Perhaps fortunately this gave me insomnia, because the only other sleep I remember was dreaming that I had a Taenia solium larval cyst in my CNS and was experiencing epileptic progression. Fortunately the course lecturer is excellent, and I think that, after this term, very, very little will turn my stomach.

The thing that gets me about parasitic diseases is how little is being done to counter them, especially given the global prevalence of the problem. Above I mentioned Ascaris, which has about 1.4 billion infections worldwide. That falls into the broader category of  geohelminths, which has an estimated 3 billion infections, with 4.5 billion people at risk. There are only 6.76 billion people in the world. Now consider a few other major helminth diseases: schistosomiasis at 200+ million infections, onchocerciasis at 18 million infections, and lymphatic filariasis at 120 million infections, and the picture gets ugly. At risk, respectively, are 600 million, 120 million, and 1 billion people.* There aren’t even any cestodes on that list. Of course, a lot of these infections overlap, and that just makes them worse. Moreover, most (and by far the worst) infections are in children.

Drug-wise, between 1975 and 1995, although 1,223 new types of drugs were introduced worldwide, only 14 were for tropical diseases. In truth, most of those were developed for veterinary medicine, and came to people second.** There is no vaccine nor acquired immunity to infection, so chemotherapy would have to be administered to each infected individual every 6 months. Even with inexpensive drugs like benzamidazoles and ivermectin, that’s terrifically expensive, and a logistic nightmare. The only way we can effectively control or eliminate helminth diseases is with sanitation and piped water for washing and drinking. Give those two things to a community, and helminth infections pretty much spontaneously disappear. However, that’s a tall order for the developing world, which hasn’t the funding or the infrastructure to do much of anything. Currently, treatment relies solely on the charity of a few pharmaceutical companies and nonprofits. I keep asking myself why, and I never like my answers.

*taken from Watkins, B.M. (2004). Trends in Parasitology 19:477-8

world population count is from the US Census Bureau

**Dr. Phil Whitfield. “HB.0317 Parasitology of Tropical Disease.” Lecture series given in room 1.10 Franklin Wilkins Building, King’s College London. 15 January, 2009.

With the 5 hour time difference, the inauguration yesterday was late afternoon for me, so while our new president was enjoying his luncheon, I went and cooked supper. Usually I cook sans recipe with whatever happens to smell good at the time, and this meal was no exception. However, it did turn out exceptionally well, so I thought I might share:

Melt a slice of butter in a skillet with a liberal amount of garam masala and a few dashes of turmeric, and roll it around so it coats the pan and the spices are well mixed. Dump in one of those 180g packs of “Seafood Selection” from the grocery store (mussels, king prawns, and squid suckers) along with a sprinkling of salt, a little black pepper, and a dash or two (or three, if you’re me) of cayenne. Sauté it until the seafood is done and the spices are beginning to get a little crispy, then serve hot.

Although I didn’t have any on hand, I’d serve this on a bed of israeli couscous with a glass of dry white wine or passion fruit juice. What I had at the time, however, was a bottle of Chateauneuf du Pape for toasting Obama. It worked just fine. 🙂

Well, important history just happened again, and although I’m still trying to wrap my mind around it, I can’t say enough how proud I am to be an American, today of all days. 

Cheers from overseas!

And take care, Ted.

***

…and NASA got the last spot on the parade. Brilliant!

Today is the day! Fortunately, I have no modules scheduled for tomorrow, because I’m going to be up late watching the proceedings and doing some festivating of my own. It’ll probably all be streaming on my laptop, but I may be able to find someplace with a high-def screen that would consider switching from football to the inauguration. Of course I’m wearing my Mayanists for Obama shirt as part of the good times. Anyway, WOOOOOOOOT!

This is a bit of what I wrote just after election day. It is a bit long because of a (perhaps foolish) desire to capture every detail, so I don’t expect you to read it all. Skip to the end, if you’d like. As you can tell, despite a few days’ distance, I was still euphoric. The same is true after a few months, so I thought I’d fish it out for today:

Well, it was an exciting day from the outset. First thing I recall was waking up at 7:30 and hearing Sherri come back in the suite. She said she went down to Wohl at 6:40 to vote, but the line was already two hours long. I had worn my Obama shirt the day before, so I just left my Obama pin where it’s been all semester on my backpack and went to class. There were signs everywhere! Chalk on the sidewalks, signs on the doors, t-shirts, signs on the trashcans even, stickers, and blue soap on the door of the Subway reading “Vote Obama.” Every single window of the new polysci building had an Obama Missouri voters’ rights poster on it. That was a beautiful sight. I got stickered on my way to class with the I Voted Today one, and again when a friend and I were talking in the library with the Yes We Can one. The voting sticker I gave to Steph, who actually had voted that day in person, but had missed the stickering.

Work that day was markedly unexciting, since my boss wasn’t there and all we had to do was clean. Of the three of us, two were decked out for Obama (Ellen hadn’t been to the polls yet). Class was more interesting. Clay and Hirsch and I were early – we’re always half an hour early on Tues/Thurs – so we talked about whatever came to mind. It’s funny, but I don’t remember how much we actually talked about the election. There was the sense for me, at least, that the future was now out of my hands. I had cast my ballot weeks before, although, with the way our commissioner was acting, I was worried that it might not count. Now there was nothing to do but wait and see.

While we were all talking, there was a rustling outside the windows. It was a little, shriveled up, sour prune-faced old hag in dark glasses looking in at us from the shrubbery and scowling as if young people in class were the most disgusting aberration in the world. After giving us all a start, she proceeded to tear down the voters’ rights bills from the windows and skulk off through the bushes. I wondered aloud who peed in her cheerios. As it turned out, the university apparently got quite mad at all the flyering and spent much of the day neglecting its own recycling policies by filling the waste bins with pictures of Obama. Given their recent honoring of Phyllis Schlafly and the exorbitant amount of Student Union funds that went towards bringing Alberto Gonzalez and Karl Rove to campus – not to mention rejecting Obama’s request to speak (for free) on campus about his book – I can’t say I’m surprised. I wonder if they would have torn down McCain posters.

I hardly remember lab, though it must have been preparing our PCR products for the gel and beginning PCR on the T. sax. hindlegs. Mara and I decided to study together in Ursa’s for Wednesday’s bio exam. Between leaving lab and meeting in the café, I think I primarily watched the New York Times’ desktop monitor. I tried CNN, but they were mostly interviewing and doing nothing much useful. When Virginia’s results began coming in, I called my parents and continued wasting time fretting over what was outside my control. I just couldn’t escape the feeling that this was the most important night of my life, and I wanted to remember it, not waste it studying for some piddling assessment of my memorization skills.

As it happened, Mara and I didn’t get terribly much studying done. We tried, and we did work out a few problems, but my mind was on the neck and neck race in Virginia. Pennsylvania was already called for Obama, but if he could get Virginia…Then Mom called, saying NPR had just called Virginia for Obama. Damn, but that was a fine moment! For the first time since John F. Kennedy, my state had gone blue! At this point, I knew Obama had the election. Mara knew by my side of the conversation something was up, so I kept trying to balance two conversations at once with no success. When I hung up and could get the facts strung together, Mara and I pretty much abandoned pretense of work. CNN took a few minutes longer, but there was much cheering when they called my state. I was so proud! Steven, a staunch Republican, came over to study with us, which was pretty depressing for him. We and the staff were all so excited we couldn’t muster convincing sympathy for his despondency. And when they called the election! We were whooping and shouting and laughing. That’s what I remember most: I couldn’t stop laughing. All those months of fear and hope and dreamwalking after so many years (for one as young as I) of fear and shame and despair, all gone in a few minutes. It felt like the world had been lifted from my shoulders!

We nodded sagely through McCain’s concession and then the camera shifted to Chicago. It was the same sense of joyous expectation as on the finest of Christmas Eves as a child, and then he came on! And he spoke so beautifully while we were still laughing and floating in the air. We stayed fairly quiet through the acceptance speech, but when it was done, we just lost it. Looking at the international news reports the next day, I realized just how much the wording was geared to a global audience. It was perfect for us, and it was a powerful statement to the world at large. With that election and that one speech, I think we have done much to counter the last eight years.

After the speech, we figured studying was pointless, so Mara and I packed up and danced out the door. (Steven left just after the speech, since he was pretty down about it.) There were people whooping it up all over campus. I went up to my room to study more and was met with an extremely excited Sherri. I picked her up and we twirled around the room shouting “We won!” and “Barack my socks!” and the like and laughing all the while. I tried to sit down and focus, but with the screaming and pounding of hundreds of students celebrating on the swamp, that didn’t last long. I decided to run down to the swamp and join in for a bit, but they had moved into the dorms mostly, so I ran around part of the swamp, then out on Wydown to the park and back to the dorms – about 5k in all. And yes, I was still laughing.

A friend told me that someone over in the old dorms started playing We Are the Champions at full volume on their patio thrice through, and that the 500 person crowd eventually went back to the swamp (which I did hear several times throughout the night). It was a loud night, and I don’t know how I managed to touch the ground long enough to shower and sleep. All the while the laughter bubbled up and I thought my heart would burst. Even now, when I think back on it, I can hardly believe my own memories. I’m so proud of our country! I can hardly wait for the next four years. Mom and Dad and Charlie are going to the inauguration in January, the lucky people. I guess I’ll stay up and watch it on tv. For so many of us, for so many reasons, this was a great and triumphant moment. For so many of us, too, it was our first step in politics, and what a step! What shall we accomplish next?

So, what will it be? I have no idea, but I’ve got a few hopes, and today might make something of them. For the moment, though I’m just going to have some fun. Go out and enjoy your holiday!

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